**1. Introduction**

p38α was identified by three groups in 1994 as a 38 kDa polypeptide that was phosphorylated after exposure to lipopolysaccharide (LPS), hyperosmolarity, or interleukin 1 (IL-1), that directly phosphorylates and activates the upstream kinase MAPKAP-K2 [1–3]. Later, three additional isoforms were described: p38β [4], p38γ (also called SAPK3 and ERK6) [5], and p38δ (also called SAPK4) [6]. The p38 family members are encoded by different genes, located tandemly in two chromosomes. The p38β (*Mapk11*) and p38γ (*Mapk12*) genes are located together on one chromosome (15 in mice and 22 in humans), whereas p38δ (*Mapk13*) and p38α (*Mapk14*) genes are located on another (17 in mice and six in humans). *Mapk12* is proposed to have arisen from tandem duplication of *Mapk11*, and the *Mapk13-Mapk14* gene unit is thought to have originated in a segmental duplication of the *Mapk11-Mapk12* unit [7].

The p38 family can be subdivided into two subsets, with p38α and p38β in one group and p38γ and p38δ in the other. This classification is based partly on amino-acid sequence identity; p38α and p38β are 75% identical, whereas p38γ and p38δ are 62% and 61% identical to p38α, respectively, while sharing 70% sequence identity with each other. The two p38 subsets also differ in their susceptibility to inhibitors, with in vitro and in vivo assays demonstrating that only p38α and p38β are inhibited by pyridinyl imidazoles (SB202190 and SB203580). A third difference between the p38 subgroups is substrate selectivity, with p38γ sharing common substrates with p38δ, and p38α with p38β [8–10].

p38 activity is regulated by phosphorylation at the end of a cascade composed of a MAPK kinase (MKK) and an MKK kinase (MEKK) [11–13]. The cascade is initiated by one of several MKK-phosphorylating MAP3Ks in cell-type- and stimulus-dependent manner. These MAP3Ks include mixed-lineage kinases (MLK), TGF β-activated kinase 1 (TAK1), MAPK/ERK kinase kinases (MEKK), TAO1 and TAO2, and apoptosis signal-regulating kinase-1 (ASK1) [14]. The p38s are activated by MKK-mediated dual phosphorylation of tyrosine and threonine residues in the conserved Thr–Xaa–Tyr motif (in p38, kinases Xaa is glycine, whereas in JNKs, it is proline in ERKs glutamic acid) [14]. Phosphorylation by MKKs is highly selective due to the specificity of the phosphorylation motif and the interaction of the MKK N-terminal region with di fferent docking sites on the p38s. In addition, in T cells, p38 is activated by autophosphorylation [15] and also through AMPK-TAB1 [16], an alternative pathway that has been shown also in adipose tissue [17].

p38 activation is further tightly regulated by a group of inactivating phosphatases [11,14]. All p38 family members are widely expressed and considered ubiquitous, although p38β is most abundantly expressed in brain and adipose tissue, p38 γ in skeletal muscle, and p38δ in secretory glands [5,6,18,19]. While all four p38s are expressed in the heart, the predominant family members in cardiomyocytes are p38 α and p38 γ. Extensive research into cardiac p38 function has suggested both protective and deleterious roles in the stressed myocardium. Which outcome predominates seems to depend in part on the model system under study and on the identity of the activated p38 family member. However, understanding remains limited of how the di fferent p38 family members control cardiac physiology and respond to cardiac stress. In this review, we summarize current knowledge of p38 function in the heart and discuss recent advances.

## **2. Cardiovascular Development**

In 2000, three groups independently showed that p38 α is essential for normal cardiovascular development. Allen M. et al. demonstrated that genetic disruption of the p38 α gene *Mapk14* was embryonically lethal [20]. Four months later, Adams R. et al. confirmed the essential requirement for p38 α during early mouse development, showing that p38 α deletion correlated with a massive reduction in myocardium formation and the appearance of blood-vessel malformations in the head region [21]. These authors suggested that p38 α is necessary for placental organogenesis but is not necessary for other aspects of mammalian embryonic development [21]. Mudgett, J. et al. showed that p38 α is required for the vascular remodeling associated with placental angiogenesis and trophoblast development [22].

Although p38 has been shown to play a key role in skeletal muscle development [23], less attention has been paid to its role in cardiac development. Several in vitro studies point to a possible role of p38 in cardiac development. For example, p38 α activity is required for cardiomyocyte di fferentiation of P19CL6 cells, which is mediated via the activation of the transcription factor AP-1 [24]. p38 α has also been shown to promote cardiogenesis over neurogenesis in ES cells [25]. Unfortunately, despite the strong suggestion of a cardiogenic role of p38 α from cell-culture studies, in vivo data supporting this hypothesis are scarce. While embryos lacking p38 α die due to defects in placental angiogenesis, cardiac-specific deletion of p38 α results in normal development of the heart [26].

Several studies have shown that p38 kinases play an important role in di fferent aspects of cardiogenesis, such as the regulation of cardiomyocyte di fferentiation and apoptosis. The role of p38 in cardiomyocyte di fferentiation was first suggested by studies using a specific inhibitor of p38 α and p38β (SB203580), which demonstrated that p38 activity regulates important mitotic genes in cardiomyocytes. Neonatal mice lacking p38 α have increased cardiomyocyte mitosis, suggesting that p38 α acts as a negative regulator of cardiomyocyte proliferation 2. In adult cardiomyocytes, SB203580 and fibroblast growth factor 1 (FGF1) act synergistically to induce the expression of genes involved in proliferation and regeneration [27,28], indicating that the combination of FGF1 stimulation and p38 α inhibition might rescue cardiac structure and function after injury [28]. The importance of p38 in cardiac di fferentiation was evident by the finding that p38 α inhibition or gene deletion were su fficient to block cardiomyogenesis, suggesting that p38 α activation constitutes an early switch in embryonic stem cell commitment to cardiomyogenesis [25]. The deletion or inhibition of p38 α reduces expression of myocyte enhancer factor 2C (MEF2C), an important transcription factor acting on many genes encoding cardiac structural proteins [29]. The inhibition of p38 correlates with decreases in other cardiac transcription factors and MEF2C targets, such as atrial natriuretic factor (ANF) and myocardin, all of which contribute to the proper activation of the cardiac differentiation program during the early stages of development. p38α also regulates sarcomere assembly through the phosphorylation of ventricular myosin light chain 2 (MLC-2v), as well as the accumulation of α-actinin and its incorporation into sarcomeric units [29]. The lack of MEF2C activation upon p38 inhibition suppresses the expression of bone morphogenetic protein 2 (BMP-2), a key regulator of early cardiac cell development [30]. Most studies of cardiovascular development have focused on p38α; however, it is important to also define the role of other p38 family members in cardiac development. Mice with combined deletion of p38α and p38β display diverse developmental defects at mid-gestation, together with major cardiovascular abnormalities [31]. Embryos that express p38β only under the control of the p38α promoter display a similar heart phenotype as the double-knockout embryos, suggesting that heart development requires endogenous p38β expression [31]. Moreover, p38α and p38β have synergistic roles and specific functions in the regulation of cardiac gene expression during development, suggesting that some specific functions could be explained by differences in expression patterns [31]. It has been demonstrated the selective activation of p38 in the right ventricle during neonatal development and simultaneous inactivation in the left ventricle in neonatal mouse heart [32]. Cardiac-specific deletion of p38α and p38β in mice showed an abnormal gross morphology of the heart, developed right ventricle-specific enlargement dilation and, in consequence, a significant increase in cardiomyocyte proliferation, hypertrophy and a reduction in apoptosis without changes in the left ventricle. Furthermore, p38 inactivation induces XBP1 activity via IREα in the regulation of neonatal cardiomyocyte proliferation [32]. Finally, the role of p38γ and p38δ in cardiomyocyte development have not been assessed; however, animals lacking these kinases have smaller hearts at birth while conserving a normal number of cardiomyocytes, suggesting that these kinases contribute to the control of cardiomyocyte hypertrophy [33].

