Question: Mice without IFN-γ or its receptor are resistant to EAM induced with α-MyHC/CFA.

Title: CD11b+ monocytes abrogate Th17 CD4+ T cell-mediated experimental autoimmune myocarditis. Content: Experimental autoimmune myocarditis (EAM) represents a Th17 T cell-mediated mouse model of postinflammatory heart disease. In BALB/c wild-type mice, EAM is a self-limiting disease, peaking 21 days after alpha-myosin H chain peptide (MyHC-alpha)/CFA immunization and largely resolving thereafter. In IFN-gammaR(-/-) mice, however, EAM is exacerbated and shows a chronic progressive disease course. We found that this progressive disease course paralleled persistently elevated IL-17 release from T cells infiltrating the hearts of IFN-gammaR(-/-) mice 30 days after immunization. In fact, IL-17 promoted the recruitment of CD11b(+) monocytes, the major heart-infiltrating cells in EAM. In turn, CD11b(+) monocytes suppressed MyHC-alpha-specific Th17 T cell responses IFN-gamma-dependently in vitro. In vivo, injection of IFN-gammaR(+/+)CD11b(+), but not IFN-gammaR(-/-)CD11b(+), monocytes, suppressed MyHC-alpha-specific T cells, and abrogated the progressive disease course in IFN-gammaR(-/-) mice. Finally, coinjection of MyHC-alpha-specific, but not OVA-transgenic, IFN-gamma-releasing CD4(+) Th1 T cell lines, together with MyHC-alpha-specific Th17 T cells protected RAG2(-/-) mice from EAM. In conclusion, CD11b(+) monocytes play a dual role in EAM: as a major cellular substrate of IL-17-induced inflammation and as mediators of an IFN-gamma-dependent negative feedback loop confining disease progression.

Title: the Development of Autoimmune Myocarditis in Mice by an Content: BACKGROUND Interleukin (IL)-12 exerts a potent proinflammatory effect by stimulating T-helper (Th) 1 responses. This effect is believed to be mediated primarily through the activation of STAT4 and subsequent production of interferon (IFN)-gamma. Methods and Results- We examined the role of IL-12 receptor (IL-12R) signaling in the development of murine experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization. Both IL-12Rbeta1-deficient mice and STAT4-deficient mice were resistant to the induction of myocarditis. Treatment with exogenous IL-12 exacerbated disease. We questioned whether IFN-gamma is required for the disease-promoting activity of IL-12. On the contrary, we found that IFN-gamma suppresses EAM. Lack of IFN-gamma due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Spleens from IFN-gamma-deficient mice immunized with cardiac myosin showed increased cellularity; greater numbers of CD3+, CD4+, CD8+, and IL-2-producing cells; and heightened ability to produce cytokines on stimulation in vitro. Treatment of mice with recombinant IFN-gamma suppressed the development of myocarditis. CONCLUSIONS IL-12/IL-12R/STAT4 signaling promotes the development of EAM. In contrast, IFN-gamma plays a protective role. The disease-limiting effects of IFN-gamma might be explained by its ability to control the expansion of activated T lymphocytes.

Title: Myeloid differentiation factor-88/interleukin-1 signaling controls cardiac fibrosis and heart failure progression in inflammatory dilated cardiomyopathy. Content: RATIONALE The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self-antigen-presenting cells and promotes autoreactive CD4(+) T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. OBJECTIVE The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. METHODS AND RESULTS Using alpha-myosin heavy chain peptide (MyHC-alpha)-loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88(-/-) mice with similar distributions of heart-infiltrating cell subsets and comparable CD4(+) T-cell responses. Injection of complete Freund's adjuvant (CFA) or MyHC-alpha/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88(-/-) mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow-derived cells was critical for development of cardiac fibrosis during progression to heart failure. CONCLUSIONS Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

Title: Lethal autoimmune myocarditis in interferon-gamma receptor-deficient mice: enhanced disease severity by impaired inducible nitric oxide synthase induction. Content: BACKGROUND Interferon-gamma (IFN-gamma) is an essential cytokine in the regulation of inflammatory responses in autoimmune diseases. Little is known about its role in inflammatory heart disease. METHODS AND RESULTS We showed that IFN-gamma receptor-deficient mice (IFN-gammaR(-/-)) on a BALB/c background immunized with a peptide derived from cardiac alpha-myosin heavy chain develop severe myocarditis with high mortality. Although myocarditis subsided in wild-type mice after 3 weeks, IFN-gammaR(-/-) mice showed persistent disease. The persistent inflammation was accompanied by vigorous in vitro CD4 T-cell responses and impaired inducible nitric oxide synthase expression, together with evidence of impaired nitric oxide production in IFN-gammaR(-/-) hearts. Treatment of wild-type mice with the nitric oxide synthetase inhibitor N:-nitro-l-arginine-methyl-ester enhanced in vitro CD4 T-cell proliferation and prevented healing of myocarditis. CONCLUSIONS Our data provide evidence that IFN-gamma protects mice from lethal autoimmune myocarditis by inducing the expression of inducible nitric oxide synthase followed by the downregulation of T-cell responses.

Title: Transgenic Interleukin 10 Prevents Induction of Experimental Autoimmune Encephalomyelitis Content: The effectiveness of interleukin 10 (IL-10) in the treatment of autoimmune-mediated central nervous system inflammation is controversial. Studies of the model system, experimental autoimmune encephalomyelitis (EAE), using various routes, regimens, and delivery methods of IL-10 suggest that these variables may affect its immunoregulatory function. To study the influence of these factors on IL-10 regulation of EAE pathogenesis, we have analyzed transgenic mice expressing human IL-10 (hIL-10) transgene under the control of a class II major histocompatibility complex (MHC) promoter. The hIL-10 transgenic mice are highly resistant to EAE induced by active immunization, and this resistance appears to be mediated by suppression of autoreactive T cell function. Myelin-reactive T helper 1 cells are induced but nonpathogenic in the IL-10 transgenic mice. Antibody depletion confirmed that EAE resistance is dependent on the presence of the transgenic IL-10. Mice expressing the hIL-10 transgene but not the endogenous murine IL-10 gene demonstrated that transgenic IL-10 from MHC class II–expressing cells is sufficient to block induction of EAE. This study demonstrates that IL-10 can prevent EAE completely if present at appropriate levels and times during disease induction.

Title: Mice deficient in CD4 T cells have only transiently diminished levels of IFN-gamma, yet succumb to tuberculosis. Content: CD4 T cells are important in the protective immune response against tuberculosis. Two mouse models deficient in CD4 T cells were used to examine the mechanism by which these cells participate in protection against Mycobacterium tuberculosis challenge. Transgenic mice deficient in either MHC class II or CD4 molecules demonstrated increased susceptibility to M. tuberculosis, compared with wild-type mice. MHC class II-/- mice were more susceptible than CD4-/- mice, as measured by survival following M. tuberculosis challenge, but the relative resistance of CD4-/- mice did not appear to be due to increased numbers of CD4-8- (double-negative) T cells. Analysis of in vivo IFN-gamma production in the lungs of infected mice revealed that both mutant mouse strains were only transiently impaired in their ability to produce IFN-gamma following infection. At 2 wk postinfection, IFN-gamma production, assessed by RT-PCR and intracellular cytokine staining, in the mutant mice was reduced by >50% compared with that in wild-type mice. However, by 4 wk postinfection, both mutant and wild-type mice had similar levels of IFN-gamma mRNA and protein production. In CD4 T cell-deficient mice, IFN-gamma production was due to CD8 T cells. Thus, the importance of IFN-gamma production by CD4 T cells appears to be early in infection, lending support to the hypothesis that early events in M. tuberculosis infection are crucial determinants of the course of infection.

Title: Beta interferon controls West Nile virus infection and pathogenesis in mice. Content: Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.

Title: An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection Content: Tuberculosis, a major health problem in developing countries, has reemerged in recent years in many industrialized countries. The increased susceptibility of immunocompromised individuals to tuberculosis, and many experimental studies indicate that T cell-mediated immunity plays an important role in resistance. The lymphokine interferon gamma (IFN-gamma) is thought to be a principal mediator of macrophage activation and resistance to intracellular pathogens. Mice have been developed which fail to produce IFN-gamma (gko), because of a targeted disruption of the gene for IFN-gamma. Upon infection with Mycobacterium tuberculosis, although they develop granulomas, gko mice fail to produce reactive nitrogen intermediates and are unable to restrict the growth of the bacilli. In contrast to control mice, gko mice exhibit heightened tissue necrosis and succumb to a rapid and fatal course of tuberculosis that could be delayed, but not prevented, by treatment with exogenous recombinant IFN-gamma.

Title: Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy Content: Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Title: Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction. Content: Induction of the interferon (IFN)-alpha/beta gene transcription in virus-infected cells is an event central to innate immunity. Mice lacking the transcription factor IRF-3 are more vulnerable to virus infection. In embryonic fibroblasts, virus-induced IFN-alpha/beta gene expression levels are reduced and the spectrum of the IFN-alpha mRNA subspecies altered. Furthermore, cells additionally defective in IRF-7 expression totally fail to induce these genes in response to infections by any of the virus types tested. In these cells, a normal profile of IFN-alpha/beta mRNA induction can be achieved by coexpressing both IRF-3 and IRF-7. These results demonstrate the essential and distinct roles of thetwo factors, which together ensure the transcriptional efficiency and diversity of IFN-alpha/beta genes for the antiviral response.

Title: Dual role of the IL-12/IFN-gamma axis in the development of autoimmune myocarditis: induction by IL-12 and protection by IFN-gamma. Content: IL-12 and IFN-gamma positively regulate each other and type 1 inflammatory responses, which are believed to cause tissue damage in autoimmune diseases. We investigated the role of the IL-12/IFN-gamma (Th1) axis in the development of autoimmune myocarditis. IL-12p40-deficient mice on a susceptible background resisted myocarditis. In the absence of IL-12, autospecific CD4(+) T cells proliferated poorly and showed increased Th2 cytokine responses. However, IFN-gamma-deficient mice developed fatal autoimmune disease, and blockade of IL-4R signaling did not confer susceptibility to myocarditis in IL-12p40-deficient mice, demonstrating that IL-12 triggers autoimmunity by a mechanism independent of the effector cytokines IFN-gamma and IL-4. In conclusion, our results suggest that the IL-12/IFN-gamma axis is a double-edged sword for the development of autoimmune myocarditis. Although IL-12 mediates disease by induction/expansion of Th1-type cells, IFN-gamma production from these cells limits disease progression.

Title: Retinoic acid-related orphan receptors α and γ: key regulators of lipid/glucose metabolism, inflammation, and insulin sensitivity Content: Retinoic acid-related orphan receptors RORα and RORγ play a regulatory role in lipid/glucose homeostasis and various immune functions, and have been implicated in metabolic syndrome and several inflammatory diseases. RORα-deficient mice are protected against age- and diet-induced obesity, hepatosteatosis, and insulin resistance. The resistance to hepatosteatosis in RORα-deficient mice is related to the reduced expression of several genes regulating lipid synthesis, transport, and storage. Adipose tissue-associated inflammation, which plays a critical role in the development of insulin resistance, is considerably diminished in RORα-deficient mice as indicated by the reduced infiltration of M1 macrophages and decreased expression of many proinflammatory genes. Deficiency in RORγ also protects against diet-induced insulin resistance by a mechanism that appears different from that in RORα deficiency. Recent studies indicated that RORs provide an important link between the circadian clock machinery and its regulation of metabolic genes and metabolic syndrome. As ligand-dependent transcription factors, RORs may provide novel therapeutic targets in the management of obesity and associated metabolic diseases, including hepatosteatosis, adipose tissue-associated inflammation, and insulin resistance.

