As baicalin could attenuate ASC speck formation upon NLRP3 activation, we next explore whether the suppression of ASC speck formation by baicalin could also be reversed by blocking the PKA activity. The PKA inhibitor H89 increased the ASC speck formation induced by ATP, and it could partly reversed the inhibitory effect of baicalin on ATP-induced ASC speck formation (**Figures 9A,B**), further confirming that PKA signaling had been involved in the action of baicalin on suppressing NLRP3 inflammasome activation.

Therefore, we next explored whether baicalin could enhance the Ser/Thr phosphorylation of NLRP3 on PKA-specific sites, which has been reported to prevent NLRP3 inflammasome activation in macrophages (37, 38). To this end, NLRP3 was immunoprecipitated, and its phosphorylation on Ser/Thr residues of PKA substrate motifs was evaluated by Western blotting. Indeed, the PKA-specific phosphorylation on Ser/Thr residues of NLRP3 was greatly increased by baicalin pretreatment. The PKA inhibitor H89 blocked such phosphorylation, further

verifying that baicalin-enhanced NLRP3 phosphorylation was mediated by PKA signaling (**Figures 9C,D**). Altogether, these results suggested that baicalin inhibited NLRP3 inflammasome activation at least partly through augmenting PKA signaling.

# Baicalin Treatment Increases Mouse Survival in Bacterial Sepsis

As ATP-induced NLRP3 inflammasome activation has critical roles in bacterial sepsis (5), we explored whether baicalin could ameliorate sepsis in a mouse model of bacterial infection in the peritoneal cavity. Mice were administered once intragastrically with baicalin solution (100 or 200 mg/kg body weight) or vehicle 3 h before intraperitoneal injection of viable *E. coli* (2 × 109 CFU/mouse). One hour after the injection of bacteria, the mice were administered once again with baicalin or vehicle. Only 10% of mice survived the period of observation (120 h) in the vehicle group, whereas baicalin administration significantly increased their survival rates when compared with the vehicle group (**Figure 10A**). Moreover, baicalin treatment significantly decreased IL-1β levels in the sera of bacterial infected mice as compared with vehicle (**Figure 10B**), indicating that baicalin suppressed systemic inflammation in the mice, which was consistent with the *in vitro* studies showing baicalin's inhibitory effects on NLRP3 activation. Therefore, this result highlights the potential application of baicalin in the treatment of inflammatory diseases including bacterial sepsis.

# DISCUSSION

In this study, we found that baicalin markedly inhibited NLRP3 inflammasome activation in LPS-primed macrophages upon ATP or nigericin triggering, thereby blocking the activation of caspase-1 and the release of mature IL-1β and HMGB1. This inhibitory action of baicalin is in part mediated by augmentation of PKA signaling as the adenyl cyclase inhibitor MDL12330A or PKA inhibitor H89 could partly reverse such an effect. Indeed,

supernatant.

was used to assess the expression levels of indicated proteins in the cell lysates and culture supernatants, respectively. β-Tubulin was used as a loading control for cell lysates. (D) Histograms showing the relative intensity of high-mobility group box-1 (HMGB1) band in the culture supernatants in panel (C) (*n* = 3). The intensity of HMGB1 band in nigericin group was set to 1.0, and those of the other groups were calculated relative to the nigericin group. The experiments were performed three times independently, with one representative experiment shown. Data are shown as mean ± SD (*n* = 3). *\*\*\*P* < 0.001; BA, baicalin; Sup, supernatant.

baicalin enhanced PKA-mediated phosphorylation of NLRP3, thereby reducing NLRP3's capacity to recruit the adaptor ASC and the formation of ASC oligomerization. In line with the finding that baicalin inhibited NLRP3 inflammasome activation *in vitro*, baicalin administration *in vivo* significantly improved the survival of mice in bacterial sepsis.

During bacterial infection, ATP can be released either from the host's monocytes/macrophages or from bacterial cells (5, 49, 50). By acting on the purinergic P2X7 receptor on the plasma membrane, extracellular ATP can induce the efflux of K+, thus triggering the NLRP3 inflammasome leading to the activation of caspase-1 (51–53). The activated caspase-1 further leads to pyroptosis and release of inflammatory cytokines including IL-1β and HMGB1 (7, 8, 13, 46, 47), which greatly exacerbate the inflammatory responses during bacterial infections and even leading to organ damage and septic shock (54–56). We showed in this study that baicalin markedly inhibited NLRP3 inflammasome activation in murine macrophages upon ATP stimulation, suggesting that baicalin may attenuate the severity of bacterial sepsis. Consistent with this notion, we found that baicalin could significantly improve the survival of mice in a model of bacterial sepsis. This result is in line with several previous studies showing that baicalin can prolong the survival of mice in both CLP-induced sepsis and LPS-induced endotoxemia (28–30). Consistent with our results, these previous reports showed that baicalin did reduce the levels of HMGB1 in septic mice and suppress the release of IL-1β and HMGB1 from macrophages (29), even though not clearly pointing to NLRP3 inflammasome activation. In support of our findings, two separate studies provided evidence that baicalin could suppress the NLRP3 inflammasome pathway in piglet mononuclear phagocytes in response to LPS stimulation (36) or *Haemophilus parasuis* infection (35). Those previous studies and our data indicated that baicalin robustly inhibited NLRP3 inflammasome activation to attenuate inflammatory responses during infections such as bacterial sepsis.

More recently, there is evidence indicating that PKA signaling negatively regulates NLRP3 activation. For example, bile acid induces an increase in intracellular cAMP levels through acting on its receptor TGR5, leading to PKA activation and PKA-mediated phosphorylation of NLRP3, which suppresses

NLRP3 inflammasome activation in macrophages (37). It has also been shown that prostaglandin E2 can inhibit NLRP3 inflammasome activation by inducing PKA signaling (38). In addition, dopamine negatively regulates NLRP3 inflammasome *via* a cAMP-dependent manner (57). Consistent with these studies, we in this study provided evidence that baicalin suppressed NLRP3 activation by modulating PKA activity. Baicalin greatly increased PKA-mediated Ser/Thr phosphorylation of NLRP3 and prevented ATP-induced formation of ASC specks, but these effects of baicalin could be counteracted by blocking the PKA signaling pathway with its specific inhibitors. This indicated that baicalin prevented NLRP3 inflammasome assembly by enhancing the NLRP3 phosphorylation on PKA-specific sites. In further support of this, PKA inhibitors also counteracted baicalin's effects on suppressing ATP-induced caspase-1p10 and IL-1β release. In addition, ATP-induced NLRP3 inflammasome activation was further enhanced by PKA inhibitor, which is in line with previous studies (37, 38). Collectively, these data demonstrated that

baicalin can suppress NLRP3 inflammasome activation by, at least partly, modulating PKA signaling.

Furthermore, blocking cAMP formation by inhibiting the adenyl cyclase (AC) activity also significantly reversed baicalininduced suppression of NLRP3 activation, highlighting the possibility that baicalin may modulate upstream components of PKA signaling. It was likely that baicalin might have increased cAMP levels by modulating AC activity, although we had not assayed the cAMP levels. In support of this notion, a previous study showed that baicalin administration could increase cAMP levels *in vivo* (48). However, the intracellular levels of cAMP are also regulated by its degradation enzyme phosphodiesterases (PDEs) (58). A large panel of PDEs has been shown to regulate cAMP levels thus modulating the PKA activity and NLRP3 activation. For example, the PED4 inhibitor Rolipram has been shown to inhibit NLRP3 inflammasome activation in submandibular gland cells (59). Although we had shown that AC inhibitor could reverse baicalin-mediated suppression of NLRP3, our data does not

exclude the possibility that baicalin may regulate PKA signaling by affecting the activity of PDEs. Recently, it has been shown that the phosphorylation of NLRP3 on Ser 3 (in mouse, which corresponds Ser 5 in human) has a critical role in modulating NLRP3 activation and that the protein phosphatase PP2A is involved in dephosphorylation and activation of NLRP3 inflammasome (60). As the motif surrounding Ser 3 (MTS\*) is different from the PKA substrate motif [(K/R)(K/R)XS\*/T\*], it is still unknown whether Ser/Thr phosphorylation of NLRP3 on PKA-specific sites is regulated by PP2A. Besides, although H89 did inhibit the action of PKA on NLRP3 as revealed by abrogating the phosphorylation on PKA-specific sites, it should be noted that H89 may have other action targets beyond PKA (61). Therefore, further investigation is warranted to elucidate the precise mechanism by which baicalin modulates the cAMP/PKA signaling pathway.

As aforementioned, NLRP3 inflammasome activation requires two signals: the priming (first signal by LPS, etc.) and triggering (second signal by ATP or nigericin, etc.) (3, 62). Baicalin has been shown to exhibit anti-inflammatory activity by suppressing the NF-κB pathway, thus suppressing the expression of inflammatory cytokines and NLRP3 inflammasome components under various circumstances (31–36). It is worth noting that in those studies such inhibitory effects of baicalin appear to be attributed to the first signal (priming) of NLRP3 inflammasome (35, 36), thus leading to the downregulation of NLRP3 activation since the full activation of this inflammasome is relying on a marked upregulation of this key protein. However, it was unclear whether baicalin could inhibit NLRP3 inflammasome activation at the triggering step (second signal). In this study, we found that baicalin had minimal effect on the expression of

were measured by cytometric bead array assay. Data are shown as mean ± SD (*n* = 3). \**P* < 0.05; \*\**P* < 0.01; \*\*\**P* < 0.001; ns, not significant; BA, baicalin.

NLRP3 inflammasome components, including the constitutive expression of ASC and pro-caspase-1 as well as induced expression of NLRP3 and pro-IL-1β in LPS-primed macrophages. Instead, baicalin could block NLRP3 inflammasome activation upon ATP or nigericin triggering, indicative of its action on the second signal for this inflammasome. Such inhibitory effects of baicalin are likely mediated by augmenting the PKA signaling leading to increased phosphorylation of NLRP3 on PKA-specific sites, which in turn prevents the adaptor ASC recruitment into the inflammasome. Therefore, apart from its inhibitory effects on NLRP3 transcription as revealed by previous studies (36, 37), baicalin can also inhibit NLRP3 inflammasome activation in macrophages upon ATP or nigericin triggering.

Adenosine triphosphate and nigericin have been widely used as activators to stimulate NLRP3 inflammasome activation in LPS-primed macrophages (6, 37, 38, 44), accompanied by caspase-1-dependent cell death, which is regarded as pyroptotic cell death (2, 3). Supporting this, one recent study elegantly showed that gasdermin D, NLRP3, and caspase-1 are all needed for nigericin to induce cell death and IL-1β secretion in LPS-primed RAW 264.7 cells expressing ASC, indicating that nigericin does induce pyroptosis in LPS-primed macrophages (11). However, it has been found that a diverse array of NLRP3 activators, including ATP and nigericin, induced necrosis in human THP-1 cells irrespective of LPS stimulation (63). One possible explanation for this discrepancy is that the cell death types induced by NLRP3 activators may be dependent on experimental settings (such as incubation time and stimulator concentrations). For example, in most of studies (6, 37, 38, 44), ATP (2–5 mM) and nigericin (10 µM) were incubated for 0.5–1 h to activate the NLRP3 inflammasome, respectively. But longer nigericin incubation time (4–6 h) and higher ATP concentrations (≥30 mM for 4–6 h) had been shown to trigger necrosis (63). In this study, we used nigericin stimulation for 1 h and found that baicalin significantly inhibited nigericininduced inflammasome activation and cell death in LPS-primed macrophages, suggesting that baicalin could inhibit NLRP3 mediated pyroptosis. Similarly, as LPS plus ATP treatment induced caspase-1 activation and IL-1β release, it was likely that the accompanied cell death was pyroptosis. Further studies are warranted to uncover whether baicalin inhibits ATP- or nigericin-induced necrosis under the condition without LPS priming.

Figure 9 | Baicalin inhibited ASC speck formation by upregulating protein kinase A (PKA) signaling. Bone marrow-derived macrophages (BMDMs) were treated as described in Figure 8. (A) Representative immunofluorescence images showing ASC subcellular distribution. Yellow arrows indicate ASC specks. The enlarged insets showed cells with an ASC speck. Scale bars, 20 µm. (B) The ASC speck formation was quantified by the cells with ASC specks relative to the total cells from five random fields each containing ~50 cells. (C) LPS-primed BMDMs were incubated with the PKA inhibitor H89 (20 µM) for 30 min and then treated with baicalin (0.6 mM) for 1 h without adenosine triphosphate (ATP) treatment. NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) immunoprecipitation was analyzed for phosphorylation on PKA-specific sites (p-Ser/Thr PKA substrate). (D) The intensity of p-Ser/Thr phosphorylation on PKA-specific sites of NLRP3 was quantified relative to total NLRP3 in panel (C). \**P* < 0.05; \*\**P* < 0.01; \*\*\**P* < 0.001; ns, not significant; BA, baicalin.

In conclusion, we found that baicalin robustly inhibited ATP or nigericin-induced NLRP3 inflammasome activation in mouse macrophages, at least partly *via* augmenting PKA signaling. Furthermore, we also showed that baicalin administration significantly prolonged mouse survival in a model of bacterial sepsis, suggestive of suppression of systemic inflammation. Although future research is warranted to unravel the precise mechanism of baicalin's action on the PKA pathway and investigations in human macrophages may provide more informative data, our studies highlight that baicalin can be used for the treatment of NLRP3-related inflammatory diseases including bacterial sepsis.

### ETHICS STATEMENT

All animal experiments were performed according to the guidelines for the care and use of animals approved by the Committee on the Ethics of Animal Experiments of Jinan University.

#### REFERENCES

# AUTHOR CONTRIBUTIONS

C-GL, LY, F-YM, and Y-YJ performed *in vitro* studies; L-HX, Z-JS, and Q-BZ conducted animal studies; C-GL, LY, and Z-JS analyzed the data; D-YO and X-HH supervised the study; X-HH, D-YO, and C-GL wrote the paper.

### FUNDING

This work was supported by the grants from the National Natural Science Foundation of China (No. 81773965, No. 81673664 and No. 81373423).