## **3. Cardiac Hypertrophy**

The role of p38 kinases in cardiac hypertrophy was first suggested by the hypertrophic responses induced upon overexpression in cardiomyocytes of active forms of the upstream activators MKK3 and MMK6 [34–36]. The p38 pathway is also activated in cardiomyocytes exposed to hypertrophic stimuli, and hypertrophic growth is blocked by the SB203580-mediated inhibition of p38α/β [35]. However, MKK3 overexpression in cardiomyocytes also increased apoptosis [34]. The differences between the effects of MKK3 and MKK6 might point to distinct roles of p38 family members, a possibility supported by the finding that p38β activation in cultured cardiomyocytes induces characteristic features of hypertrophy [36], whereas p38α activation promotes cardiomyocyte apoptosis [37]. Consistent with this result, cardiac-specific knockout revealed a critical role for p38α in the cardiomyocyte survival pathway triggered by pressure overload, whereas hypertrophic growth was unaffected [37]. Although some groups have reported a role of p38α in the regulation of cardiac hypertrophy, most of those studies were based on the non-physiological overexpression of dominant-negative p38α or indirect strategies that might alter the function of other p38 family members [38,39].

The cardiomyocyte expression and subcellular localization of p38 isoforms was characterized by Dharmendra Dingar et al. in physiological conditions and in response to chronic pressure overload induced by transverse aorta constriction (TAC) [40]. TAC did not induce changes in the amount of p38α mRNA, whereas p38β and p38δ mRNA increased within 1 day of pressure overload and remained, while p38γ mRNA increased initially before returning to baseline levels by day 7. Despite these increases in mRNA abundance, the overall protein levels of p38β, p38γ, and p38δ were unaltered [40]. Confocal immunofluorescence analysis detected p38α and p38γ in the cytoplasm and nucleus at baseline; however, after chronic pressure overload, p38γ accumulated in the nucleus, whereas p38α distribution was unaffected. These localization differences would result in access to different substrates, and hence distinct functional effects [40].

The lack of pharmacological inhibitors of p38γ and p38δ has limited the study of these family members, though they were recently shown to control physiological and pathological cardiomyocyte growth. p38γ and p38δ are activated by angiotensin II and phosphorylate the mTOR inhibitor DEPTOR, inducing its degradation by the proteasome. Once DEPTOR is degraded, mTOR is released and activated, triggering protein synthesis and cardiomyocyte growth [33]. Mice lacking p38γ and p38δ have smaller hearts than controls and a reduced cardiomyocyte area [33]. Reduced hypertrophy capacity in these mice resulted in partial protection against angiotensin treatment [33]. Further experiments are needed to assess the therapeutic effects of p38γ and p38δ modulation during pathological hypertrophy.

## **4. Cardiac Regeneration**

The best model for studying the molecular mechanisms of cardiac regeneration is the zebrafish, in which injury induces a cardiomyocyte proliferation that can overcome scar formation, thus allowing cardiac muscle regeneration [41]. Little attention has been paid to the role of p38 in zebrafish cardiac regeneration. p38α activation negatively regulates the proliferation of adult zebrafish cardiomyocytes [42], as also occurs in mammals [27]. During heart regeneration in adult zebrafish, the induction of p38α activity blocks cardiomyocyte proliferation, suggesting that p38α activity must be switched off in order to trigger cardiomyocyte proliferation and myocardial regeneration [42].

Mammalian hearts have a very low or non-existent regenerative capacity after cardiac injury. Nevertheless, in principle, signals that acutely trigger cardiomyocyte survival or modulate myoblast activity could be manipulated to promote cardiac regeneration and avoid heart failure. There is evidence implicating FGF1-upregulated genes in cardiac regeneration and cell-cycle control. The inhibition of p38 and stimulation of FGF1 act together to induce the expression of specific genes involved in proliferation and regeneration, such as cytokinesis regulator Ect2, cell-cycle-regulated protein 1 (CRP1), Ki67, cdc2, cyclin A, and the cell-cycle inhibitor p27 [27] (Figure 1A). Moreover, p38 inhibition and FGF1 induction lead to the phosphorylation of the key cell-cycle regulator Rb. These findings suggest that the promotion of cardiomyocyte proliferation by combined treatment with FGF1 and a p38 inhibitor could provide an alternative approach to the rescue of cardiac function after injury [28]. Studies of p38 isoforms in muscle regeneration have shown that p38β, p38γ, and p38δ are not required for efficient adult muscle regeneration and growth after injury [43], suggesting that p38α, in the absence of the other isoforms, especially p38γ, is sufficient to maintain satellite-cell-mediated myogenesis in vivo and in vitro [43].

The actions of p38 kinases in skeletal muscle regeneration have received much more attention than their roles in the heart. However, given the similarities between cardiac and skeletal muscle, the results of skeletal muscle studies can shed valuable light on the role of p38 in cardiac regeneration. Much research has focused on the potential role in adult myogenesis of p38γ, which is highly expressed in skeletal muscle. p38γ-deficient mice have a low muscle regeneration capacity after injury, with a reduced number of satellite cells that express myogenin prematurely and proliferate poorly. Mechanistically, p38γ phosphorylates MyoD, enhancing its occupancy of the myogenin promoter and thereby suppressing its expression. p38γ thus acts in opposition to p38α, blocking premature differentiation by inducing a repressive MyoD transcriptional complex during satellite-cell–mediated muscle growth and muscle regeneration [44] (Figure 1B). MyoD activates proliferation-associated genes but not differentiation genes, whose regulatory regions are repressed by ZEB1. Macrophages present in the injured muscles of Zeb1-deficient mice have low phosphorylation levels of p38, and forced p38 activation alleviates muscle damage and improves muscle regeneration [45]. In a later study, Brien P et al. demonstrated that a lack of p38α results in increased p38γ activation [46], suggesting that p38γ hyperactivation is involved in muscle regeneration.