Title: Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway Content: Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Title: Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses. Content: The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.

Title: An integrin β3–KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition Content: Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

Title: Transdifferentiation of glioblastoma cells into vascular endothelial cells. Content: Glioblastoma (GBM) is the most malignant brain tumor and is highly resistant to intensive combination therapies and anti-VEGF therapies. To assess the resistance mechanism to anti-VEGF therapy, we examined the vessels of GBMs in tumors that were induced by the transduction of p53(+/-) heterozygous mice with lentiviral vectors containing oncogenes and the marker GFP in the hippocampus of GFAP-Cre recombinase (Cre) mice. We were surprised to observe GFP(+) vascular endothelial cells (ECs). Transplantation of mouse GBM cells revealed that the tumor-derived endothelial cells (TDECs) originated from tumor-initiating cells and did not result from cell fusion of ECs and tumor cells. An in vitro differentiation assay suggested that hypoxia is an important factor in the differentiation of tumor cells to ECs and is independent of VEGF. TDEC formation was not only resistant to an anti-VEGF receptor inhibitor in mouse GBMs but it led to an increase in their frequency. A xenograft model of human GBM spheres from clinical specimens and direct clinical samples from patients with GBM also showed the presence of TDECs. We suggest that the TDEC is an important player in the resistance to anti-VEGF therapy, and hence a potential target for GBM therapy.

Title: A crucial role for interleukin (IL)-1 in the induction of IL-17–producing T cells that mediate autoimmune encephalomyelitis Content: It was recently demonstrated that interleukin (IL)-23–driven IL-17–producing (ThIL-17) T cells mediate inflammatory pathology in certain autoimmune diseases. We show that the induction of antigen-specific ThIL-17 cells, but not T helper (Th)1 or Th2 cells, by immunization with antigens and adjuvants is abrogated in IL-1 receptor type I–deficient (IL-1RI−/−) mice. Furthermore, the incidence of experimental autoimmune encephalomyelitis (EAE) was significantly lower in IL-1RI−/− compared with wild-type mice, and this correlated with a failure to induce autoantigen-specific ThIL-17 cells, whereas induction of Th1 and Th2 responses was not substantially different. However, EAE was induced in IL-1RI−/− mice by adoptive transfer of autoantigen-specific cells from wild-type mice with EAE. IL-23 alone did not induce IL-17 production by T cells from IL-1RI−/− mice, and IL-23–induced IL-17 production was substantially enhanced by IL-1α or IL-1β, even in the absence of T cell receptor stimulation. We demonstrate essential roles for phosphatidylinositol 3-kinase, nuclear factor κB, and novel protein kinase C isoforms in IL-1– and IL-23–mediated IL-17 production. Tumor necrosis factor α also synergized with IL-23 to enhance IL-17 production, and this was IL-1 dependent. Our findings demonstrate that IL-1 functions upstream of IL-17 to promote pathogenic ThIL-17 cells in EAE.

Title: P0 glycoprotein peptides 56–71 and 180–199 dose-dependently induce acute and chronic experimental autoimmune neuritis in Lewis rats associated with epitope spreading Content: Two synthetic peripheral nerve myelin P0 protein peptides, an immunodominant (amino acids 180-199) and a cryptic (amino acids 56-71) one, induced an acute or chronic course of experimental autoimmune neuritis (EAN) in Lewis rats, when given at low dose (50-100 microg/rat) or high dose (250 microg/rat), respectively. Corresponding to the different clinical course, pathological changes and immune responses were found: (1) Onset of clinical signs of P0 peptide 56-71 (P0 56-71) induced EAN was 1-3 days later than in P0 peptide 180-199 (P0 180-199) induced EAN at all immunizing doses, whereas the peak of the disease occurred at a similar time point post immunization (p.i.), i.e. at days 14-16 p.i. in P0 56-71 induced EAN and at day 16 p.i. in P0 180-199 induced EAN. (2) Intramolecular epitope spreading as assessed by delayed type hypersensitivity response occurred in P0 56-71 induced EAN at both low and high antigen doses and in P0 180-199 induced EAN at high antigen dose (250 microg/rat) only. (3) P0 180-199 stimulated higher levels of interferon-gamma production in P0 180-199 induced EAN than in P0 56-71 induced EAN and vice versa. (4) Histopathologic evaluation revealed a similar grade of mononuclear cell infiltration in the sciatic nerves of both types of EAN, but more severe demyelination was found in P0 180-199 induced EAN compared to P0 56-71 induced EAN. The results support the hypothesis that high dose autoantigen immunization induces extensive determinant spreading and chronic course of autoimmune diseases.

Title: Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors Content: The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-γ (IFN-γ)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1β (IL-1β). The priming of IFN-γ–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3−/−) or caspase-1–deficient (Casp-1−/−) mice unless exogenous IL-1β is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7−/− or Nlrp3−/− or Casp1−/− hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Title: The Dual Immunoregulatory function of Nlrp12 in T Cell-Mediated Immune Response: Lessons from Experimental Autoimmune Encephalomyelitis Content: Although the etiology of multiple sclerosis (MS) remains enigmatic, the role of T cells is unquestionably central in this pathology. Immune cells respond to pathogens and danger signals via pattern-recognition receptors (PRR). Several reports implicate Nlrp12, an intracellular PRR, in the development of a mouse MS-like disease, called Experimental Autoimmune Encephalomyelitis (EAE). In this study, we used induced and spontaneous models of EAE, as well as in vitro T cell assays, to test the hypothesis that Nlrp12 inhibits Th1 response and prevents T-cell mediated autoimmunity. We found that Nlrp12 plays a protective role in induced EAE by reducing IFNγ/IL-4 ratio in lymph nodes, whereas it potentiates the development of spontaneous EAE (spEAE) in 2D2 T cell receptor (TCR) transgenic mice. Looking into the mechanism of Nlrp12 activity in T cell response, we found that it inhibits T cell proliferation and suppresses Th1 response by reducing IFNγ and IL-2 production. Following TCR activation, Nlrp12 inhibits Akt and NF-κB phosphorylation, while it has no effect on S6 phosphorylation in the mTOR pathway. In conclusion, we propose a model that can explain the dual immunoregulatory function of Nlrp12 in EAE. We also propose a model explaining the molecular mechanism of Nlrp12-dependent regulation of T cell response.

Title: Obesity induces a phenotypic switch in adipose tissue macrophage polarization. Content: Adipose tissue macrophages (ATMs) infiltrate adipose tissue during obesity and contribute to insulin resistance. We hypothesized that macrophages migrating to adipose tissue upon high-fat feeding may differ from those that reside there under normal diet conditions. To this end, we found a novel F4/80(+)CD11c(+) population of ATMs in adipose tissue of obese mice that was not seen in lean mice. ATMs from lean mice expressed many genes characteristic of M2 or "alternatively activated" macrophages, including Ym1, arginase 1, and Il10. Diet-induced obesity decreased expression of these genes in ATMs while increasing expression of genes such as those encoding TNF-alpha and iNOS that are characteristic of M1 or "classically activated" macrophages. Interestingly, ATMs from obese C-C motif chemokine receptor 2-KO (Ccr2-KO) mice express M2 markers at levels similar to those from lean mice. The antiinflammatory cytokine IL-10, which was overexpressed in ATMs from lean mice, protected adipocytes from TNF-alpha-induced insulin resistance. Thus, diet-induced obesity leads to a shift in the activation state of ATMs from an M2-polarized state in lean animals that may protect adipocytes from inflammation to an M1 proinflammatory state that contributes to insulin resistance.

Title: Cord factor and peptidoglycan recapitulate the Th17-promoting adjuvant activity of mycobacteria through mincle/CARD9 signaling and the inflammasome. Content: Although adjuvants are critical vaccine components, their modes of action are poorly understood. In this study, we investigated the mechanisms by which the heat-killed mycobacteria in CFA promote Th17 CD4(+) T cell responses. We found that IL-17 secretion by CD4(+) T cells following CFA immunization requires MyD88 and IL-1β/IL-1R signaling. Through measurement of Ag-specific responses after adoptive transfer of OTII cells, we confirmed that MyD88-dependent signaling controls Th17 differentiation rather than simply production of IL-17. Additional experiments showed that CFA-induced Th17 differentiation involves IL-1β processing by the inflammasome, as mice lacking caspase-1, ASC, or NLRP3 exhibit partially defective responses after immunization. Biochemical fractionation studies further revealed that peptidoglycan is the major component of heat-killed mycobacteria responsible for inflammasome activation. By assaying Il1b transcripts in the injection site skin of CFA-immunized mice, we found that signaling through the adaptor molecule caspase activation and recruitment domain 9 (CARD9) plays a major role in triggering pro-IL-1β expression. Moreover, we demonstrated that recognition of the mycobacterial glycolipid trehalose dimycolate (cord factor) by the C-type lectin receptor mincle partially explains this CARD9 requirement. Importantly, purified peptidoglycan and cord factor administered in mineral oil synergized to recapitulate the Th17-promoting activity of CFA, and, as expected, this response was diminished in caspase-1- and CARD9-deficient mice. Taken together, these findings suggest a general strategy for the rational design of Th17-skewing adjuvants by combining agonists of the CARD9 pathway with inflammasome activators.

Title: Enhanced pathological angiogenesis in mice lacking β3 integrin or β3 and β5 integrins Content: Inhibition of αvβ3 or αvβ5 integrin function has been reported to suppress neovascularization and tumor growth, suggesting that these integrins are critical modulators of angiogenesis. Here we report that mice lacking β3 integrins or both β3 and β5 integrins not only support tumorigenesis, but have enhanced tumor growth as well. Moreover, the tumors in these integrin-deficient mice display enhanced angiogenesis, strongly suggesting that neither β3 nor β5 integrins are essential for neovascularization. We also observed that angiogenic responses to hypoxia and vascular endothelial growth factor (VEGF) are augmented significantly in the absence of β3 integrins. We found no evidence that the expression or functions of other integrins were altered as a consequence of the β3 deficiency, but we did observe elevated levels of VEGF receptor-2 (also called Flk-1) in β3-null endothelial cells. These data indicate that αvβ3 and αvβ5 integrins are not essential for vascular development or pathological angiogenesis and highlight the need for further evaluation of the mechanisms of action of αv-integrin antagonists in anti-angiogenic therapeutics.