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01409/ full#supplementary-material.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Li, Yan, Mai, Shi, Xu, Jing, Zha, Ouyang and He. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Tumor necrosis Factor-alpha Targeting can Protect against arthritis with low sensitization to infection

*Nadia Belmellat1,2, Luca Semerano1,2,3, Noria Segueni 4 , Diane Damotte5 , Patrice Decker1,2, Bernhard Ryffel4,6, Valérie Quesniaux4 , Marie-Christophe Boissier1,2,3\* and Eric Assier1,2*

*1UMR 1125 INSERM, Bobigny, France, 2Sorbonne Paris Cité Université Paris 13, Bobigny, France, 3Service de Rhumatologie, Groupe Hospitalier Avicenne—Jean Verdier—René Muret, APHP, Bobigny, France, 4 INEM UMR7355, CNRS, University of Orléans, Orléans, France, 5Service de pathologie Hôpitaux Universitaires Paris Centre, APHP, Université Paris Descartes, Paris, France, 6 IDM, University of Cape Town, Cape Town, South Africa*

#### *Edited by:*

*Guixiu Shi, Xiamen University, China*

#### *Reviewed by:*

*Dipyaman Ganguly, Indian Institute of Chemical Biology (CSIR), India Dong Li, Jilin University, China*

*\*Correspondence:*

*Marie-Christophe Boissier<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 01 August 2017 Accepted: 27 October 2017 Published: 14 November 2017*

#### *Citation:*

*Belmellat N, Semerano L, Segueni N, Damotte D, Decker P, Ryffel B, Quesniaux V, Boissier M-C and Assier E (2017) Tumor Necrosis Factor-Alpha Targeting Can Protect against Arthritis with Low Sensitization to Infection. Front. Immunol. 8:1533. doi: 10.3389/fimmu.2017.01533*

Tumor necrosis factor-alpha (TNF-α) blockade is an effective treatment for rheumatoid arthritis (RA) and other inflammatory diseases, but in patients, it is associated with reduced resistance to the infectious agents *Mycobacterium tuberculosis* and *Listeria monocytogenes*, among others. Our goal was to model infection and arthritis in mice and to compare etanercept, a currently used anti-TNF-α inhibitor, to an anti-TNF-α vaccine. We developed a murine surrogate of the TNF-α kinoid and produced an anti-murine TNF-α vaccine (TNFKi) composed of keyhole limpet hemocyanin conjugated to TNF-α, which resulted in anti-TNF-α antibody production in mice. We also used etanercept (a soluble receptor of TNF commonly used to treat RA) as a control of TNF neutralization. In a mouse model of collagen-induced arthritis, TNFKi protected against inflammation similar to etanercept. In a mouse model of acute *L. monocytogenes* infection, all TNFKi-treated mice showed cleared bacterial infection and survived, whereas etanercept-treated mice showed large liver granulomas and quickly died. Moreover, TNFKi mice infected with the virulent H37Rv *M. tuberculosis* showed resistance to infection, in contrast with etanercept-treated mice or controls. Depending on the TNF-α blockade strategy, treating arthritis with a TNF-α inhibitor could result in a different profile of infection suceptibility. Our TNFKi vaccine allowed for a better remaining host defense than did etanercept.

Keywords: tumor necrosis factor, vaccine, rheumatoid arthritis, infection, host-defense

# INTRODUCTION

Tumor necrosis factor-α (TNF-α) is a central mediator of inflammation, tumor growth control, autoimmunity, and immune response to infection (1). In rheumatoid arthritis (RA) and spondyloarthritis, two frequent and severe diseases (2), TNF-α mediates a wide variety of effector functions, including the production of pro-inflammatory cytokines and chemokines, activation of immune cells and angiogenesis.

Two types of TNF-α antagonists are used for treating rheumatic diseases, including RA and spondyloarthritides: the soluble TNF-α receptor 2 etanercept and monoclonal antibodies such as infliximab, adalimumab, golimumab, or pegylated-IgG-Fc fragments such as certolizumab. Anti-TNF-α drugs have greatly changed the treatment of RA in terms of clinical control and articular damage prevention. However, these treatments have limitations, such as high cost, frequent therapeutic failures (only 20% of RA patients achieve sustained remission) (3), and increased risk of serious infections, such as reactivation of pulmonary and extrapulmonary tuberculosis (4, 5).

Recently, we developed an alternative strategy of TNF neutralization by a vaccination approach. A human TNF-α vaccine (hTNF-K) was obtained by chemically coupling the human cytokine to the carrier protein keyhole limpet hemocyanin (KLH) (6). Preclinical studies showed that vaccination with hTNF-K reduced arthritis development in a human TNF-α transgenic mouse (TTG) model, with a dose effect depending on level of anti-TNF-α antibody production (7–9). Anti-TNF-α antibody production was reversible and could not be stimulated by TNFα administration, because this approach did not induce any cellular-mediated immunity against TNF-α (10). A first clinical trial was conducted with RA patients resistant to TNF antagonists and showed promising results (11).

In previous studies, we demonstrated that anti-human TNFα vaccination activates resting autoreactive B cells to produce anti-TNF-α antibodies. The carrier protein (KLH) possesses the T epitopes able to activate T cells, thereby providing the necessary help for anti-TNF-α antibody production. No T cell being directly sensitized to TNF-α has two major consequences. First the antibody production is self-limited, with serum antibody titers showing a bell-shaped curve, with peak concentration around day 50 in mice and between day 112 and 140 in humans. Second, any increase in self-produced TNF-α (e.g., during infection) does not elicit any anti-TNF-α antibody response in immunized subjects. However, the risk of infection with the hTNF-K treatment could not be easily studied in the TTG mice because antihuman TNF-α antibodies are usually species-specific and do not cross-react with murine TNF-α.

To evaluate the risk of infection using anti-TNF-α strategies, we compared a vaccine directed against murine TNF-α we developed by coupling murine TNF-α to KLH (TNFKi) and etanercept, the anti-TNF-α treatment associated with the lowest occurrence of infections, namely iatrogenic tuberculosis (12, 13). Our data reveal the protective effect of TNFKi in two mouse models of arthritis. Furthermore, we show that, in contrast to etanercept, TNFKi had limited effects on the host resistance to *Listeria monocytogenes* and *Mycobacterium tuberculosis* infection, as reflected by a differential local immune response to the pathogens. Hence, depending on the anti-TNF-α inhibition strategy, TNF-α inhibition could control chronic inflammation without modifying the pathogen-induced immune response and, therefore, have no deleterious effect.

#### MATERIALS AND METHODS

#### Vaccine Design

Murine TNF-α was from PeproTech (315-01A-1000UG, PeproTech France). KLH (77600, Thermo Scientific Pierce), GMBS (N-[g-maleimidobutyryloxy]), succinimide ester (22309 Pierce), and SATA [*N*-succinimidyl *S*-acetylthioacetate (26102 Pierce)] were from Thermo Scientific Pierce, France. TNFKi resulted from the chemical coupling of murine TNF-α to the carrier protein KLH, by using heterobifunctional cross-linkers that react with primary amines. TNFKi was prepared as follows: 4 mg murine TNF-α was derivatized with 1.2 mg SATA for 2 h at room temperature. In parallel, 20 mg KLH was derivatized with 1.76 mg GMBS for 30 min. Cross-linker quantities were calculated to modify half of the lysine residues. For coupling, 1 mol KLH-GMBS was incubated with 20 mol TNF-SATA for 2 h at room temperature in the presence of 5 mM hydroxylamide (23225 Thermo Scientific Pierce). The heterocomplex (TNF-KLH) was dialyzed overnight against phosphate-buffered saline (PBS) by use of a Microcon tube (YM100 Millipore). TNFKi was sterilized by centrifugation in an Ultrafree-CL 0.22-µm tube (FC40GV 0S Millipore), then stored at 4°C. Three batches were prepared successively for different experiments. They were validated biochemically by ELISA and western blot analysis. Batches were systematically tested *in vivo* in the collagen-induced arthritis (CIA) model.

#### Mice

For CIA, 36 DBA/1 male mice (6 weeks old) were used, and for collagen antibody-induced arthritis (CAIA), 24 C57Bl/6 male mice (6 weeks old) were used, all purchased from Janvier Laboratory (France). Mice were randomly assigned to treatment groups and randomly distributed to cages. Body weight was monitored weekly. *L. monocytogenes* (LM) infection experiments involved 40 C57Bl/6 female mice (8 weeks old, Janvier Laboratory) and 10 TNF-α−/− mice (TNF−/−) (14) backcrossed at least 8–10 times on a C57BL/6 genetic background, bred, and housed in the Transgenose Institute animal facility (CNRS UPS44, Orleans, France). *M. tuberculosis* (Mt) infection experiments involved 60 C57Bl/6 female mice (8 weeks old, Janvier Laboratory) and 7 TNF−/− mice. Mice were kept in isolators in a biohazard animal unit. Infected mice were monitored every day for clinical status and weighed twice weekly. Mice were randomly assigned to treatment groups and randomly distributed to cages (except for naïve uninfected mice).

#### Arthritis Models

DBA/1 mice used for CIA were injected with a 1:1 emulsion of bovine type II collagen (CIIb, 50 μg/mice) and complete Freund's adjuvant (Difco, France) on day 0 (total volume injected in the tail: 100 µl). A boost was given on day 21 with 100 µl of a 1:1 emulsion of CIIb (50 μg/mice) and incomplete Freund's adjuvant (IFA, Difco, France). Arthritis onset occurs at about 30–35 days after the first collagen injection (15).

C57Bl/6 mice used for CAIA were intraperitoneally injected with 5 mg of a cocktail of five monoclonal anti-collagen antibodies on day 0 (53100 Arthrogen-CIA Arthritogenic Monoclonal Antibody, Gentaur, Belgium) and lipopolysaccharide (50 µg, *E. coli* 0111:B4 strain) on day 3. Arthritis onset occurs at about 3–4 days after anticollagen antibody injection and peaks at about day 7–10 (16).

Clinical signs of arthritis were evaluated with blinding to treatment three times per week. Arthritis was monitored in all four paws. For each mouse, the clinical severity of arthritis was scored as 0, normal; 1, erythema; 2, swelling; 3, deformity; and 4, ankylosis in 10 joints or groups of joints: three joints of the two hind limbs (toes, tarsus, ankle) and two joints of the two forelimbs (digits, wrist). The maximum score for each of the 10 joints was 4, so the maximum individual arthritis clinical score on a given day was 40 (17). The mean arthritic score on each day of clinical observation was calculated for each treatment group.

For histology, both left hind limbs of each mouse were collected, fixed, decalcified, dehydrated, and transferred to paraffin blocks. Slides of 7 µm thickness were stained with hematoxylin and eosin before optical microscopy observation. At least 10 fields per section were evaluated with blinding to treatment. In each joint, two variables were separately assessed on a 4-point scale (0–3, 0 indicating a normal joint, and 3, maximally severe arthritis). The first variable was inflammation, as reflected by synovial membrane thickness (synovial proliferation) and inflammatory cell infiltration. Inflammation was scored as 0, no synovial proliferation and no inflammatory cell infiltration; 1, limited or absent synovial proliferation with inflammatory cells infiltrating ≤5% of synovial membrane; 2, synovial proliferation with inflammatory cells infiltrating between 5 and 50% of synovial membrane; and 3, massive synovial proliferation with inflammatory cells infiltrating >50% of synovial membrane. The second variable was joint destruction (bone erosions, cartilage thickness, and cartilage unevenness). Joint destruction was quantified as 0, no bone erosion, smooth cartilage surface with conserved thickness; 1, presence of cartilage erosion or unevenness or thinning involving ≤50% of cartilage surface with absent or single bone erosion; 2, multiple cartilage erosions or cartilage thinning involving >50% of cartilage surface and/or >1 bone erosions involving ≤50% of articular surface; and 3, complete derangement of articular structure. For determining prevalence, histological inflammation or destruction was defined as inflammation or destruction score ≥0.5 (17).

For clinical and histological evaluation of arthritis, blinding was ensured by a random distribution of treatments inside cages, limited by the need to have each treatment group represented, and by the use of tables including only cages and mouse numbers.

#### Anti-TNF Immunization in CIA Model

Immunization involved the anti-mouse TNF-α vaccine (TNFKi) emulsified in IFA. DBA/1 mice (*n* = 6) were vaccinated intramuscularly with different doses of TNFKi (20, 10, or 5 µg) on days −21, −7, and 7. In the same experiment, one group of mice (*n* = 6) were intraperitoneally injected three times/week with 30 mg/kg etanercept (PAA0252P1, Pfizer) from day 22. Negative controls were mice receiving intraperitoneal injections of PBS or intramuscular injections of KLH (10 µg) emulsified in IFA. KLH and PBS were administered on the same time schedule as TNFKi or etanercept treatment, respectively.

#### Infection Protocols

*Listeria monocytogenes* (L028) was grown in BHI medium (Brain Heart Infusion, Difco Laboratories). Aliquots of *M. tuberculosis* H37Rv kept frozen at −80°C were thawed, diluted in sterile saline containing 0.05% Tween 20, and clumping was disrupted by 30 repeated aspirations through a 26-G needle (Omnican, Braun, Germany). Pulmonary infection involved delivering 2250 colony formation units (CFU) of *M. tuberculosis* H37Rv/lung into nasal cavities of mice under xylazine–ketamine anesthesia, as verified by determining bacterial load in the lungs on day 1 postinfection.

Mice were vaccinated before infection with four TNFKi intramuscular injections (10 µg) on days −44, −31, −17, and −4. One group of mice (*n* = 10) received intraperitoneal injections of etanercept (30 mg/kg) twice a week from days −4 to day 52 before and during infection. Controls were untreated (naïve) mice, vaccinated with KLH, or received intraperitoneal injections of PBS. All groups comprised 10 mice per group except for TNF−/− mice (*n* = 7). On day 25 postinfection for all TNF−/− mice or on day 28 for other groups, 5 animals were killed; lung, livers, and spleens were harvested; and the number of viable bacteria in organ homogenates was determined by plating serial dilutions in duplicate onto Middlebrook 7H11 (Difco) agar plates containing 10% OADC and incubating at 37°C. Colonies were counted at 3 weeks and results are expressed as log10 CFU per organ. Similar analyses were performed on day 56 (established infection) with the remaining mice.