**Figure 1.** p38 in cardiovascular regeneration. **(A)** p38α blocks cardiovascular regeneration by inhibiting the expression of genes involved in cardiomyocyte proliferation and regeneration, such as *Ect2, Crp1, ki67, cdc2, cyclin A*, and p27, and reducing Rb phosphorylation to block cell-cycle progression. FGF1 stimulation has the opposite effect. **(B)** p38γ activates differentiation and myogenesis in satellite cells by phosphorylating MyoD and activating proliferation. p38α prevents p38γ activation in satellite cells, blocking regeneration. ↑ increase, ↓ decrease.

Aging is characterized by a general decline in metabolic activity and function, with a loss of both skeletal and cardiac muscle accompanied by marked functional and structural impairment. Skeletal muscle has an outstanding regenerative capacity provided by its resident satellite cells. These normally quiescent cells are activated after injury to promote skeletal muscle regeneration [47]. Cardiomyocytes and cardiac muscle have long been thought to have lost these satellite cells and appear to lack the capacity for self-renewal and repair, preventing full recovery. Given the fundamental role of p38 in skeletal muscle regeneration, it is reasonable to postulate a causal link between cardiac muscle loss-of-function and p38.

To identify potential regeneration strategies based on satellite cells, a number of studies have investigated the effect of manipulating p38 signaling on aged satellite cells. For example, the manipulation of satellite cells enhances fibroblast growth factor receptor 1 (FGFR1) signaling and reduces p38α/β activation in satellite cells, increasing self-renewal and stimulating skeletal muscle regeneration [48]. Another study improved skeletal muscle repair and regeneration in rats using gold and gold-silver nanoparticles (AuNPs and Au-AgNPs, respectively) [49]. These nanoparticles regulate MyoD gene expression and activate p38α signaling, enhancing myoblast myogenic differentiation and promoting skeletal muscle regeneration. The effects of AuNPs and AuAgNPs were blocked with SB203580, suggesting that p38α is essential for myogenic differentiation [49]. These studies indicate an important role for p38 signaling in muscle regeneration; however, the underlying molecular mechanisms need further investigation to define the antagonic roles between p38α and p38γ. It will also be important to determine whether this antagonistic relationship operates in cardiac muscle.

#### **5. p38 in Ischemia–Reperfusion Injury**

Ischemic heart disease, the leading cause of death worldwide, is normally produced by a coronary artery occlusion that impairs cardiac blood flow. The ensuing decrease in oxygen delivery can lead

to myocardial infarction. Reperfusion consists of the restoration of blood flow after the ischemia, and although this step is essential to avoid cardiomyocyte death, it also causes further damage associated with increased oxidative stress and inflammation [50,51]. Short ischemic episodes have been shown to protect the heart from a later ischemic insult, a process known as preconditioning or postconditioning, depending on whether it happens before or after the ischemia [52].

The p38 pathway is activated in response to ischemia–reperfusion and during preconditioning, producing a variety of results and cardiovascular scenarios [53]. In perfused rat hearts, p38 activation during ischemia and reperfusion is associated with a poor cardiac outcome. In contrast, during repetitive preconditioning treatments, p38 is maximally activated in the first episode, and activation gradually is reduced during sustained ischemia–reperfusion, improving cardiac functional recovery [53]. Interestingly, p38 activation is compartmentalized, with ischemia activating p38 in mitochondria, whereas during reperfusion p38 is activated in all cell compartments [54]. It would be interesting to determine whether different p38 family members are activated and localized in different cell compartments during these processes. Experiments performed in PC12 cells showed that moderate hypoxia (5% O2) increases p38γ and p38α phosphorylation, suggesting that ischemia might activate more than one p38 family member [55]. Most studies have examined p38α and β, both of which are inhibited by the widely used inhibitor SB203580. There is evidence to suggest that these two kinases have opposite roles, with p38α activation during ischemia triggering apoptosis, whereas p38β is responsible for pro-survival signaling during preconditioning [56]. Therefore, SB203580-mediated blockade of both isoforms during preconditioning results in loss of cardioprotective effects, whereas inhibition during I/R is beneficial [53].

The activation of p38 during preconditioning seems to be a consequence of adenosine release, which triggers the opening of ATP-sensitive potassium channels (KATP) during hypoxia [57,58]. The cardioprotective effects of p38 are thought to be due to phosphorylation of the downstream target Hsp27 and the subsequent enhancement of cytoskeletal stabilization during hypoxic stress [59]. In line with this idea, reactive oxygen species (ROS)-induced activation of p38α during hypoxia stabilizes hypoxia-inducible factor 1 (HIF-1) [60]. Moreover, the lack of dual specificity protein phosphatase 4 (DUSP4) leads to p38 hyper-phosphorylation and apoptosis [61]. Preclinical and clinical studies [62–64] with antioxidants highlight the importance of ROS as mediators of cardiac stress injury during I/R. ROS are potent p38 activators, but in vitro results with HL-1 cardiomyocytes also showed that p38 activation drives elevated ROS levels during ischemia–reperfusion [65]. In this analysis, the damaging effects of ROS were abolished by the p38 pan inhibitor BIRB796 [65]. Moreover, p38 inhibition with an antioxidant during ischemia–reperfusion is associated with improved cardiac recovery, decreased infarct size, and reduced apoptosis [66]. This was related to increased endogenous anti-oxidative enzyme activity and inhibition of oxidative stress [67]. The antioxidant Peroxiredoxin 1 and the ROS scavenger N-acetyl-l-cysteine have been also shown to decrease oxidative stress and block the activation of p38 and JNK, thus reducing apoptosis during ischemia–reperfusion [68]. Moreover, oxidative stress can directly regulate p38α activity and protein interactions by affecting the oxidation state of cysteines. Treatment with H2O2 induced p38-MKK3 disulfide dimer formation in isolated rat hearts and in an ischemia–reperfusion model, and dimer formation was abolished when the redox-sensitive cysteines were mutated or sterically inaccessible [69]. Further research is needed to determine whether p38 activation triggers ROS production, as proposed by Ashraf et al. [53,65], whether ROS induce p38 activation, or, more likely, there is reciprocal ROS–p38 regulation.

p38 has also been linked to cardiac inflammation through its promotion of the expression of regenerating islet-derived 3γ (Reg3γ), a protein associated with cardiac inflammatory signaling [70]. Moreover, treatment with the anti-inflammatory compound gamboge protects against infarction-induced inflammation by targeting the NF-κB–p38 pathway [71]. p38α activation is also associated with increased fibrosis during ischemia-induced cardiac remodeling [72]. Supporting this idea, inhibition of the p38 substrate MK2 impairs fibrotic scar formation after myocardial infarction [73]. The profibrotic effect of p38α was confirmed by conditional p38α deletion in myofibroblasts, which demonstrated that a lack of p38 α blocks cardiac fibroblast di fferentiation into myofibroblasts, reducing fibrosis in response to ischemic injury [74]. p38 pathway activation by MKK6 overexpression results in interstitial and perivascular cardiac fibrosis [74], and p38 inhibition may underlie the beneficial e ffects of some statins on cardiac remodeling after myocardial infarction [75,76]. Supporting this idea, protease inhibitors induce cardioprotection in models of ischemia–reperfusion, in part by attenuating p38 phosphorylation, leading to reductions in injury, ROS levels, and infarct size [77].