Title: Curcumin attenuates inflammatory response in IL-1beta-induced human synovial fibroblasts and collagen-induced arthritis in mouse model. Content: Curcumin, a major component of turmeric, has been shown to exhibit anti-oxidant and anti-inflammatory activities. The present study was performed to determine whether curcumin is efficacious against both collagen-induced arthritis (CIA) in mice and IL-1beta-induced activation in fibroblast-like synoviocytes (FLSs). DBA/1 mice were immunized with bovine type II collagen (CII) and treated with curcumin every other day for 2weeks after the initial immunization. For arthritis, we evaluated the incidence of disease and used an arthritis index based on paw thickness. In vitro proliferation of CII- or concanavalin A-induced splenic T cells was examined using IFN-gamma production. Pro-inflammatory cytokines TNF-alpha and IL-1beta were examined in the mouse ankle joint and serum IgG1 and IgG2a isotypes were analyzed. The expression levels of prostaglandin E(2) (PGE(2)), cyclooxygenase-2 (COX-2), and matrix metalloproteinases (MMPs) in human FLSs were also determined. The results showed that compared with untreated CIA mice, curcumin-treated mice downregulated clinical arthritis score, the proliferation of splenic T cells, expression levels of TNF-alpha and IL-1beta in the ankle joint, and expression levels of IgG2a in serum. Additionally, by altering nuclear factor (NF)-kappaB transcription activity in FLSs, curcumin inhibited PGE(2) production, COX-2 expression, and MMP secretion. These results suggest that curcumin can effectively suppress inflammatory response by inhibiting pro-inflammatory mediators and regulating humoral and cellular immune responses.

Title: Crucial Role of Interferon Consensus Sequence Binding Protein, but neither of Interferon Regulatory Factor 1 nor of Nitric Oxide Synthesis for Protection Against Murine Listeriosis Content: Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF)1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP−/− and to a lesser degree also IRF2−/− mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-γ in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-γ stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-γ–mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

Title: Regulation of myeloid cell function through the CD200 receptor. Content: Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.

Title: Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion Content: Interferon-gamma (IFN-γ) is a pleiotropic molecule with associated antiproliferative, pro-apoptotic and antitumor mechanisms. This effector cytokine, often considered as a major effector of immunity, has been used in the treatment of several diseases, despite its adverse effects. Although broad evidence implicating IFN-γ in tumor immune surveillance, IFN-γ-based therapies undergoing clinical trials have been of limited success. In fact, recent reports suggested that it may also play a protumorigenic role, namely, through IFN-γ signaling insensitivity, downregulation of major histocompatibility complexes, and upregulation of indoleamine 2,3-dioxygenase and of checkpoint inhibitors, as programmed cell-death ligand 1. However, the IFN-γ-mediated responses are still positively associated with patient's survival in several cancers. Consequently, major research efforts are required to understand the immune contexture in which IFN-γ induces its intricate and highly regulated effects in the tumor microenvironment. This review discusses the current knowledge on the pro- and antitumorigenic effects of IFN-γ as part of the complex immune response to cancer, highlighting the relevance to identify IFN-γ responsive patients for the improvement of therapies that exploit associated signaling pathways.

Title: Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Content: We show that NF-kappaB and transcriptional targets are activated in liver by obesity and high-fat diet (HFD). We have matched this state of chronic, subacute 'inflammation' by low-level activation of NF-kappaB in the liver of transgenic mice, designated LIKK, by selectively expressing constitutively active IKK-b in hepatocytes. These mice exhibit a type 2 diabetes phenotype, characterized by hyperglycemia, profound hepatic insulin resistance, and moderate systemic insulin resistance, including effects in muscle. The hepatic production of proinflammatory cytokines, including IL-6, IL-1beta and TNF-alpha, was increased in LIKK mice to a similar extent as induced by HFD in in wild-type mice. Parallel increases were observed in cytokine signaling in liver and mucscle of LIKK mice. Insulin resistance was improved by systemic neutralization of IL-6 or salicylate inhibition of IKK-beta. Hepatic expression of the IkappaBalpha superrepressor (LISR) reversed the phenotype of both LIKK mice and wild-type mice fed an HFD. These findings indicate that lipid accumulation in the liver leads to subacute hepatic 'inflammation' through NF-kappaB activation and downstream cytokine production. This causes insulin resistance both locally in liver and systemically.

Title: Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML Content: Cancer stem cells have been proposed to be important for initiation, maintenance and recurrence of various malignancies, including acute myeloid leukemia (AML). We have previously reported that CD34+CD38− human primary AML stem cells residing in the endosteal region of the bone marrow are relatively chemotherapy resistant. Using a NOD/SCID/IL2rγnull mouse model of human AML, we now show that the AML stem cells in the endosteal region are cell cycle quiescent and that these stem cells can be induced to enter the cell cycle by treatment with granulocyte colony-stimulating factor (G-CSF). In combination with cell cycle-dependent chemotherapy, G-CSF treatment significantly enhances induction of apoptosis and elimination of human primary AML stem cells in vivo. The combination therapy leads to significantly increased survival of secondary recipients after transplantation of leukemia cells compared with chemotherapy alone.

Title: Tumor-specific Th17-polarized cells eradicate large established melanoma. Content: CD4+ T cells can differentiate into multiple effector subsets, but the potential roles of these subsets in anti-tumor immunity have not been fully explored. Seeking to study the impact of CD4+ T cell polarization on tumor rejection in a model mimicking human disease, we generated a new MHC class II-restricted, T-cell receptor (TCR) transgenic mouse model in which CD4+ T cells recognize a novel epitope in tyrosinase-related protein 1 (TRP-1), an antigen expressed by normal melanocytes and B16 murine melanoma. Cells could be robustly polarized into Th0, Th1, and Th17 subtypes in vitro, as evidenced by cytokine, chemokine, and adhesion molecule profiles and by surface markers, suggesting the potential for differential effector function in vivo. Contrary to the current view that Th1 cells are most important in tumor rejection, we found that Th17-polarized cells better mediated destruction of advanced B16 melanoma. Their therapeutic effect was critically dependent on interferon-gamma (IFN-gamma) production, whereas depletion of interleukin (IL)-17A and IL-23 had little impact. Taken together, these data indicate that the appropriate in vitro polarization of effector CD4+ T cells is decisive for successful tumor eradication. This principle should be considered in designing clinical trials involving adoptive transfer-based immunotherapy of human malignancies.

Title: Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress Content: During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.

Title: Persistent LCMV infection is controlled by blockade of type I interferon signaling. Content: During persistent viral infections, chronic immune activation, negative immune regulator expression, an elevated interferon signature, and lymphoid tissue destruction correlate with disease progression. We demonstrated that blockade of type I interferon (IFN-I) signaling using an IFN-I receptor neutralizing antibody reduced immune system activation, decreased expression of negative immune regulatory molecules, and restored lymphoid architecture in mice persistently infected with lymphocytic choriomeningitis virus. IFN-I blockade before and after establishment of persistent virus infection resulted in enhanced virus clearance and was CD4 T cell-dependent. Hence, we demonstrate a direct causal link between IFN-I signaling, immune activation, negative immune regulator expression, lymphoid tissue disorganization, and virus persistence. Our results suggest that therapies targeting IFN-I may help control persistent virus infections.

Title: Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance Content: Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Title: Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse. Content: The role of IL-10 in the pathogenesis of autoimmune diabetes mellitus was assessed in the nonobese diabetic (NOD) mouse. In these studies the effect of IL-10 was determined on three parameters of diabetes: The development of hyperglycemia, the development of insulitis, and the production of insulin by beta cells. Initial experiments investigated the effect of anticytokine antibodies on the development of disease. These results indicated that monoclonal anti-IFN-gamma antibody greatly reduced the incidence of hyperglycemia in female NOD mice, while anti-IL-4, IL-5, and IL-10 were ineffective. In subsequent studies, daily subcutaneous administration of IL-10, a known potent inhibitor of IFN-gamma production by TH1 T cells, to 9 and 10-week-old NODs was shown to delay the onset of disease and significantly reduce the incidence of diabetes. Histopathology performed on pancreatic tissue demonstrated that treatment with IL-10 reduced the severity of insulitis, prevented cellular infiltration of islet cells, and promoted normal insulin production by beta cells. Taken together these results indicate IL-10 suppresses the induction and progression of autoimmune pathogenesis associated with diabetes mellitus and suggest a potential therapeutic role for this cytokine in this autoimmune disease.

Title: Antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to Clostridium difficile infection Content: Antibiotics can have significant and long-lasting effects on the gastrointestinal tract microbiota, reducing colonization resistance against pathogens including Clostridium difficile. Here we show that antibiotic treatment induces substantial changes in the gut microbial community and in the metabolome of mice susceptible to C. difficile infection. Levels of secondary bile acids, glucose, free fatty acids and dipeptides decrease, whereas those of primary bile acids and sugar alcohols increase, reflecting the modified metabolic activity of the altered gut microbiome. In vitro and ex vivo analyses demonstrate that C. difficile can exploit specific metabolites that become more abundant in the mouse gut after antibiotics, including the primary bile acid taurocholate for germination, and carbon sources such as mannitol, fructose, sorbitol, raffinose and stachyose for growth. Our results indicate that antibiotic-mediated alteration of the gut microbiome converts the global metabolic profile to one that favours C. difficile germination and growth.

Title: Apoptosis and chemoresistance in transgenic cancer models Content: Multidrug resistance remains an unresolved problem in clinical oncology. Over a decade ago genes encoding cellular efflux pumps were shown to confer resistance to a broad spectrum of biochemically unrelated anticancer drugs even before the compounds reached their intracellular targets. More recently it has become apparent that many drugs induce a common apoptotic program, such that mutations in this program can also produce multidrug resistance. However, a thorough evaluation of the contribution of apoptotic defects to this "postdamage" drug resistant phenotype is technically complicated, and this has led to uncertainty about the overall significance of apoptosis in therapy-induced cell death. For example, correlative analyses using patient specimens are limited by unknown background mutations in the biopsy material, and assays using cancer cell lines can be biased by unphysiological conditions. We sought to circumvent these restrictions by utilizing a tractable transgenic cancer model to examine the impact of apoptosis on treatment outcome. Here we discuss potential caveats of cell culture based assays, highlight features of genetically engineered mice as potential model systems, and describe a tractable transgenic mouse model to study drug responses in a series of primary lymphomas with genetically defined lesions treated at their natural site.

Title: Modification of kidney barrier function by the urokinase receptor Content: Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of αvβ3 integrin. Mice lacking uPAR (Plaur−/−) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active β3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate αvβ3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of αvβ3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Title: Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing. Content: Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.