A similar vaccination schedule was used before *L. monocytogenes* infection, and one group of mice (*n* = 9) received intraperitoneal injection of etanercept (30 mg/kg) on days −4, −2, 0, and 3 before and after infection. Controls were treated with KLH (*n* = 9 mice) or PBS (*n* = 10) on the same time schedule as TNFKi vaccination or etanercept treatment, respectively. All groups and TNF−/− mice (*n* = 10) underwent intraperitoneal injection on day 0 with 104 CFU *L. monocytogenes*. On day 4 postinfection, four mice/group were killed, then livers and spleens were harvested and the number of viable bacteria in organ homogenates was determined by plating serial dilutions on trypticase soy broth agar plates (Biovalley) and incubating overnight at 37°C, followed by counting CFU. Survival during infection was evaluated until day 11 (*n* = 5–6 mice/group of treatment).

### ELISA for Anti-KLH and Anti-TNF Antibodies in Serum

Blood samples were collected at different times in accordance with the duration of experiments: 19 and 41 days after the third TNFKi vaccination in the CIA study; 7 days after the third and fourth TNFKi vaccination in the *Listeria* study; 7 days after the third and 32 days after the fourth TNFKi vaccination in the *Mycobacterium* study. Serum was obtained and tested for anti-KLH and anti-TNF-α antibody content. Specific anti-mTNF-α and anti-KLH antibody content was determined by direct ELISA. Precoated ELISA plates with 50 ng per well mTNF-α (PeproTech) or KLH (Sigma) were incubated with serial dilutions of serum from immunized and control mice. Specific antibodies were detected by using phosphatase alkaline-conjugated rabbit antimouse IgG (IgG-PA; Sigma-Aldrich A1902). Substrate PNP (Sigma-Aldrich) was added for 40 min. The optical density was measured at 405 nm.

#### Tissue Preparation for Histology and Immunohistochemistry

Lungs and livers from *M. tuberculosis*-infected mice collected on days 25–28 and 56 were fixed in 4% buffered formalin and paraffin-embedded, and 4 µm sections were stained with hematoxylin and eosin (H&E) or underwent the Ziehl–Neelsen coloration. Sections were observed by light microscopy at 400× magnification with a Zeiss Observer D1 microscope equipped with a motorized stage connected to Histolab and Archimed software (Microvision, Les Ulis, France). The number and size of granulomas were quantified by using Archimed.

For immunohistochemistry, lung and liver sections were fixed in acetone–ethanol (5 min), and endogenous peroxidase activity was blocked with PBS and 3% H2O2. Endogenous biotin in the liver was blocked by using an avidin-biotin kit (Vector Laboratories, SP-2001). Tissue sections were incubated for 2 h at room temperature or overnight at 4°C with the primary antibody CD45R (AbD Serotec, MCA1258G) or inducible nitric oxide synthase (iNOS; Abcam, 15323). Sections were then incubated for 30 min at 37°C with the appropriate biotinylated secondary antibody. Avidine biotin peroxidase complexes were added to the sections for 30 min (ABC Vector kit; Vector Laboratories, PK-6100), then incubated with diaminobenzidine substrate (Imm PACT DAB, Vector: SK-4105, Biovalley). After a rinsing in water, sections were counterstained with hematoxylin before mounting and examined by use of a Zeiss Observer D1 microscope as described previously.

For *Listeria* infection, four mice per group were euthanized on day 4 postinfection. Livers were treated as described previously; H&E-stained sections were observed by light microscopy and images were obtained by using a nanozoomer. The number and size of microabcesses were assessed by using NDPview (Hamamatsu, Japan). Immunohistology of livers, macrophages (F4/80, AbD Serotec, MCA497GA), and neutrophils (Ly6G, Abcam, 25377) infiltration was performed as described for CD45R and iNOS staining.

#### Statistics

For arthritis studies, serial measurements of clinical variables were analyzed by the area under the receiver operating characteristic curve for each mouse as a summary measure, then as raw data for inter-group comparisons (ANOVA). Differences in arthritis onset were analyzed by ANOVA. Posttest data were compared by data distribution (Student–Newman–Keuls). For *Listeria* infection studies, evaluation of survival involved the Kaplan–Meier method (log-rank test), and bacterial burden data were analyzed by one-way ANOVA with Newman–Keuls posttest. *p* < 0.05 was considered statistically significant.

#### Study Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. For arthritis models, all procedures were approved by the Animal Care and Use Committee of the University of Paris 13 (ethical approval ID: Ce5/2010/036). Infectious protocols were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans (CCO) (no. CLE CCO 2015-1071). We were particularly concerned by a strict application of the 3R rules (18).

#### RESULTS

### TNFKi Reduced Clinical and Histological Signs of Arthritis

To investigate the anti-inflammatory effect of TNFKi *in vivo*, we used the CIA model with DBA/1 mice. Mice were vaccinated three times with 5, 10, or 20 μg/mice TNFKi (TNFKi-5, TNFKi-10, and TNFKi-20 groups). Control groups were vaccinated with KLH or PBS. Etanercept was a positive control. The first signs of arthritis appeared in all control groups at 33 ± 2 days, as expected, but in TNFKi groups at 38 ± 2 days and in the etanercept group at 41 ± 3 days. All anti-TNF treatments resulted in significantly lower clinical arthritis scores as compared with controls (**Figure 1A**).

Maximal clinical arthritis index (Amax) was significantly lower for mice with 10 µg TNFKi or etanercept treatment than other mice (Table S1 in Supplementary Material). Despite this, the incidence and onset of arthritis were not significantly delayed with TNFKi or etanercept treatment as compared with KLH and PBS controls (Table S1 in Supplementary Material). The weights of TNFKi-vaccinated mice were comparable to that of PBS and KLH controls during the experiment (data not shown).

Histology of paws of PBS and KLH control mice showed significant inflammation, with cell infiltration associated with cartilage destruction (**Figures 1B–E**). By contrast, TNFKi-10 or etanercept treatment significantly reduced the inflammatory signs of arthritis (**Figure 1F**). TNFKi-10 or etanercept treatment protected against joint destruction (**Figure 1G**). In addition, histology showed protection of joints with TNFKi vaccination in the CAIA arthritis model in C57Bl/6 mice (Figure S1 in Supplementary Material).

Thus, TNFKi was effective in protecting against TNF-mediated arthritis in mice.

#### TNFKi Did Not Impair Defense to *L. monocytogenes* as Compared with Etanercept

Mice were vaccinated on days −44, −31, −17, and −4 with TNFKi or intraperitoneally injected with etanercept on days −4, −2, 0, and 3 before (or after) infection with *L. monocytogenes*. Controls received PBS or KLH, and TNF−/− mice were a genetic control. All mice were injected intraperitoneally with *L. monocytogenes* (104 CFU) on day 0. Blood was collected before (day −10) and during infection (day 3) to evaluate anti-TNF-α antibody levels. ELISA revealed the production of anti-TNF-α and anti-KLH antibodies in serum from mice with TNFKi vaccination, with an increase in level in responder mice between day −10 and day 3 (Figure S2 in Supplementary Material).

Survival of mice was monitored for 11 days postinfection. All TNF−/− mice died within 5 days, and 80% of mice with etanercept treatment died by day 6, as expected; by contrast, mice vaccinated with TNFKi did not die due to infection during this period (**Figure 2A**). Hence, unlike etanercept, TNF-α neutralization with TNFKi protected against death due to *L. monocytogenes* infection, with a clear survival benefit. The increase in bacterial load in the liver and spleen homogenates seen on day 4 in

differents doses of TNFKi (20, 10, 5 µg) at days −21, −7, 7 before and during collagen-induced arthritis (CIA) induced by two injections of CIIb (50 μg/mice; day 0 and 21). Treatment with etanercept (30 mg/kg, two times/week) or phosphate-buffered saline (PBS) began from day 22. (A) Clinical arthritis was measured by using the sum of arthritis scores (0–4) from four paws (*n* = 6/group). Data are mean ± SEM. \**p* < 0.05 vs. both PBS and KLH during the whole experiment by ANOVA. The area under the curve (AUC) of clinical scores for each animal are the raw value. (B–E) Histology of paws for treatment groups. (B) TNFKi-20, (C) TNFKi-10, (D) TNFKi-5 (20, 10, 5 µg, respectively), (E) KLH shows inflammatory synovitis (blue arrow) and articular surface (black arrow). (F) Histological inflammation score and (G) joint destruction in treatment groups. Horizontal bar is mean, outer bars are SEM, and whiskers are range. \**p* < 0.05 for TNFKi and \*\**p* < 0.01 for etanercept vs PBS (Newman–Keuls test with subsequent *post hoc* comparisons). CIA inhibition with TNFKi was observed in three independent experiments.

TNF−/− mice and those with etanercept treatment was not present in either organ of TNFKi-vaccinated mice or in KLH or PBS controls (**Figures 2B,C**).

We further investigated histological changes in mouse livers on day 4 postinfection (**Figures 2D–H**). Etanercept-treated and TNF−/− mice showed typical and abundant hepatic microabscesses (**Figures 2E,F**) characterized by diffuse infiltration and necrotic areas, whereas liver microabscesses in mice vaccinated with TNFKi appeared smaller and similar to those from KLH and PBS control mice (**Figures 2D,G,H**). Hepatic microabscesses were significantly larger for TNF−/− mice than other groups, except with etanercept treatment (**Figure 2J**). In addition, the number of microabcesses was significantly larger in TNF−/− mice (**Figure 2I**), with no difference among TNFKi, KLH, and PBS groups.

Immunohistochemistry of liver sections to investigate the cellular composition of microabcesses after 4 days of infection revealed an increased accumulation of neutrophils (Ly6G) in TNF−/− and etanercept-treated mice but not TNFKi, KLH, and PBS mice (**Figure 3A**). Macrophage staining (F4/80) appeared more dispersed and less localized inside microabcesses in etanercept-treated and TNF−/− mice as compared with TNFKi, KLH, and PBS treatment (**Figure 3B**).

#### TNFKi Did Not Affect the Defense against *M. tuberculosis* Infection

We assessed the consequences of TNFKi vaccination during early and established *M. infection*. Mice were vaccinated on days −44, −31, −17, and −4 with TNFKi (10 μg/mice) or treated with etanercept (30 mg/kg twice a week from day −4 to day 52 before or after infection). Control groups were mice with KLH vaccination or PBS treatment. TNF−/− mice, which are very sensitive to virulent *M. tuberculosis* H37Rv infection, were used as a positive control of total infection in the absence of functional TNF. Except for five naïve mice, all mice were infected on day 0 by intranasal instillation of *M. tuberculosis* H37Rv (2,250 CFU). TNF−/− mice lost weight rapidly and had to be euthanized on day

5 days postinfection. Four etanercept-treated mice died on day 6 (*n* = 5) and two mice treated with KLH (*n* = 5). All mice treated with TNFKi (*n* = 6) and PBS (*n* = 6) survived during this period. Survival of TNF−/− and etanercept groups was significantly reduced as compared with PBS, TNFKi, and KLH mice (*Logrank test for trend*, *p* < 0.0001). Colony-forming units (CFUs) evaluated in liver (B) and spleen (C) 4 days after infection in mice (\**p* < 0.05; \*\**p* < 0.01, \*\*\**p* < 0.001, ANOVA). Liver lesions were studied 4 days after infection with *Listeria* after staining sections with hematoxylin-eosin. Liver sections (D–H) showing size and number of lesions (black arrow), with necrotic areas (red arrow) and *Listeria* accumulation (green arrow). Quantification of hepatic lesion number (I) and surface area (j) in slides involved use of NDPview software (Hamamatsu, Japan) (\**p* < 0.05, \*\**p* < 0.01, ANOVA).

25 postinfection, but the other mice did not experience body weight loss (data not shown) and were euthanized during early infection (day 28) or established infection (day 56).

Blood was collected before (day −10) and during infection (day 28) to evaluate levels of anti-TNF-α antibodies. As expected, ELISA revealed a sustained serum production of anti-TNF-α antibodies in mice vaccinated with TNFKi (Figures S3A,B in Supplementary Material).

At euthanasia, weights of lungs, spleens, and livers were evaluated as indicators of inflammation. During early infection, all TNF−/− mouse organs showed a marked increase in weight as compared with the other groups, with the exception of lung and spleen of etanercept-treated mice (**Figures 4A–C**). At day 56, established infection, organ weights were similar between all infected mice (**Figures 4D–F**).

Liver and lung bacterial loads were then assessed in vaccinated and TNF−/− and etanercept-treated mice. During early infection, total CFU were greatly increased in organs of TNF−/− mice as compared with all other groups (§ *p* < 0.001 TNF−/− vs all groups in lung and liver, ANOVA) (**Figures 5A,B**). At the same time, CFU per organ were similar between TNFKi, etanercept, and control groups (KLH and PBS). At day 56, established infection, total CFU were similar in lungs of all infected mice (**Figure 5C**).

To determine the effect of TNFKi on organ integrity during early and established infection, we investigated histological changes in mouse lung and liver. Indeed, during early infection, TNF−/− mice showed inability to control *M. tuberculosis* infection, with rapid, exacerbated lung inflammation, and many granulomas with large necrotic areas (**Figure 6D**). In the lungs of TNFKi, KLH, and PBS groups, granulomas were more discrete, with the typical

Figure 4 | Organ weights during early and established *M. tuberculosis* infection. C57Bl/6 mice were vaccinated with keyhole limpet hemocyanin (KLH) (*n* = 10) or 10 µg TNFKi (*n* = 10) at days −44, −31, −17, and −4 before infection. One group of mice received 30 mg/kg of etanercept (*n* = 10) twice a week from day 4 to day 52. Except for naïve mice (*n* = 5), all treated mice and TNF−/− mice (*n* = 7) were infected intranasally with 2,250 CFU of *M. tuberculosis* on day 0. Five mice per treatment (day 28) and all TNF−/− mice (day 25) were euthanized and lung, spleen, and liver were weighed. Survival was monitored until day 56 (4–5 mice/group) and organs were weighed at euthanasia. Relative organ weights were calculated as the ratio of absolute organ weights to body weights. Organ weights of mice during early infection (A–C) and established infection (D–F). Organ weights were expressed as % of body weight. Horizontal bar is mean, outer bars are SEM, and whiskers are range. \**p* < 0.05 vs TNFKi, KLH, phosphate-buffered saline (PBS), \*\*\**p* < 0.001 vs naïve mice; Newman–Keuls test with *post hoc* comparisons. &*p* < 0.05 naïve vs TNFKi, KLH, PBS, and etanercept.

granuloma structure at day 56 (**Figures 6A–H**). Etanercepttreated mice showed granulomas on day 28, but these were more developed and less structured by day 56 (**Figures 6C,G**). With Archimed software quantification, during early infection, the lung granuloma surface area was increased but not significantly in TNF−/− and etanercept-treated mice (**Figure 6I**). At day 56, the granuloma surface area of TNFKi- and KLH-vaccinated mice appeared decreased, contrary to etanercept treatment (\**p* < 0.05 vs etanercept group, ANOVA) (**Figure 6J**). Granuloma numbers were similar between mouse groups at both phases (data not shown). During both early and established infection, all infected mice showed less free alveolar space as compared with naïve mice (**Figures 6K,L**), which was most prominent with etanercept treatment on day 56 (**Figure 6L**).