The ischemia–reperfusion injury response is crucially determined by mitochondrial function and activity because mitochondria control cell metabolic status, intracellular calcium influx, oxidative stress, and apoptotic pathways, among other processes. p38 inhibition during ischemia–reperfusion decreases mitochondrial swelling, protects against ultra-structure alterations, and mitigates mitochondrial membrane depolarization [78]. There is also evidence that p38 activation during ischemia–reperfusion contributes to cardiac damage by triggering intracellular Ca2+ overload [79]. Pharmacological inhibition of p38 during ischemia–reperfusion induces cardioprotection by promoting phospholamban phosphorylation, increasing the activity of sarcoplasmic reticulum Ca2+-ATPase (SERCA2), and decreasing Ca2+ overload [80]. The role of p38 in the control of intracellular Ca2+ was corroborated in H9c2 cells: phosphodiesterase-inhibitor–mediated reduction in ERK1/2, JNK, and p38 activation reduced ischemia-induced apoptosis and restored normal calcium influx, oxidative stress levels, and eNOS expression [81]. p38 inhibition also potentiates the metformin-induced reduction in myocardial ischemia–reperfusion injury in non-obese type 2 diabetic rats [82].

Ischemia–reperfusion injury also increases the risk of arrhythmia. p38 inhibition with SB203580 decreases ventricular tachycardia and ventricular fibrillation when administered to adult Wistar rats before or during ischemia, but not at the onset of reperfusion [83]. Mechanistically, this could be due to the ROS-dependent activation of p38 by ASK1 [84]. ASK1 would respond to the moderate increase in ROS during ischemia, but not to the higher levels of ROS observed in ischemia/reperfusion, acting as a redox sensor to mediate ROS-dependent signaling to p38 [84]. These findings highlight the importance of determining the optimal timing of p38 inhibition in order to achieve an e fficient therapeutic response (Figure 2).

**Figure 2.** Dual role of p38 activation during preconditioning and ischemia–reperfusion injury. Activation of p38β during preconditioning triggers pro-survival signaling pathways, whereas decreased p38α activation during the ischemic episode leads to cardioprotection. On the other hand, ROS-induced p38α activation during the ischemic insult triggers HIF1-α stabilization; increases (↑) fibrosis, arrhythmias, and inflammation; and disrupts mitochondrial homeostasis. SB203580 administration during preconditioning increases myocardial injury, whereas administration during ischemia–reperfusion improves cardiac outcome. Indirect p38 downregulators, such as gamboge, statins, and antioxidants, seem to have beneficial e ffects when administered during or after the ischemia. Further research is needed to determine the precise reciprocity of ROS–p38 regulation. ↑ increase.

#### **6. p38 in Heart Failure and Cardiac Arrhythmia**

Heart failure (HF) is a major cardiac pathology and a global pandemic that a ffects more than 37 million people worldwide [85,86]. The p38 pathway is activated in HF, and specifically in the pathological cardiac remodeling that can lead to cardiac arrhythmia in the failing heart [87–90]. p38 plays an important role in the regulation of cardiac remodeling and cardiac contractility. Most studies suggest a negative role of p38 activation in extracellular matrix remodeling and the development of cardiac fibrosis, processes related to the development of HF [91–93]. Studies using transgenic animals with cardiac-specific expression of the activated p38 upstream kinases MKK3bE and MKK6bE showed that p38 pathway activation promotes cardiac interstitial fibrosis and increased expression of embryonic gene markers, similar to the expression profile observed in HF [89,94]. The profibrotic e ffect of p38 activation may be due to the induction in cardiomyocytes of TNFα and IL-6 [89], which are closely associated with the development of fibrosis, adverse cardiac remodeling, and HF [95,96]. The e ffect of p38 activation on cardiac fibrosis is not limited to cardiomyocytes and also a ffects cardiac fibroblasts. The specific activation of the p38 pathway in cardiac fibroblasts leads to maladaptive cardiac remodeling with a profibrotic and hypertrophic phenotype and the activation of TGF-β signaling [97], a key cytokine involved in cardiac fibrosis and HF [95,96,98,99]. p38 is also necessary for the di fferentiation of fibroblasts into myofibroblasts, and specific deletion of p38 α in cardiac fibroblasts or myofibroblasts reduces cardiac fibrosis in response to cardiac injury [74]. This is consistent with results showing that p38 inhibition decreases cardiac fibrosis and pro-inflammatory cytokine production [74,90], suggesting that p38 blockade is a possible treatment in HF. However, mice with cardiac-specific p38 α deletion have a worse outcome to TAC-induced pressure overload, characterized by extensive cardiac fibrosis, dysfunction, and dilatation [37]. These opposite results might indicate that another family member is responsible for the protective e ffects of inhibitors or that p38 has opposite roles in cardiomyocytes versus other cardiac cells. Moreover, by promoting non-specific phosphorylations, overexpression of activated kinases may produce artificial cardiac structural and functional phenotypes. Further research is needed to determine the specific roles played by the di fferent p38 family members in cardiac fibrosis and HF, since most studies have focused on p38 α or the p38 pathway in general.

p38 can also control cardiomyocyte contractility, a predominant target of therapeutic strategies to treat HF [100]. The existing evidence indicates that p38 activation has an anti-inotropic e ffect in cardiac muscle [101–106]. Di fferent mechanisms have been proposed for the negative e ffect of p38 on cardiac contractility. For example, p38 has been proposed to mediate the anti-inotropic effects of angiotensin II and ROS, which are also increased during HF, desensitizing the response of myofilaments to Ca2+ [102,105,107]. The mechanism underlying p38-mediated dampening of Ca2+ responsiveness is unknown, but two main possibilities have been proposed: modification of intracellular pH or phosphorylation of contractile proteins. However, studies have disproved the involvement of pH modification in myofilament sensitivity to Ca2+ [105,107], leaving phosphorylation of contractile proteins as the more likely mechanism. Analysis by Liao et al. did not detect increased p38-mediated troponin I phosphorylation, which is known to reduce myofilament responsiveness to Ca2+ [102]. Later work by Vahebi et al. showed that, rather than phosphorylation, p38 activation promotes the dephosphorylation of α-tropomyosin and troponin I. This was accompanied by a depression of cardiac and myofilament function and a decrease in maximum ATPase activity [106]. In agreement with this finding, p38 inhibition was found to promote troponin I phosphorylation [108]. p38-mediated dephosphorylation of α-tropomyosin and troponin I appears to be mediated by the protein phosphatases PP2Cα and PP2C-β, since p38, PP2Cα, and PP2C-β were found in the same protein complex in the sarcomere [106].