Title: IL-18 induction of osteopontin mediates cardiac fibrosis and diastolic dysfunction in mice. Content: Osteopontin (OPN), a key component of the extracellular matrix, is associated with the fibrotic process during tissue remodeling. OPN and the cytokine interleukin (IL)-18 have been shown to be overexpressed in an array of human cardiac pathologies. In the present study, we determined the role of IL-18 in the regulation of cardiac OPN expression and the subsequent interstitial fibrosis and diastolic dysfunction. We demonstrated parallel increases in IL-18, OPN expression, and interstitial fibrosis in murine models of left ventricular pressure and volume overload. Exogenous recombinant (r)IL-18 administered for 2 wk increased cardiac OPN expression, interstitial fibrosis, and diastolic dysfunction. Stimulation of the T helper (Th)1 lymphocyte phenotype with a selective toll-like receptor (TLR)9 agonist induced cardiac IL-18 and OPN expression, which was associated with increased cardiac fibrillar collagen concentrations and interstitial fibrosis resulting in diastolic dysfunction. rIL-18 induced OPN expression and protein levels in primary of cardiac fibroblast cultures. Conditioned media from TLR9-stimulated T lymphocyte cultures induced IL-18 and OPN expression in cardiac fibroblasts, while blockade of the IL-18 receptor with a neutralizing antibody abolished the increase in OPN expression. Furthermore, a mutation in the transcriptional factor interferon regulatory factor (IRF)1 or IRF1 small interfering RNA (siRNA) resulted in the decreased expression of IL-18 and OPN in cardiac fibroblasts. With pressure overload, IRF1-mutant mice showed downregulation of IL-18 and OPN expression in cardiac tissue, reduced cardiac fibrotic development, and increased left ventricular function compared with wild type. These results provide direct evidence that the induction of IL-18 regulates OPN-mediated cardiac fibrosis and diastolic dysfunction.

Title: Efficacy of Beta1 Integrin and EGFR Targeting in Sphere-Forming Human Head and Neck Cancer Cells Content: BACKGROUND Resistance to radiotherapy continues to be a limiting factor in the treatment of cancer including head and neck squamous cell carcinoma (HNSCC). Simultaneous targeting of β1 integrin and EGFR was shown to have a higher radiosensitizing potential than mono-targeting in the majority of tested HNSCC cancer models. As tumor-initiating cells (TIC) are thought to play a key role for therapy resistance and recurrence and can be enriched in sphere forming conditions, this study investigated the efficacy of β1 integrin/EGFR targeting without and in combination with X-ray irradiation on the behavior of sphere-forming cells (SFC). METHODS HNSCC cell lines (UTSCC15, UTSCC5, Cal33, SAS) were injected subcutaneously into nude mice for tumor up-take and plated for primary and secondary sphere formation under non-adhesive conditions which is thought to reflect the enrichment of SFC and their self-renewal capacity, respectively. Treatment was accomplished by inhibitory antibodies for β1 integrin (AIIB2) and EGFR (Cetuximab) as well as X-ray irradiation (2 - 6 Gy single doses). Further, flow cytometry for TIC marker expression and cell cycling as well as Western blotting for DNA repair protein expression and phosphorylation were employed. RESULTS We found higher primary and secondary sphere forming capacity of SAS cells relative to other HNSCC cell lines, which was in line with the tumor up-take rates of SAS versus UTSCC15 cells. AIIB2 and Cetuximab administration had minor cytotoxic and no radiosensitizing effects on SFC. Intriguingly, secondary SAS spheres, representing the fraction of surviving SFC upon passaging, showed greatly enhanced radiosensitivity compared to primary spheres. Intriguingly, neither AIIB2 nor Cetuximab significantly altered basal sphere forming capacity and radiosensitivity. While an increased accumulation of G0/G1 phase cells was observable in secondary SAS spheres, DNA double strand break repair indicated no difference on the basis of significantly enhanced ATM and Chk2 dephosphorylation upon irradiation. CONCLUSIONS In the HNSCC model, sphere-forming conditions select for cells, which are unsusceptible to both anti-β1 integrin and anti-EGFR inhibitory antibodies. With regard to primary and secondary sphere formation, our data suggest that both of these SFC fractions express distinct survival strategies independent from β1 integrin and EGFR and that future work is warranted to better understand SFC survival and enrichment before and after treatment to untangle the underlying mechanisms for identifying novel, druggable cancer targets in SFC.

Title: A tumor necrosis factor-α–mediated pathway promoting autosomal dominant polycystic kidney disease Content: Autosomal dominant polycystic kidney disease (ADPKD) is caused by heterozygous mutations in either PKD1 or PKD2, genes that encode polycystin-1 and polycystin-2, respectively. We show here that tumor necrosis factor-α (TNF-α), an inflammatory cytokine present in the cystic fluid of humans with ADPKD, disrupts the localization of polycystin-2 to the plasma membrane and primary cilia through a scaffold protein, FIP2, which is induced by TNF-α. Treatment of mouse embryonic kidney organ cultures with TNF-α resulted in formation of cysts, and this effect was exacerbated in the Pkd2+/− kidneys. TNF-α also stimulated cyst formation in vivo in Pkd2+/− mice. In contrast, treatment of Pkd2+/− mice with the TNF-α inhibitor etanercept prevented cyst formation. These data reveal a pathway connecting TNF-α signaling, polycystins and cystogenesis, the activation of which may reduce functional polycystin-2 below a critical threshold, precipitating the ADPKD cellular phenotype.

Title: PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis Content: Osteogenesis during bone modeling and remodeling is coupled with angiogenesis. A recent study showed that a specific vessel subtype, strongly positive for CD31 and endomucin (CD31hiEmcnhi), couples angiogenesis and osteogenesis. Here, we found that platelet-derived growth factor-BB (PDGF-BB) secreted by preosteoclasts induces CD31hiEmcnhi vessel formation during bone modeling and remodeling. Mice with depletion of PDGF-BB in the tartrate-resistant acid phosphatase–positive cell lineage show significantly lower trabecular and cortical bone mass, serum and bone marrow PDGF-BB concentrations, and fewer CD31hiEmcnhi vessels compared to wild-type mice. In the ovariectomy (OVX)-induced osteoporotic mouse model, serum and bone marrow levels of PDGF-BB and numbers of CD31hiEmcnhi vessels are significantly lower compared to sham-operated controls. Treatment with exogenous PDGF-BB or inhibition of cathepsin K to increase the number of preosteoclasts, and thus the endogenous levels of PDGF-BB, increases CD31hiEmcnhi vessel number and stimulates bone formation in OVX mice. Thus, pharmacotherapies that increase PDGF-BB secretion from preosteoclasts offer a new therapeutic target for treating osteoporosis by promoting angiogenesis and thus bone formation.

Title: β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat. Content: Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

Title: Suppression of interferon-mediated anti-HBV response by single CpG methylation in the 5′-UTR of TRIM22 Content: Objective Interferons (IFNs) mediate direct antiviral activity. They play a crucial role in the early host immune response against viral infections. However, IFN therapy for HBV infection is less effective than for other viral infections. Design We explored the cellular targets of HBV in response to IFNs using proteome-wide screening. Results Using LC-MS/MS, we identified proteins downregulated and upregulated by IFN treatment in HBV X protein (HBx)-stable and control cells. We found several IFN-stimulated genes downregulated by HBx, including TRIM22, which is known as an antiretroviral protein. We demonstrated that HBx suppresses the transcription of TRIM22 through a single CpG methylation in its 5′-UTR, which further reduces the IFN regulatory factor-1 binding affinity, thereby suppressing the IFN-stimulated induction of TRIM22. Conclusions We verified our findings using a mouse model, primary human hepatocytes and human liver tissues. Our data elucidate a mechanism by which HBV evades the host innate immune system.

Title: SOCS1/JAB is a negative regulator of LPS-induced macrophage activation. Content: Bacterial lipopolysaccharide (LPS) triggers innate immune responses through Toll-like receptor (TLR) 4. We show here that the suppressor of cytokine-signaling-1 (SOCS1/JAB) is rapidly induced by LPS and negatively regulates LPS signaling. SOCS1(+/-) mice or SOCS1(-/-) mice with interferon-gamma (IFNgamma)-deficient background were more sensitive to LPS-induced lethal effects than were wild-type littermates. LPS-induced NO(2)(-) synthesis and TNFalpha production were augmented in SOCS1(-/-) macrophages. Furthermore, LPS tolerance, a protection mechanism against endotoxin shock, was also strikingly reduced in SOCS1(-/-) cells. LPS-induced I-kappaB and p38 phosphorylation was upregulated in SOCS1(-/-) macrophages, and forced expression of SOCS1 suppressed LPS-induced NF-kappaB activation. Thus, SOCS1 directly suppresses TLR4 signaling and modulates innate immunity.

Title: Interleukin 6 plays a key role in the development of antigen-induced arthritis. Content: To investigate the direct role of interleukin (IL) 6 in the development of rheumatoid arthritis, IL-6-deficient (IL-6 -/-) mice were backcrossed for eight generations into C57BL/6 mice, a strain of mice with a genetic background of susceptibility for antigen-induced arthritis (AIA). Both histological and immunological comparisons were made between IL-6-deficient (IL-6 -/-) mice and wild-type (IL-6 +/+) littermates after the induction of AIA. Although all IL-6 +/+ mice developed severe arthritis, only mild arthritis was observed in IL-6 -/- mice. Safranin O staining demonstrated that articular cartilage was well preserved in IL-6 -/- mice, whereas it was destroyed completely in IL-6 +/+ mice. In addition, comparable mRNA expression for both IL-1beta and tumor necrosis factor alpha, but not for IL-6, was detected in the inflamed joints of IL-6 -/- mice, suggesting that IL-6 may play a more crucial role in cartilage destruction than either IL-1beta or tumor necrosis factor alpha. In immunological comparisons, both antigen-specific in vitro proliferative response in lymph node cells and in vivo antibody production were elicited in IL-6 -/- mice, but they were reduced to less than half of that found in IL-6 +/+ mice. Lymph node cells of IL-6 -/- mice produced many more Th2 cytokines than did IL-6 +/+ mice with either antigen-specific or nonspecific stimulation in in vitro culture. Taken together, these results indicate that IL-6 may play a key role in the development of AIA at the inductive as well as the effector phase, and the blockade of IL-6 is possibly beneficial in the treatment of rheumatoid arthritis.

Title: Tissue-Specific Knockout of the Insulin Receptor in Pancreatic β Cells Creates an Insulin Secretory Defect Similar to that in Type 2 Diabetes Content: Dysfunction of the pancreatic beta cell is an important defect in the pathogenesis of type 2 diabetes, although its exact relationship to the insulin resistance is unclear. To determine whether insulin signaling has a functional role in the beta cell we have used the Cre-loxP system to specifically inactivate the insulin receptor gene in the beta cells. The resultant mice exhibit a selective loss of insulin secretion in response to glucose and a progressive impairment of glucose tolerance. These data indicate an important functional role for the insulin receptor in glucose sensing by the pancreatic beta cell and suggest that defects in insulin signaling at the level of the beta cell may contribute to the observed alterations in insulin secretion in type 2 diabetes.

Title: Blimp-1 attenuates Th1 differentiation by repression of ifng, tbx21, and bcl6 gene expression. Content: T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.

Title: Lipoatrophy and severe metabolic disturbance in mice with fat-specific deletion of PPARγ. Content: Adipose tissue is an important metabolic organ, the dysfunction of which is associated with the development of obesity, diabetes mellitus, and cardiovascular disease. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is considered the master regulator of adipocyte differentiation and function. Although its cell-autonomous role in adipogenesis has been clearly demonstrated in cell culture, previous fat-specific knockouts of the murine PPARγ gene did not demonstrate a dramatic phenotype in vivo. Here, using Adipoq-Cre mice to drive adipose-specific recombination, we report a unique fat-specific PPARγ knockout (PPARγ FKO) mouse model with almost no visible brown and white adipose tissue at age 3 mo. As a consequence, PPARγ FKO mice had hugely enlarged pancreatic islets, massive fatty livers, and dramatically elevated levels of blood glucose and serum insulin accompanied by extreme insulin resistance. PPARγ FKO mice also exhibited delayed hair coat formation associated with absence of dermal fat, disrupted mammary gland development with loss of mammary fat pads, and high bone mass with loss of bone marrow fat, indicating the critical roles of adipose PPARγ in these tissues. Together, our data reveal the necessity of fat PPARγ in adipose formation, whole-body metabolic homeostasis, and normal development of fat-containing tissues.