Horizontal bar is mean, outer bars are SEM, and whiskers are range (§

Histology of the liver at the early phase of infection (**Figures 7A–F**) revealed that granuloma surface area was significantly larger for TNF−/− and etanercept-treated mice than other mice (**Figure 7G**) (\**p* < 0.05 vs TNFKi).

TNF−/− and etanercept-treated groups showed a higher number of granulomas, although not significantly (data not shown). At day 56, liver granuloma surface area was larger for etanercepttreated mice than other groups (**Figure 7H**). Hence, histology of both lungs and livers suggested that mice vaccinated with TNFKi, similar to KLH or PBS, effectively controlled the infection during the progression of the disease as compared with etanercept.

To confirm mycobacterial infiltration in granulomas, we used Ziehl–Neelsen coloration and semiquantitative evaluation of bacterial staining in mouse lungs (**Figure 8**). Microscopy revealed TNF−/− and etanercept-treated mice with greater mycobacteria infiltration than other groups during early and established infection (**Figures 8B,C**, \**p* < 0.05 for etanercept and \*\*\**p* < 0.001 for TNF−/− mice vs TNFKi, KLH, PBS, ANOVA).

*p* < 0.001 TNF−/− vs all groups; Newman–Keuls test with subsequent *post hoc* comparisons).

We used immunohistochemistry of mouse lung sections to investigate the relationship between cellular infiltration or activation status and TNF-α neutralization. During early infection, iNOS was expressed in granulomas of all mouse groups (Figure S4A in Supplementary Material). Histology revealed lesions in TNFKi, KLH, and PBS lungs characterized by macrophage infiltration and a very low number of neutrophils. Conversely, in TNF−/− and etanercept-treated lungs, iNOS expression in macrophages co-localized with microabscesses containing altered neutrophil number. Etanercept-treated lungs showed more macrophage infiltration, higher number of foamy histiocytes expressing iNOS, and fewer neutrophils during established than early infection (Figures S4A,B and S5 in Supplementary Material). Lesions of PBS-treated lungs showed few neutrophils, and iNOS was expressed in macrophages and foamy histiocytes. Lesions of mouse lungs with KLH or TNFKi vaccination showed similar cellular infiltration as for PBS-treated lungs but a lower number of foamy histiocytes. Thus, macrophages within the granuloma

of TNFKi-vaccinated lungs retained the bactericidal function controlling infection.

We explored the effect of the TNFKi on lung infiltration by B lymphocytes during *M. tuberculosis* infection (**Figure 9**). During early infection, anti-CD45R staining revealed a massive infiltration of B lymphocytes in lungs of TNFKi- and KLH-vaccinated mice, lower infiltration with PBS, and a near absence of B lymphocytes in TNF−/− or etanercept-treated mice. Immunostaining revealed a higher perivascular accumulation of B cells with TNFKi or KLH vaccination. During late infection, with TNFKi or KLH vaccination, pulmonary granulomas were well organized, characterized by many pulmonary nodules and accumulation of B lymphocytes. The phenotype for PBS lungs was similar to that for vaccinated lungs but with fewer B lymphocytes. By contrast, etanercept treatment resulted in massive macrophage infiltration with a large necrotic area, without typical granulomas and few B lymphocytes (**Figure 9**).

Thus, TNFKi vaccination did not impair the host control of *M. tuberculosis* infection in terms of lung and liver bacterial burden, granuloma formation or inflammatory response as compared with TNF neutralization with etanercept.

#### DISCUSSION

Here, by using a surrogate of human TNF-α vaccine, mouse TNFKi, we validated the anti-inflammatory effect of the vaccine in an experimental model of RA while documenting the little perturbed control of infection in TNFKi-treated mice in two models of infection, with *L monocytogenes* and *M. tuberculosis*.

A major concern of anti-TNF-α treatments is an increased risk of serious bacterial, viral, and fungal infections. Tuberculosis is the most common opportunistic infection and, to a lesser extent, bacterial infections, such as listeriosis (19–21). Anti-TNF-α vaccination has been widely explored in mice (22–24). Clinical trials in humans were promising in the early phases of development of TNF-α vaccination with a TNF-Kinoid (11), although not considered effective at 6 months in a larger study with a single low dose of vaccine that did not result in significant production of anti-TNF-α auto-antibodies (Neovacs.fr/products/tnf-kinoid/, NCT01911234). The TNFKi vaccine used in the present study consisted of mouse TNF-α linked to the KLH carrier protein. TNFKi induced the production of anti-TNF-α polyclonal antibodies and alleviated arthritis in two experimental

models of arthritis, CIA and CAIA, which confirms the effective neutralization of TNF-α.

infection (H). Horizontal bar is mean, outer bars are SEM, and whiskers are range (\**p* < 0.05, \*\**p* < 0.01, ANOVA).

To assess to what extent this novel anti-TNF strategy would affect immunity against infectious agents, we used two experimental models of infection, with *L monocytogenes* and *M. tuberculosis*. In both cases, we compared the effect of TNFKi to that of complete lack of TNF-α (TNF−/− mice) and a current anti-TNF-α treatment (etanercept). Etanercept is considered the safest anti-TNF inhibitor in terms of the possible risk of serious infection (notably tuberculosis reactivation). It is a pharmacological reference for anti-TNF-α blockade in animal models of infection (25). With *L. monocytogenes* infection, anti-TNF-α polyclonal antibody production induced by TNFKi did not impair immunity to the bacterium, and all mice survived. However, TNF-α neutralization with etanercept enhanced the susceptibility to infection and TNF−/− mice did not show resistance to infection. TNFKivaccinated mice showed the same bacterial clearance in liver and spleen as immunocompetent mice (PBS and KLH controls), whereas etanercept-treated mice showed increased bacterial load. In agreement, the size and number of liver microabcesses were similar between TNFKi and control mice at day 4 postinfection. In contrast, TNF−/− mice showed increased number of lesions and large lesions characterized by necrotic areas. Immunization with TNFKi did not alter immune cell migration into the liver during *Listeria* infection, whereas etanercept-treated and TNF−/− mice exhibited neutrophil accumulation and reduced macrophage recruitment in lesions.

With the model of *M. tuberculosis* infection, TNF neutralization by TNFKi vaccination did not increase the sensitivity to the pathogen, but TNF−/− mice died during the early phase of infection. Previously, mice treated with a murine homolog of etanercept died due to *M. tuberculosis* infection (26). We did not observe such a drastic effect on survival with etanercept treatment; however, Zielh–Neelssen staining revealed higher bacteria loads in lungs from TNF−/− and etanercept-treated mice than TNFKi-vaccinated mice.

We reported previously and confirm here the striking lung inflammation in TNF−/− mice, including large necrotic

mycobacteria during the early (B) and established infection (C) after blinded observations of three fields per mice (*n* = 3–7 mice per group) (\**p* < 0.05 ANOVA).

granulomas (27), which were not observed in the other groups in our study. Granuloma formation and organization in lungs was not altered with established *M. tuberculosis* infection and TNFKi vaccination as compared with etanercept treatment. In addition, granulomas observed in TNFKi-vaccinated livers were similar to those in control livers. In both TNF−/− and etanercept-treated mice, liver granulomas were significantly larger.

The protective role of iNOS in infection led us to study its expression during the anti-*M. tuberculosis* response. Indeed, mice deficient in iNOS die due to tuberculosis infection within 33–45 days, and iNOS inhibition aggravates the course of murine tuberculosis (28, 29). We found that iNOS was expressed exclusively by macrophages within granulomas during the early phase of infection and by macrophages and foamy histiocytes during established infection. Strikingly, recruitment of macrophages and neutrophils seemed altered by etanercept treatment, because numerous neutrophils and few macrophages infiltrated lungs in the acute phase as compared with numerous macrophages and foamy histiocytes infiltrating lungs in the established phase. With TNFKi treatment, neutrophilic infiltration was rare, and few foamy histiocytes were observed during established infection. The adaptive immune response to *M. tuberculosis* is also mediated by B lymphocytes. Despite low risk of tuberculosis reactivation for RA patients receiving rituximab to deplete B cells (30), a recent study showed that in *M. tuberculosis*-infected macaques, rituximab altered local granulomatous response during acute infection (31). Furthermore, another study showed that TNF-α secreted by B lymphocytes is important for aggregation (32). In our study, contrary to etanercept, TNFKi vaccination did not impair B-cell recruitment. At day 56 postinfection, B cells formed nodules in lungs of all mouse groups, except with etanercept treatment. TNFKi allowed for B-cell recruitment in infected tissues and effective granuloma formation and maintenance during both early and established infection.

Why TNF-α neutralization with anti-TNF vaccination preserved the immune response against infection remains speculative. The risk of infection depends on the nature of the TNF-α blockers. Several studies have shown increased risk of reactivation

Scale bar, 10 µm.

of latent tuberculosis with infliximab and adalimumab monoclonal antibody therapy than with the recombinant soluble TNF-α receptor etanercept (33, 34). The safety profile with both types of treatments may differ in part due to their TNF-α binding property. Infliximab forms stable complexes with soluble TNF-α and TNF-α expressed at cell membranes, whereas etanercept interacts with soluble TNF-α and only weakly and reversibly with the transmembrane form (35).

3–7 mice per group. Scale bar, 100 µm. 4× magnification of red boxes below original picture (scale bar, 25 µm).

Although the importance of soluble versus membrane TNF-α has not been investigated in detail in the CIA model, the role of both forms of TNF-α were well investigated in infectious models. Indeed, mice lacking the soluble form of TNF-α but expressing a transmembrane form or expressing uncleavable membrane TNF showed control of *M. tuberculosis* infection, at least during the acute phase (36–38) or during *L. monocytogenes* infection (39). These latter mice could still form compact granulomas, which prevented bacterial spreading, whereas mice completely lacking TNF-α were severely impaired in granuloma formation. Expression of a functional transmembrane form of TNF-α also confers partial protection against *L. monocytogenes* infection (40), but TNF-α- or TNF-R1-deficient mice are highly susceptible (41).

Low levels of TNF present at the site of infection might be sufficient to control mycobacteria infection. Indeed, in TNF-deficient mice, which usually rapidly die after *Mycobacterium bovis* bacillus Calmette–Guérin (BCG) infection, the antimycobacterial immune response was restored by infection with a TNF-αsecreting recombinant *M. bovis* BCG, effectively protecting these highly susceptible mice (42).

There are some limitations to this work that warrant further studies. The exact mechanisms by which the two different anti-TNF strategies affect the immune response to infectious agents need to be further dissected. Another issue is the extrapolation of the results obtained in the animal model to humans, which is not the aim of the present study. Although we used validated models of arthritis (CIA) and infection, these models only partially mimic human disease. Moreover, differences between the human and murine immune system both in response to vaccination and infection may lead to deep discrepancies that warrant caution. No safety issues or increased risk of infection were reported when anti-TNF vaccination was administered in humans (Crohn's disease: NCT00808262; RA: NCT01040715). Nevertheless, the samples of those trials were limited and no specific studies on response to infection were performed.

In summary, arthritis improvement when targeting TNF-α does not result in a constant and uniform alteration of the host defense against infection. This alteration is variable depending on the strategies used for targeting TNF-α. TNFKi may allow for maintenance of the host defense against *M. tuberculosis* and *L. monocytogenes* infection in mice. This is a major point for the safety features of therapeutic TNF vaccination in chronic inflammatory diseases. Pathogenic levels of TNF-α might be neutralized efficiently while maintaining the minimal levels needed to control host resistance to bacterial infection.

#### ETHICS STATEMENT

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. For arthritis models, all procedures were approved by the Animal Care and Use Committee of the University of Paris 13 (ethical approval ID: Ce5/2010/036). Infectious protocols were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans (CCO) (no. CLE CCO 2015-1071). We were particularly concerned by a strict application of the 3R rules.

### AUTHOR CONTRIBUTIONS

NB performed mice immunization, evaluated clinical arthritis, performed immunoassay, *Listeria* infection, and immuno-histological analysis and helped draft the manuscript. LS performed statistical analysis, drafted and revised the manuscript. NS performed *Mycobacteria* infection, bacterial burden, and organ weight evaluations. DD participated in the immuno-histological analysis, analysis of the data, and helped to draft the manuscript. PD helped coordinate the study and helped draft the manuscript. BR and VQ established and validated the experimental infectious

#### REFERENCES

models and conceived a part of the *Listeria* and *Mycobacteria* infection study, participated in coordination and analysis of the data, and helped draft the manuscript. M-CB conceived of the study, analyzed the data, and helped to draft the manuscript. EA conceived of the study, performed coupling of TNF vaccine, participated in coordination of the study, analyzed the data, and drafted the manuscript.

#### ACKNOWLEDGMENTS

We thank Sonia Varela (animal facilities, Paris 13 University), Béatrice Marmey, Delphine Lemeiter, and Roxane Hervé for technical contributions. We also thank Matthieu Ribon, Julie Mussard, and Marjorie Bénéteau de la Prairie for their help in the project.

#### FUNDING

The pathophysiology, target, and therapies of RA laboratory received unrestricted grants from University of Paris 13 (BQR), Agence Nationale de la Recherche (ANR CYTOVAC project), INSERM (ProA), Direction Générale de l'Armement, Société Française de Rhumatologie, and Pfizer (Passerelle Grant).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at http://www.frontiersin.org/article/10.3389/fimmu.2017.01533/ full#supplementary-material.

mice immunized with TNF-α kinoid. *Clin Vaccine Immunol* (2012) 19(5):699– 703. doi:10.1128/CVI.05649-11

arthritogenic 5-clone cocktail of monoclonal anti-type II collagen antibodies. *J Immunol Methods* (2009) 343(1):49–55. doi:10.1016/j.jim.2009

cynomolgus macaques. *Infect Immun* (2016) 84(5):1301–11. doi:10.1128/ IAI.00083-16

**Conflict of Interest Statement:** BR, DD, EA, LS, M-CB, NB, NS, PD, and VQ declare no conflict of interest.

*Copyright © 2017 Belmellat, Semerano, Segueni, Damotte, Decker, Ryffel, Quesniaux, Boissier and Assier. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Regulation of neuroinflammation: what Role for the Tumor necrosis Factor-Like weak inducer of Apoptosis/Fn14 Pathway?