Another mechanism through which p38 might affect cardiac contractile function is the regulation of proteins involved in cardiomyocyte Ca2+ handling. During cardiac contraction, a depolarizing action potential promotes Ca2+ release from the sarcoplasmic reticulum, a process known as excitation-contraction coupling. Ca2+ enters the cell via L-type Ca2+ channels and, in much lower amounts, via the Na+/Ca2+ exchanger (NCX) [109]. This Ca2+ activates further sarcoplasmic reticulum Ca2+ release via the

Ca2+-triggered Ca2+ release channel ryanodine receptor (RyR). The incoming cytosolic Ca2+ binds to the thin-filament protein troponin C, initiating myocyte contraction. For relaxation, calcium is removed from the cytosol by the action of Ca2+ transporters, mainly the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) and sarcolemmal NCX [109]. Human HF is characterized by the reduced expression or activity of SERCA2 [110], resulting in increased diastolic Ca2+ concentrations due to defects in cytosolic Ca2+ removal, leading to reduced contractile force and impaired relaxation [110]. SERCA2 expression and activity are affected by p38 activation. In rat cardiomyocytes, p38 activation reduces SERCA2 mRNA expression and protein levels and reduces the activity of the SERCA2 gene promoter [111,112]. Scharf et al. showed that MK2/3 deletion results in increased protein levels and activity of SERCA2 in the heart and that SERCA2 gene expression is regulated by MK2-dependent Egr-1 transcription factor expression and promoter binding [113]. The p38 pathway regulates not only SERCA2 expression, but also its activity. For example, p38 inhibition increases the inotropic effect of endothelin-1 (ET-1) by modifying the SERCA2 inhibitory protein phospholamban (PLN). ET-1 treatment induces p38 phosphorylation [110,114] and promotes PLN phosphorylation at Ser-16 in the presence of p38 inhibition [80]. Similar results were obtained in a model of ischemia–reperfusion, which activated p38 and reduced SERCA2 expression and activity, as well as PLN phosphorylation, whereas these effects were partially reversed by p38 inhibition [113]. In MK2/3 double knockout mice, PLN phosphorylation is increased in the heart at the Ca2+/calmodulin-dependent protein kinase II (CaMKII) site (Thr-17) [113]. In the dephosphorylated state, PLN binds to and inhibits SERCA2. Phosphorylation at Ser-16 or Thr-17 relieves this inhibition, promoting Ca2+ reuptake to the sarcoplasmic reticulum and thus increasing cardiac contractility [115]. By promoting PLN dephosphorylation, p38 activation can therefore limit cardiac contractility. Although the main phosphatase responsible for PLN dephosphorylation is protein phosphatase 1 (PP1), PLN dephosphorylation can also be mediated by protein phosphatase 2A (PP2A) [116,117]. p38 activation has been reported to promote PP2A translocation and activation in myocytes [118], and p38 inhibition reduces PP2A-mediated dephosphorylation of PLN [108,118]. Furthermore, PP2A activation could be the mechanism by which p38 activation inhibits the β-adrenergic receptor-mediated contractile response in cardiomyoctes [104,118]. Kaikkonen et al. also proposed that decreased PP2A activity upon p38 inhibition could also promote PP1 inhibition, and that the p38 isoform responsible for these effects on SERCA2 regulation would likely be p38 α [108]. Given that p38 also affects the activity of PP2Cα and PP2C-β, is feasible that p38 also affects the activity of PP2Ce, a novel member of the PP2C family that has been reported to be a specific and potent PLN phosphatase [116].

p38 α also appears to participate in α-adrenergic-mediated *Ncx1* gene upregulation [119]. The overexpression of active MKK3 and MKK6 was su fficient to induce NCX1 upregulation in isolated cardiomyocytes, and this e ffect was mediated primarily by p38 α [119,120]. Additionally, chemical inhibition of NCX1 promotes the formation of an NCX1-p38 complex and p38 activation. p38 activation induced by NCX1 inhibition has been suggested to be a physiological mechanism to compensate for loss of NCX1 activity by promoting *Ncx1* gene expression [121]. NCX1 activity and expression is also increased in heart failure [110] and the increased NCX1 activity increases Ca2+ extrusion to preserve the reduced diastolic Ca2+. This may compensate in part for the reduced SERCA2 function [110]; however, it can also promote other negative e ffects on cardiac contractility. For example, high NCX1 activity increases Ca2+ release from the cell, reducing sarcoplasmic reticulum Ca2+ stores and inducing contractile dysfunction. The elevated translocation of Ca2+ across the plasma membrane also results in a higher risk of delayed afterdepolarizations, which can cause arrhythmia and sudden death [110,122]. By reducing SERCA2 and increasing NCX1 function, p38 would seem to play an important role in the development of cardiac arrhythmia. Indeed, p38 activation has been linked to arrhythmogenic cardiac and ionic channel remodeling [83,87,88,123–125]. The pharmacological inhibition of p38 reduces the incidence of arrhythmia after ischemia–reperfusion by increasing the levels of phosphorylated connexin 43 (Cx43) [83]. Connexin clusters in the plasma membrane form gap junctions, which regulate cell-to-cell electrical and metabolic coupling and are essential for normal cardiac impulse transmission [126]. Cx43 is the most abundantly expressed connexin in cardiac

myocytes, and its alteration has been linked to increased susceptibility to cardiac arrhythmia by altering action potential propagation in the heart [126,127]. There is strong evidence indicating a role for p38 in the regulation of Cx43. Several processes are involved in Cx43 regulation, including synthesis/degradation, phosphorylation/dephosphorylation at di fferent residues, and cell membrane localization [128]. Cx43 expression is increased by p38 activation induced by several stimuli [129–133], but p38 is also implicated in Cx43 degradation [134]. The e ffect of p38 activation on Cx43 expression might depend on the activating stimulus and the cell type being studied. In cardiomyocytes, p38 activation appears to promote an increase in Cx43 mRNA and protein expression [129–133]. p38 can form a complex with Cx43 [135,136], and its activation has been reported to promote both Cx43 phosphorylation [135,137,138] and dephosphorylation (via PP2A activity) [136]. Adding further complexity, the increased phosphorylation of Cx43 upon p38 activation has been suggested to promote Cx43 degradation [137]. More research is needed to clarify the role of p38 activation in the regulation of Cx43 and gap junctions. However, despite the varying e ffects of p38 activation on Cx43 phosphorylation, the outcome of p38 activation is consistent across studies. p38-induced phosphorylation and dephosphorylation of Cx43 both lead to reduced cell-to-cell communication, impaired propagation of the action potential, and the development of cardiac arrhythmia [136–138]. Furthermore p38, inhibition improves cell-to-cell communication and reduces the incidence of arrhythmia [83,138].