Title: Pharmacological correction of a defect in PPARγ signaling ameliorates disease severity in Cftr-deficient mice Content: Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (encoded by Cftr) that impair its role as an apical chloride channel that supports bicarbonate transport. Individuals with cystic fibrosis show retained, thickened mucus that plugs airways and obstructs luminal organs as well as numerous other abnormalities that include inflammation of affected organs, alterations in lipid metabolism and insulin resistance. Here we show that colonic epithelial cells and whole lung tissue from Cftr-deficient mice show a defect in peroxisome proliferator-activated receptor-gamma (PPAR-gamma, encoded by Pparg) function that contributes to a pathological program of gene expression. Lipidomic analysis of colonic epithelial cells suggests that this defect results in part from reduced amounts of the endogenous PPAR-gamma ligand 15-keto-prostaglandin E(2) (15-keto-PGE(2)). Treatment of Cftr-deficient mice with the synthetic PPAR-gamma ligand rosiglitazone partially normalizes the altered gene expression pattern associated with Cftr deficiency and reduces disease severity. Rosiglitazone has no effect on chloride secretion in the colon, but it increases expression of the genes encoding carbonic anhydrases 4 and 2 (Car4 and Car2), increases bicarbonate secretion and reduces mucus retention. These studies reveal a reversible defect in PPAR-gamma signaling in Cftr-deficient cells that can be pharmacologically corrected to ameliorate the severity of the cystic fibrosis phenotype in mice.

Title: Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2. Content: Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro. Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents. Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.

Title: Ghrelin secretion stimulated by {beta}1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice. Content: Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding beta(1)-adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective beta(1)-adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on beta(1) receptors on the ghrelin-secreting cells of the stomach.

Title: induction of early IFN-inducible genes in the absence of type Content: On recognition of influenza virus (Flu) by TLR7, plasmacytoid dendritic cells (pDCs) produce type I IFN in significant amounts. Synthetic TLR7 ligands induce the maturation of pDCs, as evidenced by the expression of costimulatory molecules and the production of proinflammatory cytokines; however, they induce only low-level production of IFN-alpha. To dissect the TLR7 signaling in pDCs and how these different profiles are induced, we studied the effects of 2 TLR7 ligands (Flu and CL097) on the activation of blood-isolated pDCs and the human GEN2.2 pDC cell line. Type I IFN production by pDCs correlates with differential interferon regulatory factor 7 (IRF7) translocation into the nucleus induced by the 2 TLR7 ligands. Surprisingly, with both activators we nevertheless observed the rapid expression of the IFN-inducible genes mxa, cxcl10, and trail within 4 hours of stimulation. This expression, controlled by STAT1 phosphorylation, was independent of type I IFN. STAT1 activation was found to be strictly dependent on the PI3K-p38MAPK pathway, showing a new signaling pathway leading to rapid expression of IFN-inducible genes after TLR7 triggering. Thus, pDCs, through this unusual TLR7 signaling, have the capacity to promptly respond to viral infection during the early phases of the innate immune response.

Title: Fibroblast Growth Factor 21 Prevents Atherosclerosis by Suppression of Hepatic Sterol Regulatory Element-Binding Protein-2 and Induction of Adiponectin in Mice Content: BACKGROUND Fibroblast growth factor 21 (FGF21) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF21 in the cardiovascular system remains elusive. METHODS AND RESULTS The roles of FGF21 in atherosclerosis were investigated by evaluating the impact of FGF21 deficiency and replenishment with recombinant FGF21 in apolipoprotein E(-/-) mice. FGF21 deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF21 protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription factor sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF21-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF21 are abrogated by hepatic expression of sterol regulatory element-binding protein-2. CONCLUSIONS FGF21 protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

Title: Blockade of class IB phosphoinositide-3 kinase ameliorates obesity-induced inflammation and insulin resistance. Content: Obesity and insulin resistance, the key features of metabolic syndrome, are closely associated with a state of chronic, low-grade inflammation characterized by abnormal macrophage infiltration into adipose tissues. Although it has been reported that chemokines promote leukocyte migration by activating class IB phosphoinositide-3 kinase (PI3Kγ) in inflammatory states, little is known about the role of PI3Kγ in obesity-induced macrophage infiltration into tissues, systemic inflammation, and the development of insulin resistance. In the present study, we used murine models of both diet-induced and genetically induced obesity to examine the role of PI3Kγ in the accumulation of tissue macrophages and the development of obesity-induced insulin resistance. Mice lacking p110γ (Pik3cg(-/-)), the catalytic subunit of PI3Kγ, exhibited improved systemic insulin sensitivity with enhanced insulin signaling in the tissues of obese animals. In adipose tissues and livers of obese Pik3cg(-/-) mice, the numbers of infiltrated proinflammatory macrophages were markedly reduced, leading to suppression of inflammatory reactions in these tissues. Furthermore, bone marrow-specific deletion and pharmacological blockade of PI3Kγ also ameliorated obesity-induced macrophage infiltration and insulin resistance. These data suggest that PI3Kγ plays a crucial role in the development of both obesity-induced inflammation and systemic insulin resistance and that PI3Kγ can be a therapeutic target for type 2 diabetes.

Title: Lipopolysaccharide-dependent prostaglandin E(2) production is regulated by the glutathione-dependent prostaglandin E(2) synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway. Content: Macrophages produce a large amount of PGE(2) during inflammation. This lipid mediator modulates various immune responses. PGE(2) acts on macrophages and inhibits production of cytokines such as TNF-alpha and IL-12. Membrane-bound glutathione-dependent PGE(2) synthase (mPGES) has been shown to be a terminal enzyme of the cyclooxygenase-2-mediated PGE(2) biosynthesis. Here we identified mPGES as a molecule that is induced by LPS in macrophages. The expression of mPGES was not induced by LPS in mice lacking Toll-like receptor 4 or MyD88. Furthermore, mice deficient in NF-IL6 showed neither induction of mPGES nor biosynthesis of PGE(2) in response to LPS, indicating that mPGES expression in response to LPS is regulated by a Toll-like receptor 4/MyD88/NF-IL6-dependent signaling pathway. We generated mPGES-deficient mice and investigated the role of mPGES in vivo. The mice showed no augmentation of the PGE(2) production in response to LPS. However, they were not impaired in the LPS-induced production of inflammatory cytokines and showed normal response to the LPS-induced shock. Thus, mPGES is critically involved in the biosynthesis of PGE(2) induced by LPS, but is dispensable for the modulation of inflammatory responses.

Title: Critical role of CD28 in protective immunity against Salmonella typhimurium. Content: Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28-/- mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA- S. typhimurium. More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.

Title: Anti-alphav integrin monoclonal antibody intetumumab enhances the efficacy of radiation therapy and reduces metastasis of human cancer xenografts in nude rats. Content: We previously reported that intetumumab (CNTO 95), a fully human anti-αv integrin monoclonal antibody, is a radiosensitizer in mice with xenograft tumors. Because intetumumab does not cross-react with mouse integrins, but has cross-reactivity with rat integrins, we next studied the potential combined use of radiation therapy and intetumumab in human cancer xenograft models in nude rats to assess effects on both tumor cells and the tumor microenvironment. Nude rats bearing human head and neck cancer and non-small cell lung cancer (NSCLC) xenografts were treated with intetumumab and fractionated local tumor radiotherapy. Effects on tumor growth and metastasis, blood perfusion, oxygenation, and gastrointestinal toxicity were studied. Intetumumab alone had a moderate effect on tumor growth. When combined with fractionated radiation therapy, intetumumab significantly inhibited tumor growth and produced a tumor response rate that was significantly better than with radiation therapy alone. Treatment with intetumumab also significantly reduced lung metastasis in the A549 NSCLC xenograft model. The oxygenation and blood perfusion in xenograft tumors measured by microbubble-enhanced ultrasound imaging were substantially increased after treatment with intetumumab. The combined use of intetumumab and radiation therapy reduced the microvessel density and increased apoptosis in tumor cells and the tumor microenvironment. Toxicity studies showed that treatment with intetumumab did not cause the histopathologic changes in the lungs and did not sensitize the sensitive gastrointestinal epithelium to the effect of radiation therapy. Intetumumab can potentiate the efficacy of fractionated radiation therapy in human cancer xenograft tumors in nude rats without increased toxicity.

Title: CNTF reverses obesity-induced insulin resistance by activating skeletal muscle AMPK Content: Ciliary neurotrophic factor (CNTF) induces weight loss and improves glucose tolerance in humans and rodents. CNTF is thought to act centrally by inducing hypothalamic neurogenesis to modulate food intake and peripherally by altering hepatic gene expression, in a manner similar to that of leptin. Here, we show that CNTF signals through the CNTFRα–IL-6R–gp130β receptor complex to increase fatty-acid oxidation and reduce insulin resistance in skeletal muscle by activating AMP-activated protein kinase (AMPK), independent of signaling through the brain. Thus, our findings further show that the antiobesogenic effects of CNTF in the periphery result from direct effects on skeletal muscle, and that these peripheral effects are not suppressed by diet-induced or genetic models of obesity, an essential requirement for the therapeutic treatment of obesity-related diseases.

Title: ROR-γ drives androgen receptor expression and represents a therapeutic target in castration-resistant prostate cancer Content: The androgen receptor (AR) is overexpressed and hyperactivated in human castration-resistant prostate cancer (CRPC). However, the determinants of AR overexpression in CRPC are poorly defined. Here we show that retinoic acid receptor-related orphan receptor γ (ROR-γ) is overexpressed and amplified in metastatic CRPC tumors, and that ROR-γ drives AR expression in the tumors. ROR-γ recruits nuclear receptor coactivator 1 and 3 (NCOA1 and NCOA3, also known as SRC-1 and SRC-3) to an AR-ROR response element (RORE) to stimulate AR gene transcription. ROR-γ antagonists suppress the expression of both AR and its variant AR-V7 in prostate cancer (PCa) cell lines and tumors. ROR-γ antagonists also markedly diminish genome-wide AR binding, H3K27ac abundance and expression of the AR target gene network. Finally, ROR-γ antagonists suppressed tumor growth in multiple AR-expressing, but not AR-negative, xenograft PCa models, and they effectively sensitized CRPC tumors to enzalutamide, without overt toxicity, in mice. Taken together, these results establish ROR-γ as a key player in CRPC by acting upstream of AR and as a potential therapeutic target for advanced PCa.

Title: G-protein-independent coupling of MC4R to Kir7.1 in hypothalamic neurons Content: The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.