*Audrey Boulamery1,2 and Sophie Desplat-Jégo1,3\**

*1Aix-Marseille University, CNRS, NICN, Marseille, France, 2AP-HM, Hôpital Sainte-Marguerite, Centre Antipoison et de Toxicovigilance, Marseille, France, 3Service d'Immunologie, Hôpital de la Conception, Marseille, France*

Observed in many central nervous system diseases, neuroinflammation (NI) proceeds from peripheral immune cell infiltration into the parenchyma, from cytokine secretion and from oxidative stress. Astrocytes and microglia also get activated and proliferate. NI manifestations and consequences depend on its context and on the acute or chronic aspect of the disease. The tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/Fn14 pathway has been involved in chronic human inflammatory pathologies such as neurodegenerative, autoimmune, or malignant diseases. New data now describe its regulatory effects in tissues or fluids from patients with neurological diseases. In this mini-review, we aim to highlight the role of TWEAK/Fn14 in modulating NI in multiple sclerosis, neuropsychiatric systemic lupus erythematosus, stroke, or glioma. TWEAK/Fn14 can modulate NI by activating canonical and non-canonical nuclear factor-κB pathways but also by stimulating mitogen-activated protein kinase signaling. These downstream activations are associated with (i) inflammatory cytokine, chemokine and adhesion molecule expression or release, involved in NI propagation, (ii) matrix-metalloproteinase 9 secretion, implicated in blood–brain barrier disruption and tissue remodeling, (iii) astrogliosis and microgliosis, and (iv) migration of tumor cells in glioma. In addition, we report several animal and human studies pointing to TWEAK as an attractive therapeutic target.

Keywords: tumor necrosis factor-like weak inducer of apoptosis, Fn14, central nervous system, neuroinflammation, multiple sclerosis, neuropsychiatric systemic lupus erythematosus, stroke, glioma

# INTRODUCTION

Initially described as an accumulation of leukocytes in multiple sclerosis (MS) brain, neuroinflammation (NI) now also applies to other central nervous system (CNS) diseases (1). Among others factors, it results from peripheral immune cell infiltration through the brain barriers, from cytokine secretion and from oxidative stress (2). NI is based upon and is regulated by bidirectional communication pathways involving especially cytokines that connect the CNS and immune system (3). However, using the generic term "NI" for such a multifaceted process could be inaccurate (1). NI features depend on disease-specific conditions and differ in CNS infection, ischemia, and malignant or autoimmune diseases. Whether NI is acute or chronic is a major point to consider. In fact, transient NI is usually beneficial and must be preserved, while chronic NI is preferentially associated with diseases resulting in neurodegeneration (4, 5).

#### *Edited by:*

*Jixin Zhong, Case Western Reserve University, United States*

#### *Reviewed by:*

*Yumin Xia, Second Affiliated Hospital of Xi'an Jiaotong University, China Lu Huang, University of Texas MD Anderson Cancer Center, United States*

*\*Correspondence:*

*Sophie Desplat-Jégo<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 21 August 2017 Accepted: 27 October 2017 Published: 16 November 2017*

#### *Citation:*

*Boulamery A and Desplat-Jégo S (2017) Regulation of Neuroinflammation: What Role for the Tumor Necrosis Factor-Like Weak Inducer of Apoptosis/Fn14 Pathway? Front. Immunol. 8:1534. doi: 10.3389/fimmu.2017.01534*

Neuroinflammation highly involves cellular components of the neurovascular unit (6, 7), which consists in microvascular endothelial cells surrounded by basal lamina, astrocytic end-feet, pericytes, and neurons. It underlies the concept of a blood–brain barrier (BBB) actively separating the parenchyma from the circulation. Resident CNS cells such as astrocytes and microglia are also involved in regulating NI and respond to CNS insults by a proliferation and an abnormal activation, respectively called astrogliosis and microgliosis. This response is not always a harmful process, it can also be a beneficial and crucial process involved in CNS repair (8–10). During NI, immune cells, microglia, and astrocytes release soluble proteins, called cytokines, which mediate cell–cell communication (11). Among them, the tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) plays a dual role in the physiological versus inflammatory pathological responses of tissues, including the CNS (12–15).

Tumor necrosis factor-like weak inducer of apoptosis is a member of TNF superfamily initially shown to induce apoptosis of malignant cells (16). It is synthesized as a transmembrane protein form (mTWEAK) and proteolytically processed as a soluble cytokine (sTWEAK). Monocytes/macrophages are the main source of sTWEAK in inflammatory tissues. Until now, mTWEAK has only been described on freshly isolated monocytes (17, 18). Unlike TNF-α, TWEAK curtails the innate immune response and attenuates the transition to adaptive Th1 immunity (19). Moreover, TWEAK inhibits TNF receptor-1 signaling that promotes inflammation (20). TWEAK signaling mainly requires binding to fibroblast growth factor inducible 14 (Fn14), a member of the TNF receptor superfamily. Although Fn14 is poorly expressed in healthy endothelial cells, neurons, astrocytes, microglia, and progenitor cells, it is highly inducible in these cells. TWEAK interaction with its Fn14 receptor induces multiple molecular events and biological responses depending on cell type and microenvironment. These downstream signaling pathways have been compiled from the literature in 2012 by Bhattacharjee et al. (21), who cataloged 46 proteins and 28 induced genes. Thus, Fn14 engagement primarily activates nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) *via* interaction with intracellular TNF-receptor-associated-factors (21, 22). mTWEAK seems to activate more efficiently the canonical NF-κB pathway while both membrane and soluble TWEAK can induce the non-canonical NF-κB pathway (23). Secondary TWEAK signaling pathways have also been described, such as the phosphatidylinositol 3-kinase/Akt pathway (14).

In the CNS, TWEAK targets endothelial cells, astrocytes, and neurons. Interestingly, in murine and human astrocytes, the TWEAK/Fn14 pathway can stimulate reactivity, i.e., when cells proliferate, are activated and produce inflammation factors (24, 25). This associated mitogenic potency is mediated by the TWEAK-induced MAPK signaling pathway (26). Besides, in an *in vitro* model of human BBB, TWEAK induced microvascular cerebral endothelial cells to display an inflammatory profile: they increased (i) their secretion of proinflammatory cytokines, (ii) their production and activation of matrix-metalloproteinase 9 (MMP-9) involved in BBB disruption, and (iii) their expression of intercellular adhesion molecule-1 implicated in leukocyte adhesion to endothelium (27).

Here, we aimed to review TWEAK role in NI modulation in neurodegenerative, autoimmune, ischemic, and malignant CNS inflammatory diseases. Considering available data about TWEAK involvement in their pathogenesis, we will successively focus on MS, neuropsychiatric systemic lupus erythematosus (NPSLE), cerebral ischemia, glioma, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and schizophrenia (**Table 1**).

#### THE TWEAK/Fn14 PATHWAY CONTRIBUTES TO TISSUE INJURY IN MS

Multiple sclerosis is a multifactorial disease involving autoimmunity against myelin components, NI, BBB disruption, tissue remodeling, and demyelination/remyelination (28).

#### TWEAK and Fn14 Are Upregulated in MS

Serafini et al. showed that both TWEAK and Fn14 were upregulated in postmortem MS brain sections (29). Furthermore, this increase was related to the degree of inflammation and demyelination. Perivascular and intrameningeal macrophages, microglia, and astrocytes were the main sources of TWEAK, with a different contribution according to lesion location and degree of inflammation. Fn14 was expressed by neurons and astrocytes in the cortex of highly infiltrated MS brains (29). The absence of TWEAK/Fn14 expression in healthy brain reinforces the idea that TWEAK/Fn 14 pathway could play a role in MS pathogeny. In blood, Desplat-Jégo et al. demonstrated that mTWEAK was expressed at the cell surface of monocytes derived from MS patients but not from non-MS patients (18). In MS, such mTWEAK-expressing monocytes could represent immune cells that infiltrate CNS by interacting with Fn14 molecules at the membrane of BBB endothelial cells. In the same study, sTWEAK serum and cerebrospinal fluid levels were similar in MS and non-MS patients (18), suggesting that sTWEAK is not a reliable diagnosis MS biomarker.

Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) is the best-characterized animal model of MS. In chronic EAE induced in C57Bl/6 mice, TWEAK transcript levels increased in the spinal cord (24). Moreover, transgenic mice overexpressing sTWEAK developed a more severe EAE than wild-type mice (24). Besides, in cuprizonetreated mice, a model of demyelination/remyelination, TWEAK and Fn14 transcription were upregulated during both demyelination and remyelination phases (30).

#### Blocking the TWEAK/Fn14 Pathway Is Effective for Treating an MS Model

Treated with cuprizone TWEAK-knock-out mice displayed a significant delay in microglia accumulation and in demyelination phases (30). Reinforcing the role of TWEAK/Fn14 in MS, our team showed that EAE severity and CNS leukocyte infiltration were reduced in mice treated with blocking monoclonal anti-TWEAK antibody after the priming phase (31). Moreover, an Fn14-TNF-related apoptosis-inducing ligand (TRAIL) fusion protein was designed to simultaneously block endogenous TWEAK and mediate TRAIL inhibitory signals on activated

*ALS, amyotrophic lateral sclerosis; BBB, blood–brain barrier; CCL-2, chemokine ligand 2; ICAM-1, intercellular adhesion molecule-1; Il, interleukin; Ig, immunoglobulin; Inos, inducible form of nitric-oxide synthase; KO, knock-out; MAPK, mitogen-activated protein kinase; MMP-9, matrix-metalloproteinase 9; MS, multiple sclerosis; NF-κB, nuclear factorκB; NPSLE, neuropsychiatric systemic lupus erythematosus; Rac1, Ras-related C3 botulinum toxin substrate 1; RANTES, regulated on activation, normal T Cell expressed and secreted; VCAM-1, vascular cell adhesion molecule-1; ZO-1, zonula occludens-1.*

T cells (32). Injected in EAE-mice, this protein reduced EAE severity (32, 33). Additionally, vaccinating mice or rats with TWEAK extracellular domain or with Fn14 induced the production of specific inhibitory antibodies. Such treatment was associated with reduced inflammatory spinal cord infiltration and with clinical amelioration during EAE (34). However, in humans, a raising question is the relevance of targeting TWEAK in MS when anti-TNF-α has failed to improve disease status (35). One could argue that the TWEAK/Fn14 pathway could also play a role in neuroprotection. In this way, Iocca et al. observed a modest but significant delay in remyelination of TWEAK-knockout mice treated with cuprizone. However, this marginal delay did not result in prolonged defect in remyelination (30). Later, Echeverry et al. described a TNF-α-dependent neuroprotective effect of TWEAK in another NI model (13), but data in MS are lacking. Further studies are then needed to definitively establish that TWEAK is a relevant therapeutic target in MS.

#### THE TWEAK/Fn14 PATHWAY CONTRIBUTES TO NPSLE

Neuropsychiatric systemic lupus erythematosus is an autoimmune disease underlied by hyperactivation of B and T lymphocytes, leading to overproduction of autoantibodies, tissue deposition of immune complexes, and increase in proinflammatory cytokines. This inflammatory state potentially targets all the organs, with marked joint, renal, hematologic and skin damages. In a significant number of patients, neuropsychiatric manifestations can also occur and thus define NPSLE. A pathogenic role of TWEAK was shown in lupus nephritis, a renal manifestation of the disease (36). Besides, evidences of TWEAK involvement in NPSLE are growing.

#### TWEAK/Fn14 Interaction Compromises the BBB in NPSLE Mouse Model

Putterman et al. observed an increased expression of TWEAK and Fn14 in the cerebral cortex of the MRL/lpr mouse strain with NPSLE (37, 38). Furthermore, knocking out Fn14 in this strain (i) significantly improved cognitive function and (ii) decreased depression and anhedonia in comparison with MRL/lpr wildtype mice. These clinical parameters were associated with decreased levels of proinflammatory cytokines and preserved BBB integrity (37). Further, CNS anatomopathological studies in these mice showed (i) a reduction of fibronectin deposition and inducible form of nitric-oxyde synthase (iNOS) production, (ii) a diminution of immune infiltrates, and (iii) a decrease of IgG deposition and complement activation (39). Finally, reduced cortex neuronal degeneration, apoptosis, and gliosis were associated with improved cognitive functions in such mice (40).

#### TWEAK Mediates Symptoms Observed in NPSLE

These results were consolidated by intracerebroventricular injection of Fc-TWEAK in non-autoimmune mice. Just like MRL/lpr strain, these mice developed NPSLE symptoms, associated with high levels of TWEAK pathway downstream components, complement and iNOS activation, IgG brain deposition, neurodegeneration, and apoptosis (39). Trysberg et al. suggested that NPSLE could be associated with high levels of MMP-9 and sTWEAK in cerebrospinal fluid (41, 42). They also found a significant correlation between intrathecal MMP-9 and levels of tau (a neuronal degeneration marker) and glial fibrillary acidic protein (an astrocytic degeneration marker) (41). These results suggest that TWEAK/Fn14 interaction might be involved in brain damages during NPSLE since it promotes synthesis and activation of MMP-9 in CNS (27). Nevertheless, a recent study concluded that TWEAK levels in serum and cerebro-spinal fluid did not seem relevant biomarkers for NPSLE (43).

Finally, these findings highly support the involvement of TWEAK/Fn14 interaction in NPSLE pathogenesis and symptom occurrence.

### TWEAK DISPLAYS A DUAL ROLE IN CEREBRAL ISCHEMIA

Cerebral ischemia or stroke is the second cause of mortality in the world. The onset of cerebral ischemia is followed by inflammatory events that affect neurological patient's outcome. Several studies implicate TWEAK in stroke.