Many aspects of the role of p38 in the development of HF and cardiac arrhythmia remain to be clarified; however, most of the evidence points to a negative e ffect of p38 activation on the onset of HF and arrhythmias. The mechanisms involved include the development of cardiac fibrosis, alterations to Ca2+ handling proteins, and the modulation of gap junctions in the cardiomyocyte (Figure 3). Future research will need to address the role of the di fferent p38 family members in these processes, since most studies have focused on p38 α or p38 α/β.

**Figure 3.** p38 in heart failure and cardiac arrhythmia. p38 activation participates in the development of heart failure and cardiac arrhythmia through three main mechanisms: **(A)** Increased cardiac fibrosis by induction of TNF-α and IL-6 in cardiomyocytes, di fferentiation of fibroblasts, and induction of TGF-β in cardiac myofibroblasts; **(B)** Reduced cardiac contractility due to dephosphorylation of a-tropomyosin and troponin I via PP2C-α/PP2C-β; **(C)** Promotion of cardiac arrhythmias due to reduced expression and activity of SERCA2 (*Atp2a2*), increased expression of NCX1 (*Ncx1*) and Cx43 (*Cnx43*), and altered Cx43 phosphorylation, inducing cardiac contractile dysfunction and altered action potential propagation. ↑ increase, ↓ decrease.

#### **7. p38 Inhibitors in Clinical Trials**

Despite the abundance of experimental evidence for the potential benefits of p38 inhibitors, clinical trials have failed to show improved cardiac outcomes after ischemia–reperfusion. The new anti-inflammatory medication losmapimod inhibits p38, and its administration to patients with non-ST-segment elevation myocardial infarction was well tolerated and improved the cardiac outcome [139]. However, in another study in acute myocardial infarction patients, losmapimod did not reduce the risk of major ischemic cardiovascular events, resulting in the withdrawal of the clinical trial [140].

Alternative approaches to p38 inhibition have been suggested in order to avoid undesirable side e ffects. The inhibition of MK2 in activated rheumatoid arthritis fibroblast-like synoviocytes avoided the modification of the secretion of chemokines like TNF-alpha, normally associated with the activation of other pro-inflammatory pathways like ERK and JNK during direct p38 inhibition [141]. Moreover, MK2−/− mice showed improved ventricular recovery after ischemia–reperfusion, as well as reductions in infarct size and apoptosis [142]. MK2 inhibition with MMI-0100 after acute myocardial infarction inhibited cardiac fibrosis by enhancing primary cardiac fibroblast cell death while inhibiting cardiomyocyte apoptosis [73].

The lack of e fficacy of p38 inhibitors in clinical trials might be due to the high similarity among p38 family members. The lack of isoform-specific inhibitors promotes the development of toxic secondary effects and probably leads to the triggering of regulatory feedback loops. Moreover, most studies used non-specific inhibitors, and very few studies have examined the specific p38 family member involved in a given action. Given the broad spectrum of undesired e ffects associated with the administration of inhibitors, the results obtained to date are hard to interpret. Further research with transgenic animal models will help to define the complex roles of p38 kinases.

**Funding:** This research was funded by the following grants to G.S: European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n◦ ERC 260464, EFSD/Lilly European Diabetes Research Programme Dr Sabio, 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation (Investigadores-BBVA-2017) IN [17]\_BBM\_BAS\_0066, MINECO-FEDER SAF2016-79126-R, PID2019-104399RB-I00, EUIN2017-85875, Comunidad de Madrid IMMUNOTHERCAN-CM S2010/BMD-2326 and B2017/BMD-3733, Fundación AECC and intramural grant 12-2016 IGP. R.R-B and A.M.S are fellows of the FPU (FPU17/03847) and FPI Severo Ochoa CNIC program (BES-2016-077635), respectively. C.F has a Sara Borrell contract (CD19/00078). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovación y Universidades (MCNU), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).

**Acknowledgments:** We thank S. Bartlett for English editing. We thank our collaborators and the students and fellows who contributed to the studies in the Sabio´s group over the years. We regret the inadvertent omission of important references due to space limitations.

**Conflicts of Interest:** The authors declare no conflict of interest.


**5. Laparoscopy in abdominal sepsis due to affections of the small bowel (mesenteric ischemia, intestinal obstruction and incarcerated hernias)**

 Peritonitis secondary to obstruction or ischemia of the small bowel is infrequent. According to the most recent consensus about obstructive intestinal processes, conservative treatment may be maintained for up to 72 hours as long as there is no evidence of strangulation or incarceration. After 3 days of expectant treatment, whether or not these signs are present, surgical exploration is obligatory (Catena et al., 2011).

Some evidence supports the use of the laparoscopic route in the lysis of abdominal adhesions and in the treatment of incarcerated hernias before the onset of necrosis and perforation of the intestinal loops. After the occurrence of these events, most authors recommend surgery by laparotomy (Saeurland et al., 2006).

The lysis of adhesions by laparotomy, the universally accepted route of access for this situation, leads to the later formation of new adhesions, to recurrent intestinal obstruction and to a new laparotomy in 10 to 30% of cases (Landercasper et al., 1993).

In animal models, laparoscopy showed a lower incidence and a smaller number of adhesions, as well as a less severe obstructive situation compared to open surgery. Thus, the laparoscopic approach, when viable, can be considered to prevent obstruction due to adhesions (Tittel et al., 2001). Other clinical and experimental studies have also shown

Laparoscopic Approach to Abdominal Sepsis 231

costs. The indications of DL include the suspicion of intra-abdominal injury maintained after an initial negative workup in closed traumas, stab wounds with proven or possible penetration of the cavity, gun-shot wounds with a possible intra-abdominal course, a diagnosis of diaphragm perforation in penetrating wounds of the thoraco-abdominal region, and the creation of a pericardiac transdiaphragmatic window to rule out heart injury

Absolute contraindications of DL are hemodynamic instability due to hemorrhagic shock or evisceration, and the relative contraindications include peritonitis, known or obvious intraabdominal injury, posterior penetrating trauma with a high probability of intestinal injury and, of course, the lack of experienced professionals and of appropriate equipment

The accuracy of DL in defining the need for laparotomy ranges from 75 to 100%. In a review, DL prevented non-therapeutic laparotomy in 17 to 89% (median: 57%) of traumatized patients. The procedure involved a 6% rate of false-positive results (0-44%). In addition to providing an etiologic diagnosis, laparoscopy permits the appropriate treatment of

A review of 37 studies including more than 1900 patients revealed a rate of DL complication of 1% (Villavicencio & Aucar, 1999). More recent reviews have revealed even lower rates close to zero. Intraoperative complications may occur during the creation of the pneumoperitoneum, the introduction of trochars, the occurrence of pneumothorax during inspection due to an unidentified diaphragmatic injury, during the perforation of hollow viscera, the laceration of solid viscera, during gas dissection in the subcutaneous layer of the peritoneum and vascular injuries (more frequently of the epigastric or epiploic arteries)