Title: Regulation of Serum Amyloid A3 (SAA3) in Mouse Colonic Epithelium and Adipose Tissue by the Intestinal Microbiota Content: The gut microbiota has been proposed as an environmental factor that affects the development of metabolic and inflammatory diseases in mammals. Recent reports indicate that gut bacteria-derived lipopolysaccharide (LPS) can initiate obesity and insulin resistance in mice; however, the molecular interactions responsible for microbial regulation of host metabolism and mediators of inflammation have not been studied in detail. Hepatic serum amyloid A (SAA) proteins are markers and proposed mediators of inflammation that exhibit increased levels in serum of insulin-resistant mice. Adipose tissue-derived SAA3 displays monocyte chemotactic activity and may play a role in metabolic inflammation associated with obesity and insulin resistance. To investigate a potential mechanistic link between the intestinal microbiota and induction of proinflammatory host factors, we performed molecular analyses of germ-free, conventionally raised and genetically modified Myd88-/- mouse models. SAA3 expression was determined to be significantly augmented in adipose (9.9+/-1.9-fold; P<0.001) and colonic tissue (7.0+/-2.3-fold; P<0.05) by the presence of intestinal microbes. In the colon, we provided evidence that SAA3 is partially regulated through the Toll-like receptor (TLR)/MyD88/NF-kappaB signaling axis. We identified epithelial cells and macrophages as cellular sources of SAA3 in the colon and found that colonic epithelial expression of SAA3 may be part of an NF-kappaB-dependent response to LPS from gut bacteria. In vitro experiments showed that LPS treatments of both epithelial cells and macrophages induced SAA3 expression (27.1+/-2.5-fold vs. 1.6+/-0.1-fold, respectively). Our data suggest that LPS, and potentially other products of the indigenous gut microbiota, might elevate cytokine expression in tissues and thus exacerbate chronic low-grade inflammation observed in obesity.

Title: IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells. Content: The role of immune responses in tumor development is a central issue for tumor biology and immunology. IL-17 is an important cytokine for inflammatory and autoimmune diseases. Although IL-17-producing cells are detected in cancer patients and tumor-bearing mice, the role of IL-17 in tumor development is controversial, and mechanisms remain to be fully elucidated. In the current study, we found that the development of tumors was inhibited in IL-17R-deficient mice. A defect in IFN-gammaR increased tumor growth, whereas tumor growth was inhibited in mice that were deficient in both IL-17R and IFN-gammaR compared with wild-type animals. Further experiments showed that neutralization of IL-17 by Abs inhibited tumor growth in wild-type mice, whereas systemic administration of IL-17 promoted tumor growth. The IL-17R deficiency increased CD8 T cell infiltration, whereas it reduced the infiltration of myeloid-derived suppressor cells (MDSCs) in tumors. In contrast, administration of IL-17 inhibited CD8 T cell infiltration and increased MDSCs in tumors. Further analysis indicated that IL-17 was required for the development and tumor-promoting activity of MDSCs in tumor-bearing mice. These data demonstrate that IL-17-mediated responses promote tumor development through the induction of tumor-promoting microenvironments at tumor sites. IL-17-mediated regulation of MDSCs is a primary mechanism for its tumor-promoting effects. The study provides novel insights into the role of IL-17 in tumor development and has major implications for targeting IL-17 in treatment of tumors.

Title: Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection. Content: CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.

Title: Radiation and Dual Checkpoint Blockade Activates Non-Redundant Immune Mechanisms in Cancer Content: Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Title: TGF-beta inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Content: Transforming growth factor-beta (TGF-beta) inhibits the growth of endothelial cells derived from various sources, including human umbilical vein, bovine aorta, and rat heart. Long-term exposure of rat heart endothelial cells to TGF-beta also induces dramatic changes in morphology that are characteristic of senescent cells. These changes are accompanied by a decrease in the number of high-affinity receptors for epidermal growth factor (EGF), with almost no change in total receptor number. Additionally, the EGF-induced expression of specific competence genes (c-myc, JE, KC) is decreased, whereas the induction of c-fos gene expression by EGF is unaltered by TGF-beta treatment. These data suggest that growth inhibitors such as TGF-beta may act by altering the cell's response to growth-stimulatory factors.

Title: brain organization Content: Transient activation of estrogen receptors (ER) in the developing brain during a limited perinatal "window of time" is recognized as a key mechanism of defeminization of neural control of reproductive function and sexual behavior. Two major ER isoforms, alpha and beta, are present in neural circuits that govern ovarian cycle and sexual behavior. Using highly selective ER agonists, this study provides the first evidence for distinct contribution of individual ER isoforms to the process of estrogen dependent defeminization. Neonatal activation of the ERalpha in female rats resulted in abrogation of cyclic ovarian activity and female sexual behavior in adulthood. These effects are associated with male-like alterations in the morphology of the anteroventral periventricular (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA), as well as refractoriness to estrogen-mediated induction of sexual receptivity. Exposure to an ERbeta-selective agonist induced persistent estrus and had a strong defeminizing effect on the hypothalamic gonadotropin "surge generator" AVPV. However, neonatal ERbeta activation failed to alter female sexual behavior, responsiveness to estrogens and morphometric features of the behaviorally relevant SDN-POA. Thus, although co-present in several brain regions involved in the control of female reproductive function, ER isoforms convey different, and probably not synergistic, chemical signals in the course of neonatal sex-specific brain organization.

Title: Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying. Content: Ghrelin is a hunger hormone with gastroprokinetic properties but the factors controlling ghrelin secretion from the stomach are unknown. Bitter taste receptors (T2R) and the gustatory G proteins, α-gustducin (gust) and α-transducin, are expressed in the gut and are involved in the chemosensation of nutrients. This study aimed to investigate whether T2R-agonists affect (i) ghrelin release via α-gustducin and (ii) food intake and gastric emptying via the release of ghrelin. The mouse stomach contains two ghrelin cell populations: cells containing octanoyl and desoctanoyl ghrelin, which were colocalized with α-gustducin and α-transducin, and cells staining for desoctanoyl ghrelin. Gavage of T2R-agonists increased plasma octanoyl ghrelin levels in WT mice but the effect was partially blunted in gust(-/-) mice. Intragastric administration of T2R-agonists increased food intake during the first 30 min in WT but not in gust(-/-) and ghrelin receptor knockout mice. This increase was accompanied by an increase in the mRNA expression of agouti-related peptide in the hypothalamus of WT but not of gust(-/-) mice. The temporary increase in food intake was followed by a prolonged decrease (next 4 h), which correlated with an inhibition of gastric emptying. The delay in emptying, which was partially counteracted by ghrelin, was not mediated by cholecystokinin and GLP-1 but involved a direct inhibitory effect of T2R-agonists on gastric contractility. This study is unique in providing functional evidence that activation of bitter taste receptors stimulates ghrelin secretion. Modulation of endogenous ghrelin levels by tastants may provide novel therapeutic applications for the treatment of weight -and gastrointestinal motility disorders.

Title: GITR, a member of the TNF receptor superfamily, is costimulatory to mouse T lymphocyte subpopulations. Content: GITR (glucocorticoid-induced TNFR family related gene) is a member of the TNFR superfamily (TNFRSF) that is expressed in different cell types, including T lymphocytes. Because of a high homology in its cytoplasmic region with other known costimulatory members of the TNFRSF, we investigated whether GITR played a costimulatory role in T lymphocyte subpopulations. Our results show that the proliferation response of CD8+ and CD4+ peripheral T cell subpopulations was potentiated when a GITR costimulus was added to an anti-CD3 stimulus. Furthermore, expression of the main activation-induced receptor (IL-2Ralpha) and production of IL-2 and IFN-gamma were increased more with a GITR costimulus than with anti-CD3 alone. GITR stimulation also enhanced anti-CD3-induced ERK phosphorylation, suggesting that GITR is involved in MAPK-pathway activation. Interestingly, CD4+CD25+ regulatory T cell (Treg cell) proliferation was triggered by the GITR costimulus; Treg cell proliferation was paralleled by the loss of the anergic phenotype and suppressor activity. Nevertheless, unstimulated GITR(-/-) CD4+CD25+ and GITR(+/+) CD4+CD25+ cells were equally able to exert suppressor activity on CD4+CD25- responder cells. These results indicate a novel function for GITR as costimulatory molecule of T cell subsets.

Title: Deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice Content: Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.

Title: Prion-induced amyloid heart disease with high blood infectivity in transgenic mice. Content: We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 10(7) 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.

Title: SIRT1 deacetylase in SF1 neurons protects against metabolic imbalance. Content: Chronic feeding on high-calorie diets causes obesity and type 2 diabetes mellitus (T2DM), illnesses that affect hundreds of millions. Thus, understanding the pathways protecting against diet-induced metabolic imbalance is of paramount medical importance. Here, we show that mice lacking SIRT1 in steroidogenic factor 1 (SF1) neurons are hypersensitive to dietary obesity owing to maladaptive energy expenditure. Also, mutant mice have increased susceptibility to developing dietary T2DM due to insulin resistance in skeletal muscle. Mechanistically, these aberrations arise, in part, from impaired metabolic actions of the neuropeptide orexin-A and the hormone leptin. Conversely, mice overexpressing SIRT1 in SF1 neurons are more resistant to diet-induced obesity and insulin resistance due to increased energy expenditure and enhanced skeletal muscle insulin sensitivity. Our results unveil important protective roles of SIRT1 in SF1 neurons against dietary metabolic imbalance.

Title: Effects of Bifidobacterium animalis administered during lactation on allergic and autoimmune responses in rodents. Content: Probiotics are promoted as being beneficial to health and positive effects on the immune system have been reported. Beneficial immune effects have been attributed to several mechanisms, including stimulating T helper 1 (Th1) immunity. To explore the effects of the probiotic Bifidobacterium animalis on Th1- and Th2-mediated immune responses, two different animal models representing either Th1- or Th2-mediated immune responses were used: a rat model for experimental autoimmune encephalomyelitis (EAE) (Th1) and a mouse model for respiratory allergy induced by ovalbumin (OVA) (Th2). B. animalis administration started when the mice or rats were 2 weeks old. Respiratory allergy or EAE were induced when the animals were 6-7 weeks old. In the allergy model, B. animalis modestly reduced the number of infiltrating eosinophils and lymphocytes in the lungs, but no effects on allergen-specific serum immunoglobulin E levels were found. Cytokine profiles assessed after culturing spleen cells with the mitogen concanvalin A (ConA) showed that B. animalis skewed the Th1/Th2 balance towards Th1 in females. However, allergen-induced cytokine production in females was not affected by B. animalis. In males, B. animalis significantly decreased ConA-induced interleukin-13 and a trend towards lower levels of OVA-induced Th2 cytokines. In the EAE model, B. animalis significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group. Our data show that B. animalis reduced several immune parameters in the allergy as well as in the autoimmunity model.

Title: Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100. Content: The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RARalpha knockin mouse were genetically modified with firefly luciferase (APL(luc)) to allow tracking by bioluminescence imaging. Coculture of APL(luc) cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL(luc) cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AML-niche interactions.