#### TWEAK/Fn14 Interaction Increases the Volume of the Ischemic Zone in Stroke

Experimental focal cerebral ischemia was associated with an increase in TWEAK and Fn14 mRNA levels (44) and with BBB disruption (45, 46). In this context, TWEAK-induced NF-κB activation resulted in both astrocytic chemokine (C-C motif) ligand-2 (CCL-2) expression, leading to polynuclear neutrophil recruitment into the damaged zone, and in upregulation of MMP-9 activity (45–47). The effective inhibition of this neuroinflammatory process by Fn14-Fc decoy injections (46, 48) was associated with a reduced ischemic zone and hastened motor function recovery (46). Stroke patients displayed significantly elevated TWEAK serum levels and, in postmortem stroke human brains, elevated Fn14 mRNA levels were associated with upregulated Fn14 immunostaining in the ischemic zone (49).

#### The TWEAK/Fn14 Signaling Contributes to the Protective Effect of Hypoxic Preconditioning

Hypoxic-preconditioning of mice or cultured neurons promotes neuron survival and reduces ischemic lesion volume. In this context, TWEAK and Fn14 levels increase. Moreover, the protective effect of hypoxic preconditioning was abrogated in TWEAK or Fn14 KO mice and restored in cultured neurons after addition of TWEAK (13). This suggests that the TWEAK/Fn14 pathway contributes to the protective effect of hypoxic preconditioning. This is likely mediated by neuronal TNF-α and the activation of the MAPK pathway, resulting in the inactivation of the Bcl-2 associated death promoter protein (13).

These results suggest that a relevant therapeutic strategy would be administration of TWEAK in order to protect the brain in patients at high risk of stroke.

### TWEAK ACTIVATES CANONICAL AND NON-CANONICAL NF-**κ**B PATHWAYS IN GLIOBLASTOMA

Glioblastoma, the most common primary brain tumor, is associated with a high mortality related to its high local invasiveness, angiogenesis, and immunosuppressive potency. The initiation and development of tumors are associated with repetitive tissue damage and are tightly linked to chronic inflammatory processes. Tumor cell migration is essential in glioblastoma malignancy progression and depends on its inflammatory microenvironment.

# TWEAK/Fn14 Promotes Glioblastoma Cell Migration and Invasion

Tumor necrosis factor-like weak inducer of apoptosis upregulates Fn14 in migrating glioblastoma cells. This overexpression correlates with tumor aggressiveness and poor outcome (50, 51). In this way, Fn14 expression may help classifying glioma histological subtype (52). Moreover, canonical NF-κB pathway, stimulated by TWEAK, promotes Rac1 and Cdc42 expression, key mediators in the regulation of glioblastoma cell migration and invasion (51, 53, 54). Interestingly, Cherry et al. showed that MMP-9 inhibition abolished TWEAK-induced glioma invasion (55).

# Targeting Fn14 Is Promising for Treating Glioblastoma

Therapeutic strategies targeting Fn14 in malignant cells have been evaluated. In these preclinical studies, manipulating Fn14 expression levels or suppressing TWEAK-Fn14-NFκB-dependent signaling decreased glioblastoma cell invasion capacity (56, 57). However, further experiments are needed to evaluate whether these strategies can significantly impact patients' survival.

 Mutations within the superoxide dismutase 1 (SOD1) are sometimes found in familial or sporadic cases. Mutant SOD1 expression was associated with inflammatory astrocytes and microglia and with skeletal muscle atrophy (58).

#### TWEAK/CD163 Interaction Induces Motor Neuron Death in a Mouse Model of ALS

Bowerman et al. used a model of transgenic mice overexpressing human mutant SOD1 and recapitulating the main traits of ALS to study TWEAK/Fn14 involvement in ALS (15). In this model, mTWEAK was upregulated in the spinal cord and it induced astrocyte proliferation and IL-6 release. TWEAK and Fn14 were also found in skeletal muscle with different patterns of expression linked to disease development. In this study, the authors demonstrated a TWEAK induced-motor neuron death (15). Surprisingly, it was mediated by TWEAK interaction with CD163, a putative alternative TWEAK receptor. This neuronal death involved caspase-3 activation and was totally independent of Fn14. This work is the first implicating CD163 in mediating TWEAK effects in an inflammatory neurological disease. CD163 is a member of the scavenger receptors and is exclusively expressed by monocytes and macrophages. Increasing *in vivo* and *in vitro* evidences suggest that the interaction between TWEAK and CD163 may affect the development of atherosclerosis and related diseases (59).

# TWEAK Promotes Astrogliosis and Microgliosis in ALS

Tumor necrosis factor-like weak inducer of apoptosis deletion reduced astrogliosis and microgliosis in the spinal cord, which substantiated the implication of the TWEAK pathway in ALS. However, muscle pathology was only partially reduced and TWEAK deletion did not prevent motor deterioration nor increase life span. In the same way, anti-TWEAK antibodies injected in ALS mice decreased microgliosis without improving motor functions. Bowerman et al. thus proposed a model combining the action of peripheral and central TWEAK, which could both contribute to the sustained activation of microglia in ALS (15).

This work suggests that blocking TWEAK in ALS is interesting but not sufficient and requires being included in a combinatory therapeutic approach.

#### TWEAK CONTRIBUTION TO PARKINSON'S DISEASE AND PSYCHIATRIC DISEASES NEEDS CONFIRMATION

#### Anti-TWEAK Antibodies Improve a Murine Model of Parkinson's Disease

Parkinson's disease is a neurodegenerative pathology characterized by movement impairment due to dopaminergic loss in the nigrostriatal pathway. It may also have a neuroinflammatory component, where NF-κB may play a dual role, both promoting and protecting against neurodegeneration (60). The role of TWEAK itself has been studied in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model and in Parkinson's disease human brain samples (61). There was no difference in substantia nigra TWEAK concentration between MPTP mice, Parkinson's disease patients, and controls. While TWEAK or Fn14-KO failed to prevent MPTP toxicity, anti-TWEAK antibodies treatment attenuated the MPTP-induced death of dopaminergic neurons (61). These apparently conflicting results could be explained in part by existing compensatory systems, including the TNF-α

pathway, since TNF is expressed in microglia of Parkinson's disease patients.

# TWEAK Plasma Levels in Schizophrenia and Bipolar Disorders Are Not Conclusive

Neuroinflammation is highly suspected to be involved in psychiatric pathologies like schizophrenia or bipolar disorder. Recently, TWEAK plasma levels of patients suffering from schizophrenia were compared with controls. Although no difference was shown globally, TWEAK levels in male patients were significantly lower than in male controls, suggesting TWEAK involvement in subgroups of schizophrenia patients (62). In bipolar disorder, conflicting results were observed, perhaps due to the heterogeneity in patients' groups: Cingi et al. measured lower TWEAK plasma levels compared to healthy controls (63), while Barbosa et al. described increased concentrations, regardless of the mood (64). Nevertheless, as the interest for studying TWEAK/Fn14 pathway in psychiatric diseases is very recent, further studies will certainly be available soon.

This mini-review reveals that the TWEAK/Fn14 pathway modulates NI in neurodegenerative, immune, ischemic, and malignant CNS inflammatory diseases. In fact, as supported by both animal and human *in vitro* and *in vivo* studies, TWEAK/ Fn14 can modulate NI by activating canonical and non-canonical NF-κB pathways but also MAPK signaling. Then, expression or release of inflammatory cytokines, chemokines, and adhesion molecules are upregulated and astrogliosis and microgliosis occur. Additionally, MMP-9 secretion is stimulated and reinforces BBB disruption and tissue remodeling. Note that monoclonal anti-TWEAK antibodies have been injected in patients in phase I clinical studies. In healthy volunteers or patients with solid tumors or rheumatoid arthritis, they (i) displayed a classical therapeutic antibody pharmacokinetic pattern, (ii) were associated with a favorable safety profile, and (iii) induced a decrease in circulating TWEAK serum level (65–68). Additionally, in these studies, TWEAK/Fn14 pathway inhibition yielded encouraging results, such as reduced clinical and biological inflammatory markers, including sTWEAK. All these data support the concept that the TWEAK/Fn14 axis represents a promising therapeutic target for modulating inflammation, including NI.

# AUTHOR CONTRIBUTIONS

AB and SD-J both collected the publications and wrote the manuscript.

# ACKNOWLEDGMENTS

We thank Nicolas Simon for reading and critical review of this paper, and Isabelle Virard for proofreading it.

# FUNDING

This work was supported by Aix-Marseille University, CNRS, and AP-HM.

# REFERENCES

non-autoimmune mice. *Brain Behav Immun* (2016) 54:27–37. doi:10.1016/j. bbi.2015.12.017

induction of NF-κB-inducing kinase (NIK) and noncanonical NF-κB signaling. *Mol Cancer* (2015) 14:9. doi:10.1186/s12943-014-0273-1

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

*Copyright © 2017 Boulamery and Desplat-Jégo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Inflammatory Processes Associated with Canine Intervertebral Disc Herniation

*Marie Monchaux1 , Simone Forterre1 , David Spreng1,2, Agnieszka Karol 3 , Franck Forterre1,2† and Karin Wuertz-Kozak 2,4,5,6,7\*†*

*1Vetsuisse Faculty, Department of Clinical Veterinary Science, University of Bern, Bern, Switzerland, 2Competence Center of Applied Biotechnology and Molecular Medicine (CABMM), University of Zurich, Zurich, Switzerland, 3Vetsuisse Faculty, Institute of Veterinary Pathology, University of Zurich, Zurich, Switzerland, 4Department of Health Sciences and Technology, Institute for Biomechanics, ETH Zurich, Zurich, Switzerland, 5Schön Clinic Munich, Harlaching, Munich, Germany, 6Spine Research Institute, Paracelsus Medical University, Salzburg, Austria, 7Department of Health Sciences, University of Postdam, Postdam, Germany*

#### *Edited by:*

*Jixin Zhong, Case Western Reserve University, United States*

#### *Reviewed by:*

*Vineesh Vimala Raveendran, King Faisal Specialist Hospital & Research Centre, Saudi Arabia Oreste Gualillo, Servicio Gallego de Salud, Spain*

*\*Correspondence:*

*Karin Wuertz-Kozak<EMAIL_ADDRESS>
*†These authors have shared senior authorship.*

#### *Specialty section:*

*This article was submitted to Inflammation, a section of the journal Frontiers in Immunology*

*Received: 03 August 2017 Accepted: 15 November 2017 Published: 04 December 2017*

#### *Citation:*

*Monchaux M, Forterre S, Spreng D, Karol A, Forterre F and Wuertz-Kozak K (2017) Inflammatory Processes Associated with Canine Intervertebral Disc Herniation. Front. Immunol. 8:1681. doi: 10.3389/fimmu.2017.01681*

Intervertebral disc herniation (IVDH) is an important pathology in humans and also in dogs. While the molecular disease mechanisms are well investigated in humans, little is known about the inflammatory mediators in naturally occurring canine IVDH. The objective of this study was to investigate whether the involved proinflammatory cytokines in human IVDH are also key cytokines in canine IVDH and thus to elucidate the suitability of the dog as a model for human trials. 59 samples from 25 dogs with surgically confirmed thoracolumbar IVDH were collected and classified in three subgroups: herniated (H), affected non-herniated (NH) disc, and adjacent non-affected (NA) disc. Discs from 11 healthy dogs acted as controls (C). Samples were analyzed for IL-1, IL-6, IL-8, and TNFα expression (qPCR/ELISA) as well as cell infiltration and activation of the MAP kinase pathways (immunohistochemistry). Gene and protein expression of all key cytokines could be detected in IVDH affected dogs. Canine IVDH was significantly associated with a higher gene expression of IL-6 (H > C, NH > C) and TNF-α (H > C, NH > C, NA > C) and a significant down-regulation of IL-1β (H < C). Dogs with spontaneous pain had significantly higher IL-6 mRNA compared to those with pain arising only upon palpation. An inter-donor comparison (H and HN relative to NA) revealed a significant increase of IL-6 gene expression (H > NA, NH > NA). IL-8 (H > C, NA > C) and TNF-α (NH > C) protein levels were significantly increased in diseased dogs while inversely, IL-6 protein levels were significantly higher in patients with better clinical outcome. Aside from resident IVD cells, mostly monocytes and macrophages were found in extruded material, with concomitant activation of extracellular signal-regulated kinase p38 in the majority of samples. Dogs with spontaneous IVDH might provide a useful model for human disc diseases. Although the expression of key cytokines found in human IVDH was also demonstrated in canine tissue, the inflammatory mechanisms accompanying canine IVDH diverges partially from humans, which will require further investigations in the future. In dogs, IL-6 seems to play an important pathological role and may represent a new potential therapeutic target for canine patients.

Keywords: intervertebral disc herniation, inflammatory mediators, IL-1**β**, IL-6, IL-8, TNF-**α**, MAP kinase pathway, canine animal model

# INTRODUCTION

Intervertebral disc degeneration (IVDD) and intervertebral disc herniation (IVDH) are considered a major cause of acute and chronic low back pain, resulting in a tremendous economic burden worldwide (1–4). IVDD is characterized by progressive structural and functional changes, including a loss of hydrostatic properties of the normally highly hydrated nucleus pulposus (NP) (5), as well as increased vascular and neuronal ingrowth that is associated with chronic back pain (6). Recent human studies have identified proinflammatory cytokines, specifically IL-1β, IL-6, IL-8, and TNF-α, as pivotal contributors in the course of the pathophysiologic inflammatory cascade of intervertebral disc diseases and the development of neuropathic pain through irritation of ingrowing nerves (7–28). A herniated disc, presented as protrusion, extrusion, or sequestration, is a localized displacement of disc tissue in the epidural space (29), resulting in mechanical compression and chemical irritation of spinal nerves, which contribute to ischemia, radicular symptoms, and stimulation of the inflammatory cascade (30–39). Alongside of heredity (40, 41), multiple factors such as age and smoking are involved in the ethiopathogenesis of human IVDD and IVDH as well as in the initiation of an inflammatory cascade (42–45). Furthermore, obesity was highlighted as an important contributor to disc pathologies as it not only increases the load on the IVD (46–48), but also promotes inflammation *via* cytokine-release from adipocytes and recruited macrophages (49).