**8. Laparoscopy in the perforation of diverticular disease of the colon**

Perforation of diverticular colon disease, generally in the sigmoid colon, with localized contamination of the abdominal cavity can be treated with antibiotics during the early stages, but abscesses larger than 5 cm must be approached surgically. Sigmoidectomy is indicated in patients who have suffered at least 2 crises of diverticulitis and in patients younger than 50 years who have suffered only one episode (Saeurland et al.,2006). Even within an urgency context, this surgery can be performed by the laparoscopic route with a surgical time and results comparable to those of laparotomy and has been performed with a

Over the last few years, there has been an increased use of peritoneal washing and drainage of the cavity by the laparoscopic route without resection, allied to antibiotic treatment during the episode of peritonitis secondary to diverticular perforation. Definite treatment by colectomy can be performed in an elective manner after the resolution of the inflammatory

In a systematic literature review of 231 cases of acute diverticulitis with purulent peritonitis treated in this manner, abdominal sepsis was effectively controlled in 95.7% of the patients. Mortality was 1.7%, morbidity was 10.4% and 1.7% of the patients required a stoma. A long recurrence-free period of time was observed in the patients not subjected to colon resection, and later elective resection of the segment involved by the laparoscopic route was possible

(Stefanidis et al., 2009).

(Stefanidis et al., 2009).

(Hori, 2008).

intracavity injuries in up to 83% of cases (Hori, 2008).

conversion rate of 10% (Tonelli et al., 2009).

in most cases (Toorenvliet et al., 2010).

process (Saeurland et al., 2006; Tonelli et al., 2009).

evidence of a lesser formation of adhesions at the surgical site and on the abdominal wall when laparoscopy is used (Gadallah et al., 2001; Gamal et al., 2001).

The lysis of adhesions by the laparoscopic route has several theoretical advantages over open surgery: 1) less intense postoperative pain, 2) more rapid resolution of the ileum, 3) shorter hospitalization, 4) earlier return to daily activities, 5) lower incidence of complications of the surgical wound, and 6) a reduced formation of postoperative adhesions (Nagle et al., 2004). However, no randomized and controlled studies comparing adhesion lysis by the laparoscopic and open route were detected. Thus, the indications and the results of the less invasive procedure continue to be unclear (Catena et al., 2011).

Today laparoscopy should be reserved for well selected cases, with the use of an open technique for the initiation of pneumoperitoneum, preferentially in the upper left quadrant of the abdomen. It is preferable to use it in case of a first obstructive episode and also when a single or a few adhesions are predicted (for example, when the previous surgery was an appendectomy). A high rate of conversion is expected and the risk of damage to bowel is higher compared to surgery by laparotomy. Findings of a bowel segment larger than 4 cm, of multiple adhesions and of findings compatible with malignant neoplasias supports the option for conversion (Catena et al., 2011).

The extent of release of adhesions is a matter of debate and divides the opinion of authors between the option for lysis of all adhesions in the cavity in an attempt to prevent a new obstructive event or sufficient release for the resolution of obstruction (Scott-Coombes et al., 2003).

Treatment of abdominal wall hernias by laparoscopy has progressed considerably over the last decades and in general this is considered to be the access route of choice in an elective situation. However, it is not possible to transfer the knowledge acquired with this practice to urgency situations such as incarceration, strangulation and bowel injury with contamination of the cavity and infection. There are isolated reports of favorable results for properly selected cases treated by experienced surgeons (Saeurland et al., 2006).

The contribution of laparoscopy to mesenteric ischemia is small. For this situation, DL is less precise than angiography and CT and has not proved to be able to reduce the number of unnecessary laparotomies. DL can detect ischemia when present but cannot rule out this diagnosis when the intestinal loops have a normal appearance upon laparoscopy (Saeurland et al., 2006).


**7. Effect of helminths on experimental autoimmunity**

#### **7.1 Experimental autoimmune encephalomyelitis (EAE)**

EAE in rodents has been widely used as an animal model of MS. In EAE, both Th1 and Th17 have been reported to be encephalitogenic and their relative importance depends on the mouse strain and MOG epitope used for immunization (El-behi et al., 2010). In addition, Th1 and Th17 have different encephalitogenic roles as demonstrated by pathological observations; e.g., distinct cellular infiltrates (macrophage predominant or neutrophil predominant) and distinct sites of inflammation (mainly in the spinal cord or mainly in the brain) (El-behi et al., 2010; Domingues et al., 2010). These observations reinforce the necessity for an antagonistic effect against both Th1 and Th17 for ideal immunomodulatory medicines.

Studies of therapeutic effects of helminths on EAE are summarized in Table 1. Antiencephalitogenic effects have been observed in all three groups of parasitic helminths; i.e. nematodes (*Heligmosomoides polygyrus* (Wilson et al., 2010), *Trichinella spiralis* (Gruden-Movsesijan et al., 2008), *Trichinella pseudospiralis* (Wu et al., 2010) ), trematodes (*Schistosoma japonicum* (Zheng et al., 2008), *Schistosoma mansoni* (Sewell et al., 2003; La Flamme et al., 2003), *Fasciola hepatica* (Walsh et al., 2009)) and cestodes (*Taenia crassiceps*) (Reyes et al., 2011). Among them, there are some distinct results in the effects of schistosomes. According to Sewell et al. (2003), the intra-peritoneal injection of schistosome eggs protected mice from EAE, whereas La Flamme et al. (2003), reported that a similar injection did not have protective effect. In general, a down-regulation of both Th17 and Th1 cytokine expression has been demonstrated (Walsh et al., 2009; Wu et al., 2010; Reyes et al., 2011) except in papers published before the emergence of the Th17 concept (Sewell et al., 2003; La Flamme et al., 2003). Regarding cellular involvement in the suppression, B cells highly expressing CD23 were shown to be responsible for EAE suppression (Wilson et al., 2010) in adoptive transfer experiments. In that study, B cells from IL-10-deficient mice as well as from wildtype mice conferred protection against EAE. The involvement of AAMΦ is also plausible, because AAMΦ markers are increased in the brain in *T. crassiceps*-infected EAE mice (Reyes et al., 2011). In addition, abrogation of schistosome-induced anti- encephalitogenic effects in STAT6-deficient mice (Sewell et al., 2003) might support the importance of AAM Φ.

↓:down-regulation, ↑:up-regulation

Table 1. Suppressive effect of parasitic helminths on EAE.