Title: T helper 2 cytokines inhibit autophagic control of intracellular Mycobacterium tuberculosis. Content: Autophagy is a recently recognized immune effector mechanism against intracellular pathogens. The role of autophagy in innate immunity has been well established, but the extent of its regulation by the adaptive immune response is less well understood. The T helper 1 (Th1) cell cytokine IFN-gamma induces autophagy in macrophages to eliminate Mycobacterium tuberculosis. Here, we report that Th2 cytokines affect autophagy in macrophages and their ability to control intracellular M. tuberculosis. IL-4 and IL-13 abrogated autophagy and autophagy-mediated killing of intracellular mycobacteria in murine and human macrophages. Inhibition of starvation-induced autophagy by IL-4 and IL-13 was dependent on Akt signaling, whereas the inhibition of IFN-gamma-induced autophagy was Akt independent and signal transducer and activator of transcription 6 (STAT6) dependent. These findings establish a mechanism through which Th1-Th2 polarization differentially affects the immune control of intracellular pathogens.

Title: TLR-Mediated Innate Production of IFN-γ by CD8+ T Cells Is Independent of Glycolysis. Content: CD8(+) T cells can respond to unrelated infections in an Ag-independent manner. This rapid innate-like immune response allows Ag-experienced T cells to alert other immune cell types to pathogenic intruders. In this study, we show that murine CD8(+) T cells can sense TLR2 and TLR7 ligands, resulting in rapid production of IFN-γ but not of TNF-α and IL-2. Importantly, Ag-experienced T cells activated by TLR ligands produce sufficient IFN-γ to augment the activation of macrophages. In contrast to Ag-specific reactivation, TLR-dependent production of IFN-γ by CD8(+) T cells relies exclusively on newly synthesized transcripts without inducing mRNA stability. Furthermore, transcription of IFN-γ upon TLR triggering depends on the activation of PI3K and serine-threonine kinase Akt, and protein synthesis relies on the activation of the mechanistic target of rapamycin. We next investigated which energy source drives the TLR-induced production of IFN-γ. Although Ag-specific cytokine production requires a glycolytic switch for optimal cytokine release, glucose availability does not alter the rate of IFN-γ production upon TLR-mediated activation. Rather, mitochondrial respiration provides sufficient energy for TLR-induced IFN-γ production. To our knowledge, this is the first report describing that TLR-mediated bystander activation elicits a helper phenotype of CD8(+) T cells. It induces a short boost of IFN-γ production that leads to a significant but limited activation of Ag-experienced CD8(+) T cells. This activation suffices to prime macrophages but keeps T cell responses limited to unrelated infections.

Title: Direct Reprogramming of Hepatic Myofibroblasts into Hepatocytes In Vivo Attenuates Liver Fibrosis. Content: Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

Title: Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile Content: The gastrointestinal tracts of mammals are colonized by hundreds of microbial species that contribute to health, including colonization resistance against intestinal pathogens. Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens. Among these, Clostridium difficile, a major cause of antibiotic-induced diarrhoea, greatly increases morbidity and mortality in hospitalized patients. Which intestinal bacteria provide resistance to C. difficile infection and their in vivo inhibitory mechanisms remain unclear. Here we correlate loss of specific bacterial taxa with development of infection, by treating mice with different antibiotics that result in distinct microbiota changes and lead to varied susceptibility to C. difficile. Mathematical modelling augmented by analyses of the microbiota of hospitalized patients identifies resistance-associated bacteria common to mice and humans. Using these platforms, we determine that Clostridium scindens, a bile acid 7α-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration, enhances resistance to infection in a secondary bile acid dependent fashion. Using a workflow involving mouse models, clinical studies, metagenomic analyses, and mathematical modelling, we identify a probiotic candidate that corrects a clinically relevant microbiome deficiency. These findings have implications for the rational design of targeted antimicrobials as well as microbiome-based diagnostics and therapeutics for individuals at risk of C. difficile infection.

Title: Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments. Content: Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17 beta-estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17 beta-Estradiol up-regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up-regulated. Treatment of estrogen receptor (ER) alpha knockout mice with 17 beta-estradiol or treatment of C57BL/6 mice with 17 alpha-estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17 beta-estradiol effects on apoE are either directly or indirectly mediated by ER alpha in the diencephalon, while the effects in the cortex may be mediated by a non-classical mechanism or by ER beta. Measurement of mRNA levels in estrogen versus placebo-treated wild-type mice indicated that the effect of 17 beta-estradiol on apoE was not associated with changes in apoE mRNA levels.

Title: Chronic hypoxia induces modification of the N-methyl-D-aspartate receptor in rat brain. Content: This study examined [3H]MK-801 binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from cerebral cortex, hippocampus and corpus striatum of 3 week old rats exposed to 10 weeks of intermittent hypobaric hypoxia (4300 m; 450 Torr) and compared results with those of normoxic controls. The cortex, hippocampus and striatum of hypoxic animals had a 36, 35 and 31% reduction in binding sites (Bmax) and a 29, 32 and 17% decrease (reflecting increased affinity) in the dissociation constant (Kd) when compared to controls. In the cerebral cortex, both glutamate (100 microM) and glycine (10 microM) enhanced 3[H]MK-801 binding by two to 3-fold. Coagonist glutamate, however, had a higher EC50 (0.44 microM) in the hypoxic cortical membranes when compared to controls (0.28 microM). No significant differences were found in the EC50 of glycine. The results show that the NMDA receptor is altered in several brain regions of rats developing in a hypoxic environment.

Title: Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality Content: Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

Title: A direct role for IFN-gamma in regulation of Th1 cell development. Content: IL-12 has been identified as a major cytokine influencing the differentiation of CD4 cells to a Th1 phenotype, whereas a role for IFN-gamma is controversial. We investigated the interrelationship between IL-12 and IFN-gamma in promoting Th1 responses using naive CD4 cells reactive with pigeon cytochrome c from TCR transgenics and memory CD4 cells derived by in vivo priming with KLH. Without exogenous rIL-12 or rIFN-gamma, primary and memory effectors induced by Ag or anti-CD3 and anti-CD28 secreted variable levels of IL-2 and IFN-gamma. The level of IFN-gamma secreted by effectors correlated with endogenous IFN-gamma produced in primary cultures, and anti-IFN-gamma largely inhibited the development of effectors producing IFN-gamma. With optimal TCR stimulation and costimulation, endogenous IFN-gamma, without IL-12, was sufficient to elicit Th1 cells via an autocrine mechanism, whereas with suboptimal stimulation, exogenous rIFN-gamma or rIL-12 was required for Th1 development. However, rIL-12 was more effective than rIFN-gamma, partially because rIL-12 greatly enhanced autocrine production of IFN-gamma, and optimal development of the Th1 phenotype was mediated by the synergistic actions of both cytokines. Thus, both IFN-gamma and IL-12 can independently regulate Th1 development, but because of IFN-gamma-mediated feedback, their relative contributions are determined by the conditions of T cell stimulation. The extent of differentiation to a Th1 phenotype may, therefore, depend on the availability of both APC-derived IL-12 and autocrine IFN-gamma consequent to the overall strength of T cell stimulation.

Title: Role of gut commensal microflora in the development of experimental autoimmune encephalomyelitis. Content: Mucosal tolerance has been considered a potentially important pathway for the treatment of autoimmune disease, including human multiple sclerosis and experimental conditions such as experimental autoimmune encephalomyelitis (EAE). There is limited information on the capacity of commensal gut bacteria to induce and maintain peripheral immune tolerance. Inbred SJL and C57BL/6 mice were treated orally with a broad spectrum of antibiotics to reduce gut microflora. Reduction of gut commensal bacteria impaired the development of EAE. Intraperitoneal antibiotic-treated mice showed no significant decline in the gut microflora and developed EAE similar to untreated mice, suggesting that reduction in disease activity was related to alterations in the gut bacterial population. Protection was associated with a reduction of proinflammatory cytokines and increases in IL-10 and IL-13. Adoptive transfer of low numbers of IL-10-producing CD25(+)CD4(+) T cells (>75% FoxP3(+)) purified from cervical lymph nodes of commensal bacteria reduced mice and in vivo neutralization of CD25(+) cells suggested the role of regulatory T cells maintaining peripheral immune homeostasis. Our data demonstrate that antibiotic modification of gut commensal bacteria can modulate peripheral immune tolerance that can protect against EAE. This approach may offer a new therapeutic paradigm in the treatment of multiple sclerosis and perhaps other autoimmune conditions.

Title: Microenvironment rigidity modulates responses to the HER2 receptor tyrosine kinase inhibitor lapatinib via YAP and TAZ transcription factors. Content: Stiffness is a biophysical property of the extracellular matrix that modulates cellular functions, including proliferation, invasion, and differentiation, and it also may affect therapeutic responses. Therapeutic durability in cancer treatments remains a problem for both chemotherapies and pathway-targeted drugs, but the reasons for this are not well understood. Tumor progression is accompanied by changes in the biophysical properties of the tissue, and we asked whether matrix rigidity modulated the sensitive versus resistant states in HER2-amplified breast cancer cell responses to the HER2-targeted kinase inhibitor lapatinib. The antiproliferative effect of lapatinib was inversely proportional to the elastic modulus of the adhesive substrata. Down-regulation of the mechanosensitive transcription coactivators YAP and TAZ, either by siRNA or with the small-molecule YAP/TEAD inhibitor verteporfin, eliminated modulus-dependent lapatinib resistance. Reduction of YAP in vivo in mice also slowed the growth of implanted HER2-amplified tumors, showing a trend of increasing sensitivity to lapatinib as YAP decreased. Thus we address the role of stiffness in resistance to and efficacy of a HER2 pathway-targeted therapeutic via the mechanotransduction arm of the Hippo pathway.

Title: Gut Microbiota Is a Key Modulator of Insulin Resistance in TLR 2 Knockout Mice Content: Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.

Title: The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor+ Innate Lymphoid Cells Content: Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-α produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.

Title: Peroxisome Proliferator Activated Receptor gamma (PPARγ) Agonist Rosiglitazone Ameliorate Airway Inflammation by Inhibiting Toll-Like Receptor 2 (TLR2)/Nod-Like Receptor with Pyrin Domain Containing 3 (NLRP3) Inflammatory Corpuscle Activation in Asthmatic Mice Content: BACKGROUND The purpose of this study was to explore the function and mechanism of peroxisome proliferator activated receptor agonist (PPARγ) in the toll-like receptor 2 (TLR2)/nod-like receptor with pyrin domain containing 3 (NLRP3) inflammatory corpuscle pathway of asthmatic mice. MATERIAL AND METHODS Eighteen female mice (C57) were randomly divided into 4 groups: the control group, the asthma model group challenged by ovalbumin (OVA), the rosiglitazone group, and the PPARγ agonist rosiglitazone treatment group. The infiltration of peribronchial inflammatory cells as well as the proliferation and mucus secretion of bronchial epithelial goblet cells were observed by hematoxylin and eosin and periodic acid-Schiff staining. Western blots were employed to detect the expression levels of TLR2, PPARγ, nuclear factor-kappa B (NF-kappaB), NLRP3, and ASC [apoptosis-associated speck-like protein containing C-terminal caspase recruitment domain [CARD]). RESULTS The number of inflammatory cells and eosinophils, and the levels of OVAs IgE, interleukin-4 (IL-4), and IL-13 were significantly higher in the C57 asthma group compared to the C57 control group and the treatment group (P<0.05). The infiltration of peribronchiolar inflammatory cells, wall thickening, goblet cell hyperplasia, and mucus secretion in the treatment group were all significantly decreased compared to those in the asthma group. PPARg expression in the treatment group was significantly higher compared to the asthma group and the control group (P<0.05). The protein expression levels of TLR2, NF-kappaB, NLRP3, and ASC were significantly lower compared to the asthma group but were higher compared to the control group (P<0.05). CONCLUSIONS PPARγ rosiglitazone ameliorates airway inflammation by inhibiting NF-kappaB expression in asthmatic mice, and further inhibits the activation of TLR2/NLRP3 inflammatory corpuscles.