Measuring cytokine levels within the diseased tissue can provide a better understanding of the pathological process. As human tissue samples for research—especially as healthy controls—are scarce, more complex pathological investigations and testing of new therapeutic approaches often require animal experiments (50). Currently established models are predominantly based on artificially induced disc pathology through mostly invasive manipulations (e.g., stab incision in rodents), which lack similarities to the human pathology (51). In contrast, animal models based on spontaneously occurring disc pathologies, such as canine IVDH, share important similarities to the clinical presentation, pathology, lesion morphology, diagnostic, treatment, and recovery with human IVDH (50, 52, 53). IVDH in dogs has an incidence of 2% of all admissions in referral clinics and occurs predominantly in chondrodystrophic breeds, such as Beagle, Dachshund, Shi-Tzu, and French Bulldog (54–59). Similar to humans, the prevalence of overweight and obesity in dogs is increasing, being as high as 34% in the US and 25% in the UK (60, 61), with a higher risk factor for disc extrusion in dogs with higher body score index (62). Despite the potential relevance of canine IVDH as a human disease model, little in depth research has been conducted to determine its pathological processes in dogs, specifically regarding the role of inflammatory mediators in disease progression and pain development. Thus far, existing data indicate that in the early phase of canine IVDH, mRNA concentration of IL-6, a possible promoter of inflammation and apoptosis of resident glial cells, was significantly upregulated, whereas returning to baseline values in later stages of the disease. In comparison, mRNA concentration of IL-8, a potent chemokine and early mediator of inflammation, was strongly upregulated in the acute and subacute onset of IVDH. A trend of higher TNF-α mRNA concentration in acute IVDH could also be shown (63). Controversially, Karli et al. demonstrated a downregulation of IL-1β, IL-6, and TNF-α mRNA concentrations over the whole course of canine IVDH, but highlighted an upregulation of IL-8 mRNA concentration in the acute stage of the disease, which decreased when treated with non-steroidal anti-inflammatory drugs (64).

Based on the conflicting data found in the current literature, the aim of this study was to identify whether the human key cytokines are expressed in canine IVDs and to determine pathological alterations in their expression by comparing normal IVDs to diseased IVDs with IVDH, with the overall goal to provide further information on the validity of the canine disc disease model. Concomitantly, we also assessed neurologic function, pain status, and clinical outcome in affected dogs.

#### MATERIALS AND METHODS

#### Patients, Controls, and Tissue Samples

Canine disc material was collected from dogs with confirmed thoracolumbar IVDH during decompressive surgical procedures at our referral institution (November 2013 to February 2015). All samples were collected from actual referred patients and not from laboratory animals. Herniated (H) and non-herniated (NH) IVD samples were both collected from the IVDH: affected level, with H = prolapsed part and NH = contained part. Non-affected (NA) IVDs were collected from an adjacent spinal level (prophylactic disc fenestration). Inclusion criteria for IVDH patients: welldocumented records of onset of clinical signs and pretreatment, neurologic findings, diagnosis, course of the disease, ethical approval, and signed owner consent.

In addition, healthy control IVDs (C) were collected from dogs included in an unrelated animal study or patients without any history or signs of spinal disease and without any antiinflammatory pretreatment euthanized for other health reasons. These samples were directly surgically collected after euthanasia within 15 min to avoid potential artifacts in measurements of the cytokines (65–67).

In the patient group, breed, age, gender, duration of neurologic signs, pretreatment, neurologic grade, severity of pain, localization of IVD extrusion, and outcome were recorded. Duration of neurologic signs were defined as the time between the onset of clinical signs and surgery and was grouped as follows: decompression within the first 24 h after onset of clinical signs (acute), between 24 h and 7 days of onset (subacute), or after more than 7 days (chronic) (68, 69). A complete neurological examination was performed at the time of admission, maximally 12 h before surgery, at the time at discharge, and at a follow up examination between 2 and 4 weeks after discharge. The neurologic condition and pain were graded as shown in **Table 1** (64, 70, 71).

### Diagnostic Imaging and Surgical Treatment

Anesthesia and analgesia were conducted under a standard clinical protocol for each patient (44). Magnetic resonance imaging (MRI) of the thoracolumbar spine was performed (Philips

TABLE 1 | Clinical scoring system.

TABLE 2 | TaqMan primers/probes and sequence accession number.

CA, USA) with 10 ng cDNA in RNAse free water (4.5 µl in total) and 0.5 µl of canine TaqMan primers/probes (TaqMan® Gene Expression Assays, Life Technologies, Van Alben Way, CA, USA) (**Table 2**).

Each sample was run in duplicate for each gene, the CT value means were calculated and the comparative CT method was used for data evaluation [relative quantification (RQ)]. Samples with gene expression lower than the detection limit were attributed an artificial maximal CT value of 43, using the selection of the median as aggregation method between samples with the same experimental condition to avoid statistical and mathematical issues (72).

For group comparisons between H, NH, NA, and C, results were calculated as 2−ΔCT values, i.e., the expression of each target gene was normalized relative to the expression of the house keeping gene [canine TATA box-binding protein (TBP)], using the classical ΔCT method and the following algorithm:

RQ = 2 identically for NH, NA, ( ) Ct H TargetGene Ct H TBP − ( ) ( )− ( C).

For interdonor calculations, an additional normalization step (ΔΔCT) was performed (ΔCT of H or NH minus ΔCT of NA, of the same animal). Results were calculated as 2−ΔΔCT values and shown as fold change in expression between affected discs (H, NH) and internal control discs (NA). The following algorithm was used:

RQ = 2− − [(Ct() () H C TargetGene t H TBP T ) ( C − − t N( ) A C argetGene t N( A )] TBP identically for NH ) ( ).

The intra-assay CV was 0.74% (*n* = 131).

# Total Protein and ELISA

ELISA samples were weighed and pulverized as described above, but with immersion in T-PER reagent (Tissue Protein Extraction Reagent, Thermo Fisher Scientific, Wilmington, DE, USA) at a ratio of 100 µl T-PER reagent per 10 mg tissue. The lysates were homogenized and purified following a standardized protocol. Briefly, samples were centrifuged at 12,000 *g* at 4°C for 10 min, the supernatant was collected and total protein was determined by the Bradford assay (Bio-Rad Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, Hercules, CA, USA), with absorbance measurement at 585 nm (Infinite® 200 Pro, Tecan Group Ltd., Männedorf, Switzerland). The protein levels of IL-1β, IL-6, IL-8, and TNF-α were detected with canine specific ELISAs (MyBioSource, San Diego, CA, USA for IL-1β, IL-6, IL-8, and Quantikine ELISA Kit, R&D Systems Inc., Minneapolis, MN, USA, for TNF-α) according to the manufacturer's instructions, with a sensitivity of 1 pg/ml for IL-1β, IL-6, and IL-8 kit and

Panorama HFO, 1.0-T open system, Philips Medical Systems Nederland B.V., The Netherlands) to define the IVD extrusion site. Immediately after MRI, patients underwent surgery for discogenic back pain and to decompress the spinal cord. A standard hemilaminectomy at the site of extrusion, fenestration of the affected and one of the adjacent discs (cranial or caudal) were performed, allowing to take three samples of disc material (H, NH, and NA). For cytokine identification, removed disc material was collected under sterile conditions, snap frozen directly within 15 s in liquid nitrogen and stored at −80°C until sample preparation. For immunohistochemical and histological analysis, the tissue was fixed in 10% buffered formalin (1–5 days), embedded in paraffin, and cut into 5 µm sections.

#### Real-time PCR (RT-PCR)

For RNA isolation, each frozen disc sample was transferred into a precooled custom-made mortar, filled with liquid nitrogen and manually grinded under RNAse free conditions. The frozen tissue powder was transferred into 500 µl Trizol (Life Technologies, Van Alben Way, CA, USA), homogenized three times for 30 s (Polytron PT 2500 E, Kinematica, Luzern, Switzerland), incubated for 5 min at RT and centrifuged at 12,000 *g* for 10 min at 4°C.

The supernatant was mixed with 250 µl chloroform, vortexed for 30 s, incubated at RT for 15 min, centrifuged at 12,000 *g* for 15 min at 4°C and the aqueous phase recovered. RNA was precipitated by adding 125 µl isopropanol and 125 µl of high salt precipitation solution (0.8 M sodium citrate, 1.2 M sodium chloride), washed with 500 µl of 75% ethanol and dissolved in 25 µl RNase free water after complete removal of ethanol. The quantity of the isolated RNA was determined spectroscopically (NanoDrop® Lite, Thermo Fisher Scientific, Wilmington, DE, USA) and the quality confirmed by the sample's OD 260/280 (1.8–2.0; samples with lower quality were excluded from further analyses). cDNA was prepared using the TaqMan Reverse Transcription kit (TaqMan® Reverse Transcription Reagent, Life Technologies, Van Alben Way, CA, USA) according to the manufacturer's instruction. RT-qPCR was conducted on the CFX96 Touch qPCR Machine (Bio-Rad Laboratories, Hercules, CA, USA) as previously described (46, 47), by mixing 5 µl of TaqMan® Fast Advanced Master Mix (Life Technologies, Van Alben Way, 4.2 pg/ml for TNF-α. Sample concentration was calculated based on a standard curve and normalized to total protein content. Results are demonstrated either as group comparisons (H, NH, NA, C: cytokine content/total protein in pg/mg) or as interdonor comparisons (H and NH relative to NA: fold change). The median intra-assay CV is 9.0% (range 5.3–15%) and the median interassay CV 10.8% (range 9.3–15%), as provided by the supplier.

#### Immunohistochemistry

Serial sections were deparaffinized, heat-mediated antigen retrieval was conducted (95°C, 20 min) and sections were incubated with primary antibodies (RT, overnight) identifying infiltrating cells (polymorphonuclear, mononuclear, macrophages, multinucleated giant cells) and MAP kinase (MAPK) pathway activation (**Table 3**). Subsequently, sections were treated with an appropriate primary antibody (Marker) and washed 3× with Tris buffer before the secondary antimouse IgG was applied (1:200 dilution, 30 min). The amino-9-ethyl-carbazole substrate kit (Dako) was employed as a chromogen. Finally, the sections were counter-stained with Gill's hematoxylin for 3 min, and cover-slipped with an aqueous mounting media (Glicerine, Sigma-Aldrich).

All slides were examined and semiquantitatively evaluated independently by two investigators for infiltrating cell types in the extruded NP as well as activation of the MAPK pathway [c-Jun NH2-terminal kinase (JNK), p38 isoforms of MAPK (p-38), extracellular signal-regulated kinases (ERKs)]. The following grades were used: (−) negative; (±) weakly (less than 5% of cells positive); (+) moderately (5–25% of cells positive); (++) strongly (more than 25% of cells positive).

#### Statistical Analysis

For all results, an outlier calculation was performed and outliers removed before statistical analysis (MedCalc 16.8/2016, http:// www.medcalc.org). Summary statistics were performed and normality assessed using D'Agostino-Pearson tests. As data were versatile regarding its distribution, nonparametric statistics were additionally used except for the interdonor comparison. A Kruskal–Wallis test was used to identify any association between gene and protein expression of cytokines with location of IVDH, duration of clinical signs, neurologic grade, pretreatment and outcome. Differences in gene or protein expression of cytokines between the samples of affected and control animals were evaluated using a Mann–Whitney *U*-test. The subgroup

comparison was carried out stepwise by a Wilcoxon signed-rank test. The Bonferoni *p*-value adjustment was used to control for multiple comparisons. For inter-donor comparisons of gene and protein expression, an independent sample *t*-test and a Mann– Whitney *U*-test was performed, respectively. A Spearman's rank correlation was used to examine the relationship between gene expression and protein level. Threshold value for statistical significance was set up to *p* < 0.05 and data are presented as boxplots, with median and 95% confidential interval (CI) unless otherwise stated.

#### RESULTS

#### Clinical Data

Twenty-five dogs with surgically confirmed thoracolumbar IVDH were included in the study. All canine patients (25 of 25 cases) were small and medium-breed dogs (<20 kg) with a median body weight at admission of 9.0 kg (range 3.6–19.0 kg). The dogs were classified as chondrodystrophic breeds with the Dachshund (*n* = 5), Cocker Spaniel, Coton de Tuléar and the French Bulldog (each *n* = 2) and the Bichon Frisé, Cavalier King Charles Spaniel, Jack Russel Terrier, Pekingese, Miniature Poodle, Pug Dog, Shi-Tzu, and the Yorkshire Terrier (each *n* = 1) or as non-chondrodystrophic or mixed breeds with the Border Collie (*n* = 1) and the Mixed Breed Dog (*n* = 5) (57, 73, 74). The median age was 5 years (range 3–13 years). Sixteen dogs were male intact, four were female intact, three were female spayed, and two were male castrated. The median duration of clinical signs until surgery was 3 days (range 1–365 days). When classified into three groups, there were 3 acute, 14 subacute, and 8 chronic cases. Ten dogs were referred without pretreatment, five had been pretreated with steroids, seven with NSAIDs, and three with a combination of both, pooled together as pretreated dogs. The neurologic status before surgery was Grade 2 in 11, Grade 3 in 6, Grade 4 in 5, and Grade 5 in 5 dogs. Pain score revealed 2 dogs with Grade 0, 14 dogs with Grade 1, and 9 animals with Grade 2. The spinal level of IVDH in the affected dogs were L2–L3 in eight dogs, Th12–Th13 in six dogs, Th11-Th12, L1–L2, L3–L4 in each three dogs, and Th10–Th11, Th13–L1 in each one dog. Fifteen dogs had a good outcome, six showed an improvement, but were not ambulatory, and four showed no improvement within 1 month after surgery. In the latter case, two of them were euthanized because of poor prognosis, a follow-up call unveiled that the other two dogs are still in physiotherapy treatment showing no improvement in the last 7 and 9 months.

Eleven euthanized dogs without signs of IVDH were included as control group. The median age of the control group was 4.5 years (2–11 years). Ten dogs were female spayed and one dog was female intact. There were 10 beagles and 1 was a mixed breed dog.

### Diagnostic Imaging and Surgical Treatment

All 25 affected dogs underwent surgical decompression of the spinal cord immediately after diagnostic imaging, including the collection of the samples. Three different samples (H, NH, and NA) were collected in 14 dogs, two different samples (H, NH) in 6 dogs, and one sample (H) in 5 dogs, resulting in a total of 59 samples. The median sample weight was 30.62 mg (range 1.7–112.3 mg). Six disc samples were excluded: four NHs and one H were excluded due to very small size and one NH sample demonstrated inconsistent housekeeping gene expression and out of range protein values. From the remaining 53 samples, 11 tissue samples weighting more than 50 mg were equally divided in samples for PCR and ELISA testing. 18 smaller samples were solely assigned to PCR testing and 24 smaller samples solely to ELISA testing. In the C group, we collected 11 samples in 11 dogs in the Th13–L1–L2 IVD space, with a median sample weight of 60.87 mg (range 30.3–133 mg). One dog was completely omitted because of RNA impurity. From the 10 remaining control samples, 4 samples weighting more than 50 mg were equally split into samples for PCR and ELISA. Three samples each were solely assigned to PCR and ELISA testing.