Title: Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor Content: The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target, suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.

Title: Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection Content: Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

Title: LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response Content: The liver X receptors (LXRs) are nuclear receptors with established roles in the regulation of lipid metabolism. We now show that LXR signaling not only regulates macrophage cholesterol metabolism but also impacts antimicrobial responses. Mice lacking LXRs are highly susceptible to infection with the intracellular bacteria Listeria monocytogenes (LM). Bone marrow transplant studies point to altered macrophage function as the major determinant of susceptibility. LXR-null macrophages undergo accelerated apoptosis when challenged with LM and exhibit defective bacterial clearance in vivo. These defects result, at least in part, from loss of regulation of the antiapoptotic factor SPalpha, a direct target for regulation by LXRalpha. Expression of LXRalpha or SPalpha in macrophages inhibits apoptosis in the setting of LM infection. Our results demonstrate that LXR-dependent gene expression plays an unexpected role in innate immunity and suggest that common nuclear receptor pathways mediate macrophage responses to modified lipoproteins and intracellular pathogens.

Title: Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo Content: Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.

Title: Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels. Content: Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins. Protein adsorption-resistant poly(ethylene glycol) (PEG) materials can be covalently modified to present precise concentrations of adhesion proteins or peptides without the unintended effects of non-specifically adsorbed proteins. Here, we describe an improved protocol that incorporates custom-engineered materials. We first reproduced the Efe et al. protocol on Matrigel-coated TCPS (the original material), reprogramming adult mouse tail-tip mouse fibroblasts (TTF) and mouse embryonic fibroblasts (MEF) to cardiomyocyte-like cells that demonstrated striated sarcomeric α-actinin staining, spontaneous calcium transients, and visible beating. We then designed poly(ethylene glycol) culture substrates to promote MEF adhesion via laminin and RGD-binding integrins. PEG hydrogels improved proliferation and reprogramming efficiency (evidenced by beating patch number and area, gene expression, and flow cytometry), yielding almost twice the number of sarcomeric α-actinin positive cardiomyocyte-like cells as the originally described substrate. These results illustrate that cellular reprogramming may be enhanced using custom-engineered materials.

Title: Behavioral characterization of neuropeptide Y knockout mice. Content: An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception.

Title: Ethanol extract of Allium fistulosum inhibits development of non-alcoholic fatty liver disease Content: BACKGROUND/OBJECTIVES Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver disease and is closely associated with metabolic syndrome. In the present study, we observed the effect of ethanol extract of Allium fistulosum (EAF) on NAFLD and have suggested the possibility of using EAF as a natural product for application in the development of a treatment for NAFLD. MATERIALS/METHODS The preventive effect on hepatic lipid accumulation was estimated by using an oleic acid (OA)-induced NAFLD model in vitro and a Western diet (high-fat high-sucrose; WD)-induced obese mouse model. Animals were divided into three groups (n = 7): normal diet group (ND), WD group, and WD plus 1% EAF group. RESULTS EAF reduced OA-stimulated lipid accumulation in HepG2 cells in the absence of cellular cytotoxicity and significantly blocked transcriptional activation of sterol regulatory element-binding protein 1 and fatty acid synthase genes. Subsequently, we investigated these effects in vivo in mice fed either ND or WD in the presence or absence of EAF supplementation. In comparison to the ND controls, the WD-fed mice exhibited increases in body weight, liver weight, epididymal fat weight, and accumulation of fat in hepatocytes, and these effects were significantly attenuated by EAF supplementation. CONCLUSIONS Allium fistulosum attenuates the development of NAFLD, and EAF elicits anti-lipogenic activity in liver. Therefore, EAF represents a promising candidate for use in the development of novel therapeutic drugs or drug combinations for the prevention and treatment of NAFLD.

Title: "Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Content: In the spleens of young, adult mice there exist naturally occurring killer lymphocytes with specificity for mouse Moloney leukemia cells. The lytic activity was directed against syngeneic or allogeneic Moloney leukemia cells to a similar extent, but was primarily expressed when tested against in vitro grown leukemia cells. Two leukemias of non-Moloney origin were resistant and so was the mastocytoma line P815. Although killer activity varied between different strains of mice, the specificity of lysis was the same as indicated by competition experiments using unlabeled Moloney or other tumor cells as inhibitors in the cytotoxic assays. Capacity to compete and sensitivy to lysis by the killer cells were found to be highly positively correlated. Analysis of the kinetics of the cytotoxic assay revealed a rapid induction of lysis within one to four hours, arguing against any conventional in vitro induction of immune response. No evidence was found of soluble factors playing any role in the cytolytic assay.

Title: Teratogen-Induced Oxidative Stress Targets Glyceraldehyde-3-Phosphate Dehydrogenase in the Organogenesis Stage Mouse Embryo Content: Exposure during the organogenesis stage of the mouse embryo to the model teratogen, hydroxyurea (HU), induces curly tail and limb malformations. Oxidative stress contributes to the developmental toxicity of HU. Reactive oxygen species (ROS) interact with polyunsaturated bilipid membranes to form α,β-unsaturated reactive aldehydes; 4-hydroxy-2-nonenal (4-HNE), one of the most cytotoxic of these aldehydes, covalently adducts with proteins, lipids, and nucleic acids. The goal of the current study is to determine if HU exposure of CD1 mice on gestation day 9 generates region-specific 4-HNE-protein adducts in the embryo and to identify the proteins targeted. The formation of 4-HNE-protein adducts was elevated in the caudal region of control embryos; HU exposure further increased 4-HNE-protein adduct formation in this area. Interestingly, three of the 4-HNE-modified proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamate oxaloacetate transaminase 2, and aldolase 1, A isoform, are involved in energy metabolism. The formation of 4-HNE-GAPDH protein adducts reduced GAPDH enzymatic activity by 20% and attenuated lactate production by 40%. Furthermore, HU exposure induced the nuclear translocation of GAPDH in the caudal region of exposed embryos; this nuclear translocation may be associated with the reactivation of oxidized proteins involved in DNA repair, such as apurinic/apyrimidinic endonuclease-1, and the stimulation of E1A-associated P300 protein/creb-binding protein (p300/CBP) activity, initiating cell death in a p53-dependent pathway. We propose that GAPDH is a redox-sensitive target in the embryo and may play a role in a stress response during development.

Title: Engraftment of engineered ES cell–derived cardiomyocytes but not BM cells restores contractile function to the infarcted myocardium Content: Cellular cardiomyoplasty is an attractive option for the treatment of severe heart failure. It is, however, still unclear and controversial which is the most promising cell source. Therefore, we investigated and examined the fate and functional impact of bone marrow (BM) cells and embryonic stem cell (ES cell)–derived cardiomyocytes after transplantation into the infarcted mouse heart. This proved particularly challenging for the ES cells, as their enrichment into cardiomyocytes and their long-term engraftment and tumorigenicity are still poorly understood. We generated transgenic ES cells expressing puromycin resistance and enhanced green fluorescent protein cassettes under control of a cardiac-specific promoter. Puromycin selection resulted in a highly purified (>99%) cardiomyocyte population, and the yield of cardiomyocytes increased 6–10-fold because of induction of proliferation on purification. Long-term engraftment (4–5 months) was observed when co-transplanting selected ES cell–derived cardiomyocytes and fibroblasts into the injured heart of syngeneic mice, and no teratoma formation was found (n = 60). Although transplantation of ES cell–derived cardiomyocytes improved heart function, BM cells had no positive effects. Furthermore, no contribution of BM cells to cardiac, endothelial, or smooth muscle neogenesis was detected. Hence, our results demonstrate that ES-based cell therapy is a promising approach for the treatment of impaired myocardial function and provides better results than BM-derived cells.

Title: Oncostatin M and leukemia inhibitory factor do not use the same functional receptor in mice. Content: Oncostatin M (OSM) and leukemia inhibitory factor (LIF) are members of the interleukin-6 (IL-6) subfamily of cytokines that use a common signal transducer gp130. Human OSM (hOSM) and LIF share a functional high-affinity receptor that is composed of gp130 and LIF receptor beta subunit (LIFRbeta). A second high-affinity receptor for hOSM was recently found to be formed by gp130 and the hOSM receptor beta subunit. However, the nature of murine OSM (mOSM) and its receptors has remained unknown. Using the recently cloned mOSM cDNA, we produced recombinant mOSM and studied its biological activity and receptor structure. Murine hematopoietic cell lines M1 and DA1.a, an embryonic stem cell line CCE, and Ba/F3 transfectants expressing gp130 and LIFRbeta responded to murine LIF (mLIF) and hOSM equally well, while these cells responded to mOSM only at a 30-fold to 100-fold higher concentration than those of mLIF and hOSM. In contrast, NIH3T3 cells responded to mOSM, but not to mLIF and hOSM. Scatchard plot analyses showed that mOSM bound to gp130 with low-affinity (kd = 2.8 to 4.2 nmol/L) and that the binding affinity did not increase in the presence of LIFRbeta. However, mOSM bound to NIH3T3 cells with high-affinity (kd = 660 pmol/L), whereas mLIF did not bind to NIH3T3 cells at all. These results indicate that unlike hOSM, mOSM and mLIF do not share the same functional receptor, and mOSM delivers signals only through its specific receptor complex. Further studies in mice will define the physiological roles of OSM.

Title: Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice Content: Endogenous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b+ cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b+ cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.

Title: Autophagy deficiency leads to protection from obesity and insulin resistance by inducing Fgf21 as a mitokine Content: Despite growing interest and a recent surge in papers, the role of autophagy in glucose and lipid metabolism is unclear. We produced mice with skeletal muscle–specific deletion of Atg7 (encoding autophagy-related 7). Unexpectedly, these mice showed decreased fat mass and were protected from diet-induced obesity and insulin resistance; this phenotype was accompanied by increased fatty acid oxidation and browning of white adipose tissue (WAT) owing to induction of fibroblast growth factor 21 (Fgf21). Mitochondrial dysfunction induced by autophagy deficiency increased Fgf21 expression through induction of Atf4, a master regulator of the integrated stress response. Mitochondrial respiratory chain inhibitors also induced Fgf21 in an Atf4-dependent manner. We also observed induction of Fgf21, resistance to diet-induced obesity and amelioration of insulin resistance in mice with autophagy deficiency in the liver, another insulin target tissue. These findings suggest that autophagy deficiency and subsequent mitochondrial dysfunction promote Fgf21 expression, a hormone we consequently term a 'mitokine', and together these processes promote protection from diet-induced obesity and insulin resistance.