### Cytokine Detectability

We could demonstrate gene and protein expression of the selected proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in IVDH in dogs.

Grade 1 = no spontaneous pain, but discomfort upon palpation of the spine. Grade 2 = spontaneous pain and excessive pain upon palpation of the spine. Asterisks indicate statistical significance between groups at *p* < 0.05 (\*).

### Gene Expression

No significant correlation between IVDH location, duration of clinical signs or outcome, and the genes of interest was demonstrated, but the gene expression of IL-6 was significantly higher (*p* = *0.043*) in pain grade 2 compared to grade 1 (**Figure 1**). Pooled results of all dogs in the respective groups (C, H, NH, NA) showed a significant downregulation of IL-1β in H compared to C, but no difference between NH or NA and C was found. IL-6 expression was significantly higher in H and NH compared to C, but not in NA. TNF-α expression showed significantly higher levels in H, NA, and NH compared to C. Gene expression of IL-8 showed no significant difference among the groups. All results are shown in **Table 4** and **Figure 2**.

As patients demonstrated differences in their basal cytokine levels, hence resulting in high donor–donor variations in subgroup comparisons, an interdonor comparison was conducted (H and HN relative to NA = internal control for each patient). A significant increase of IL-6 was observed for H and NH compared to NA. In contrast, gene expression of IL-1β, IL-8, and TNF-α were either unchanged or reduced in diseased compared to NA IVDs. All results are shown in **Table 5** and **Figure 3**.

#### Protein Expression

There were no significant correlations between cytokine protein levels and IVDH location, severity of pain, pretreatment or outcome and concentration of cytokines, but IL-6 was found to be significant higher in the clinical outcome group 2 compared to group 1 (**Figure 4**).

No significant differences of IL-1β or IL-6 protein expression were observed between the various groups. The concentration of IL-8 was significantly higher in H and NA compared to C, but no difference was found between NH and C. TNF-α showed significantly higher protein levels in NH compared to C, but not for H or NA. Results are shown in **Table 6** and **Figure 5**.

Like for gene expression, an additional inter-donor comparison was conducted, with NA as the internal control. However, no statistical differences were found between diseased (H, NH) and NA IVDs due to a level of variability and low sample numbers (data not shown).

No significant relationship was found between gene expression and corresponding protein concentration (IL-1β *r*s = 0.02; 95% CI, −0.66–0.67, *p* = 0.97; *IL-6 r*s = 0.30; 95% CI, −0.41–0.78; *p* = 0.41; IL-8 *r*s = −0.25, 95% CI, −0.85–0.62, *p* = 0.59; TNFα *r*s = −0.51, 95% CI, −0.86–0.18, *p* = 0.13). Interestingly, a significant association in gene expression between IL-6 and IL-8 (*r*s = 0.79, 95% CI 0.08–0.97, *p* = 0.036) was found in the control

between herniated (H), non-herniated (NH), non-affected (NA) as well as healthy control (C) intervertebral disc material. Data are shown as 2−ΔCT. Asterisks indicate statistical significance between groups at *p* < 0.05 (\*) and *p* < 0.01 (\*\*).

TABLE 5 | Statistical results of interdonor gene expression data (subgroup comparison, dependent samples).

group. In herniated samples, IL-1β protein correlated with IL-6 (*r*s = 0.71; 95% CI, 0.36–0.88, *p* = 0.001), IL-8 (*r*s = 0.54; 95% CI, 0.09–0.80, *p* = 0.02) as well as TNF-α concentrations (*r*s = 0.55; 95% CI, 0.11–0.81, *p* = 0.02) measured by ELISA.

#### Immunohistochemistry

Monocytes and macrophages were the most infiltrating cell population encountered in canine herniated IVD material. Sporadically, giant cells resembling notochordal cells were observed. CD 18 was highly expressed (++) in all samples, whereas detection of CD3 and vWF8 detection was semiintractable and error-prone, hence not being shown. Expression of activated (phosphorylated) ERK (++) (**Figure 6**) and p38 (+/++) could be detected in most of the examined slides, whereas pJNK (−/+) was only sporadically detectable (not shown).

betwen dogs with differing clinical outcome (*n* = 20) and shown as picogram of IL-6 per milligram total protein. Grade 0 = lack of improvement. Grade 1 = improvement of neurologic status, but not able to walk. Grade 2 = recovery of ambulation or at least one grade in neurologic grades 1 and 2. Asterisks indicate statistical significance between groups at *p* < 0.05 (\*).

#### DISCUSSION

Previous research on human IVD tissue has demonstrated that degeneration and herniation coincides with increased levels of inflammatory mediators, such as IL-1β, IL-6, IL-8, and TNF-α (8). In the present study, a comparison between diseased (herniated IVDs = H; NH) and control (C) IVDs of dogs demonstrated that disc disease was associated with significantly higher gene expression of IL-6 (H vs. C, NH vs. C) and TNF-α (H vs. C, NH vs. C). On the protein level, a significant increase of IL-8 and TNF-α in IVDs of diseased dogs compared to controls was found. A patient-specific comparison between the diseased (H, NH) and the IVDs excised from an adjacent level (NA) of the same dog revealed a pronounced pathological elevation of IL-6 (H vs. NA, NH vs. NA). In contrast, mRNA levels of IL-1β, IL-8, and TNF-α were either unchanged or reduced in diseased compared to NA IVDs of patients.

Previous studies on canine disc disease found IL-1β to be either undetectable or downregulated in dogs, which contrasts human disc pathology (64, 75). This was confirmed by our findings, showing a significant downregulation of the gene expression of IL-1β in herniated disc material (H) compared to controls, a downregulation in the affected disc material (H, NH) of the paired samples and no significant findings on the protein level.

In human IVDH, higher protein levels of the proinflammatory cytokine IL-6 were detected in different studies (10, 23, 25, 76), hence being discussed as a prognostic factor and therapeutic target. Thus far, results on IL-6 expression during canine disc disease are contradictory (63, 64, 75, 77). According to our findings, IL-6 seems to have a pivotal role, with its gene expression being significantly upregulated in the affected disc material (H, NH) of the independent and paired samples, unrelated to the duration of the clinical signs. Interestingly, we noticed a correlation between IL-6 mRNA levels and the severity of pain, with mRNA expression being significantly higher in those canine patients with basal pain than in those with pain arising only upon palpation. To the authors' knowledge, this has not been described before in canine IVDH, but might be in accordance with recent research in humans that has increasingly pointed toward IL-6 being a crucial factor in pain development (78, 79). However, IL-6 is not only a proinflammatory cytokine with significance in inflammation and diseases, but is also known to have regenerative or anti-inflammatory properties (80). Our results indicated that canine patients showing higher concentration of IL-6 protein levels in their IVD had a better outcome and were more likely to regain ambulation. This contrasting finding might be explained by the dual function of IL-6, suggesting that with time, IL-6 might control inflammation by acting as an antiinflammatory cytokine (81, 82). IL-6 originates directly from

TABLE 6 | Statistical results of protein expression data (subgroup comparison, independent samples).

IVD cells (76) and also from T-cells and macrophages (83–86), the latter constituting the main cell population of the inflammatory infiltrates in the epidural space after IVD extrusion in our samples. Based on our findings, IL-6 could potentially have also a beneficial role in the late onset of the inflammatory cascade of canine IVDH. Therefore, therapeutic blockage for IL-6 should be carefully considered.

In this study, we also analyzed the expression of IL-8, which has previously been shown to be strongly upregulated in the acute phase of canine IVDH (63, 64, 77), yet with a negative correlation between its expression and the duration of spinal cord injury secondary to IVDH (77). As IL-8 is thus thought to be an early disease marker with time-dependent expression and as our patients were mostly seen for treatment at a late stage of the disease, it explains why we could not demonstrate a significant upregulation of IL-8 mRNA, but of IL-8 protein in diseased discs. Interestingly, a significant upregulation of the protein level of IL-8 in independent samples could as also be demonstrated in NA disc material, suggesting a possible secretion of IL-8 in canine disc material before the effective herniation of the affected IVD and hence further supporting a possible early stage inflammation as a trigger for IVDH.

Together with IL-1β, TNF-α is likely the most studied cytokine in human IVDD (11, 17, 28, 87, 88), inducing matrix degrading enzyme expression and upregulation of nerve growth factor. TNF-α is hence suggested to be have an important role in the development of hyperalgesia and chronic pain after IVDH in humans (7, 16, 28, 89–92). Contradictory results have been found on TNF-α expression in canine IVDH, ranging from non-detectability (64, 75, 77) to disease-related elevation (albeit non-significant) (63). In accordance with Spitzbart et al. (63), as well as with human studies, we could demonstrate a significant

FIGURE 6 | Immunohistochemical staining for extracellular signal-regulated kinase (ERK)1/2 of extruded canine intervertebral disc material (left). Expression of ERK (brownish coloration) was detected in the majority of the examined slides. On the right, negative control and positive control (canine mammar carcinoma) are depicted.

upregulation of TNF-α on the gene expression level (H, NH) and the protein expression level (NH). Furthermore, and also similar to human NP samples in which TNF-α is known to be continuously expressed at basal levels (93), TNF-α could also be detected in adjacent discs (NA) suggesting to be a possible trigger of an early stage inflammation for IVDH.

Histological investigations demonstrated that most herniated IVDs exhibited an epidural inflammatory response, ranging from acute invasion of neutrophils to formation of chronic granulation tissue. Histological analysis of underlying inflammatory pathways demonstrated activation of the MAPKs ERK and p38 in most samples, whereas pJNK was rarely detected. MAPKs are signal transduction pathways that are activated by a multitude of stimuli, ranging from environmental, mechanical and osmotic stress to growth factors, cytokines and reactive oxygen species, with ERK and p38 controlling inflammation, catabolism as well as cell growth, differentiation and viability/death (27). The fact that the MAPKs pathways are shared between the cytokines might explain the correlation on the protein level in herniated samples of IL-1β with IL-6, IL-8, and TNF-α (94).

In accordance with our results, *in vitro* cell culture studies and rodent *in vivo* studies have previously demonstrated that p38 and ERK are activated in inflammatory environments (95–100), controlling a variety of metabolic functions associated with disc pathology, including proteoglycan degradation (96, 97). Furthermore, the decrease in osmotic pressure observed during herniation in our canine patients may also play a causative role in MAPK activation as previously demonstrated for bovine IVDs and the ERK pathway (101). Importantly, activation of p38 and ERK in our canine samples may not only induce downstream expression of additional inflammatory mediators and matrix degrading enzymes and hence play an important role in disease progression [reviewed in Ref. (27)], but may furthermore be a means to initiate resorption of non-contained herniated disc tissue (102–104).

Despite providing interesting new findings on the inflammation in canine IVDH, the present study has several limitations that should be considered. The dog size as well as the morphologic size of the herniated disc tissue imposed limitations on the number of performed tests. The quantity of surgically excised canine disc material is smaller than specimens collected in humans. Therefore, we could not divide all samples equally for PCR and ELISA testing as planned in the study design, resulting in variations in group size. Due to the non-predictable onset of canine IVDH, the group could neither be age nor gender matched, possibly influencing the results. Furthermore, most of our patients were presented at a late stage of the disease, which may explain why we might have missed the peak expression of some cytokines in the acute stage of the disease.

# CONCLUSION

Inflammation in the epidural compartment plays a central role in the pathophysiology of canine IVD herniation and significantly influences the course and outcome of the disease. Working with a clinical canine model not only provides the possibility to overcome animal experimentation, but furthermore allows (different from human studies) the analysis of adjacent, NA discs. Although expression of key cytokines found in human IVDs could also be demonstrated in canine tissue, the inflammatory mechanisms accompanying IVDH in dogs partially diverge from humans. These differences need to be considered when using dogs as a model for human medicine. In dogs, IL-6 seems to play an important pathological role, but further investigations on IL-6 as a potential therapeutic target in canine patients will be needed, especially when considering its likely ambivalent role.

#### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of "Animal Research Committee: Tierversuchsbewilligung BE 14/12, Switzerland." The protocol was approved by the "Animal Research Committee: Tierversuchsbewilligung BE 14/12, Switzerland." Furthermore, all patient-owners signed an owner consent form.

#### AUTHOR CONTRIBUTIONS

MM contributed to the conception and design, acquisition and data, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, and statistical analysis. SF contributed to the analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, and the statistical analysis. DS contributed to the conception and design, critical revision of the manuscript and intellectual content, funding, and final approval of the version to

#### REFERENCES

be published. AK contributed to acquisition and data, interpretation and analysis of data, critical revision and administrative, technical, or material support. FF and KW-K contributed equally to the conception and design, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, obtaining funding, administrative, technical or material support, supervision, and final approval of the version to be published. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. We thank Prof. Dr. Michael Stoffel (Vetsuisse Bern, Veterinary Anatomy) for his support with histology.

#### FUNDING

The study was funded and supported with 37,500 \$ by The Competence Center for Applied Biotechnology and Molecular Medicine Start-up Grant of the University of Zurich, Switzerland. Heel GmbH financially supported the salary of MM.

by the nucleus pulposus. *Eur Spine J* (2012) 21:537–45. doi:10.1007/ s00586-011-2027-8

intervertebral disc extrusion in a canine model. *Spine J* (2014) 14:2976–84. doi:10.1016/j.spinee.2014.05.021

macrophage polarization in intervertebral disc degeneration. *Sci Rep* (2016) 6:22182. doi:10.1038/srep22182

104. Park JJ, Moon HJ, Park JH, Kwon TH, Park Y-K, Kim JH. Induction of proinflammatory cytokine production in intervertebral disc cells by macrophage-like THP-1 cells requires mitogen-activated protein kinase activity. *J Neurosurg Spine* (2016) 24:167–75. doi:10.3171/2015.3.SPINE14729

**Conflict of Interest Statement:** KW-K (08/03/2017) Relationships Pertaining to Submitted Manuscript—Money payed to the institution: 37.500 \$ (CABMM Start-up Grant) Relevant financial activities outside the submitted work— Consulting Money payed to you: 85.000 \$ (Schön Clinic Group). All other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.