#### **7.2 Experimental T1D**

596 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

the finding of a critical role for IL-23 in the pathogenesis of some experimental forms of autoimmune diseases (Cua et al., 2003; Murphy et al., 2003) and subsequent discovery of a pathogenic T cell subset producing IL-17 (Th17) (Langrish et al., 2005; Park et al., 2005) has led us to revisit the "Th1/Th2 paradigm". The simplified "Th1/Th2 paradigm" still explains many immunological phenomena, but there is accumulating evidence of the importance of Th17 in the pathogenesis of autoimmunity. By using IL-17-deficient mice, essential roles of IL-17 in the pathogenesis of autoimmune disease have been demonstrated directly (Nakae et al., 2003a, 2003b; Komiyama et al., 2003). Development of the Th17 lineage is antagonized by both Th1-related cytokines (IL-12 and IFN-γ) and a Th2 cytokine (IL-4) (Park et al., 2005; Nakae et al., 2007). Conversely, IL-23 and IL-17 negatively regulate Th1 differentiation (Nakae et al., 2007). Therefore, the balance of these Th subsets is much more complex than previously believed. Complicating the situation further is the recent finding that Th17 is not a stable subset and can be changed to the Th1 phenotype; i.e. plasticity of Th17 (Kurschus et al., 2010; Dong, 2011). The fate of Th17 cells depends on their surrounding environment (Dong, 2011; Lee et al., 2009) and Th17's pathogenic nature depends on the conversion to Th1 cells, in the case of experimental T1D (Martin-Orozco et al., 2009; Bending et al., 2009). The relative importance of each Th subset to the pathogenesis may differ with the disease model, however in most cases, the pathogenicity of Th1 and Th17 is still under debate. There is a report that a transcription factor, T-bet, is essential for the encephalitogenicity of T cells rather than cytokine production (Yang et al., 2009). In contrast, T-bet seems to be a negative regulator in experimental autoimmune myocariditis (Rangachari et al., 2006). Given the unstable nature of the Th17 subset and disease heterogeneity of individual patients, antagonism of both the Th1 and Th17 subsets would be a better choice for the successful suppression of autoimmune diseases. From this viewpoint, parasitic helminths may have

ideal immunomodulatory activities for treatment of autoimmunity.

EAE in rodents has been widely used as an animal model of MS. In EAE, both Th1 and Th17 have been reported to be encephalitogenic and their relative importance depends on the mouse strain and MOG epitope used for immunization (El-behi et al., 2010). In addition, Th1 and Th17 have different encephalitogenic roles as demonstrated by pathological observations; e.g., distinct cellular infiltrates (macrophage predominant or neutrophil predominant) and distinct sites of inflammation (mainly in the spinal cord or mainly in the brain) (El-behi et al., 2010; Domingues et al., 2010). These observations reinforce the necessity for an antagonistic effect against both Th1 and Th17 for ideal immunomodulatory

Studies of therapeutic effects of helminths on EAE are summarized in Table 1. Antiencephalitogenic effects have been observed in all three groups of parasitic helminths; i.e. nematodes (*Heligmosomoides polygyrus* (Wilson et al., 2010), *Trichinella spiralis* (Gruden-Movsesijan et al., 2008), *Trichinella pseudospiralis* (Wu et al., 2010) ), trematodes (*Schistosoma japonicum* (Zheng et al., 2008), *Schistosoma mansoni* (Sewell et al., 2003; La Flamme et al., 2003), *Fasciola hepatica* (Walsh et al., 2009)) and cestodes (*Taenia crassiceps*) (Reyes et al., 2011). Among them, there are some distinct results in the effects of schistosomes. According to Sewell et al. (2003), the intra-peritoneal injection of schistosome eggs protected mice from EAE, whereas La Flamme et al. (2003), reported that a similar injection did not have

**7. Effect of helminths on experimental autoimmunity 7.1 Experimental autoimmune encephalomyelitis (EAE)**

medicines.

Non-obese diabetic (NOD) mice have been used widely as an animal model of T1D. Spontaneous destruction of pancreatic β-cells and subsequent hyperglycemia are observed in NOD mice. The pathogenesis of T1D in this mouse has been studied extensively, however, there is still considerable controversy over the relative contribution of Th1 (or IFNγ) and Th17 (or IL-17). Anti-IFN-γ treatment rendered NOD mice resistant to cyclophosphamide (CY)-accelerated diabetes (Debray-Sachs et al., 1991). In contrast, in IFNγ deficient NOD mice, neither insulitis nor diabetes was prevented although the onset was delayed (Hultgren et al., 1996; Gysemans et al., 2008). These findings suggest that IFN-γ is involved in, but not essential to, the pathogenesis. IFN-γ is not itself detrimental to β-cells, but induces apoptosis when acting with IL-1β or TNF-α (Gysemans et al., 2008). Regarding this pro-apoptotic effect, dual roles of IFN-γ in NOD mice have been indicated; i.e. IFN-γ induces β-cell destruction via STAT-1 but protects β-cells via IRF-1 (Gysemans et al., 2008). On the other hand, pathological roles of IL-17 have been also suggested in mice (Miljkovic et al., 2005: Emamaullee et al., 2009) and humans (Honkanen et al., 2010). Anti-IL-17 antibody prevented diabetes in NOD mice when administered around the time of onset (Emamaullee et al., 2009). At present, it is reasonable to conclude that both Th1 and Th17 play some role in diabetogenesis in NOD mice.

Parasitic Helminths as Potential Therapeutic Agents Against Autoimmune Disorders 599

by IL-17 and acts on osteoclast precursors to stimulate their differentiation into multinucleated osteoclasts (Okamoto & Takayanagi, 2010). An essential role for IL-17 in the pathogenesis of CIA has been demonstrated directly by using IL-17-deficient mice (Nakae et al., 2003a). IL-17 is an essential pathological factor also in other models of arthritis; e.g. IL-1 receptor antagonist-deficient mice (Nakae et al., 2003b), Ag-induced arthritis (AIA) (Irmler et al., 2007) and glucose 6-phosphate isomerase (GPI)– induced arthritis (Iwanami et al., 2008). Thus, these models could be considered Th17-dependent. In contrast, IFN-γ is now considered an ameliorating factor in CIA (Kelchtermans et al., 2009; Chu et al., 2007) and in AIA (Irmler et al., 2007). However, in proteoglycan-induced arthritis, IFN-γ (not IL-17) is responsible for the pathogenesis (Doodes et al., 2008). Taken together, models of autoimmune arthritis are mainly dependent on Th17 but exceptions do exist. In human RA, the pathological importance of TNF-α, IL-1β, IL-6 and IL-17 has been demonstrated directly by the striking efficacy of biological drugs targeting those cytokines (Nixon et al., 2007; Jones & Ding, 2010; Genovese et al., 2010). In addition, an anti-RANKL monoclonal antibody has been approved for osteoporosis and is now under clinical development for RA (Pageau,

As described in section 3, the global distribution and trends of RA do not match the hygiene hypothesis (Gabriel & Michaud, 2009; Shapira et al., 2010b). In addition, to our knowledge, there is no report of anti-RA effects of parasitic helminths in humans. Nonetheless, several investigators (including us) have found anti-arthritic effects of helminths or helminthderived products in rodents. Those studies are summarized in Table 3. We ourselves have reported suppressive effects of a blood fluke, *S. mansoni,* on mouse CIA (Osada et al., 2009). In that study, the *S. mansoni* infection reduced the severity of CIA as evaluated using scores of arthritis and numbers of arthritic paws. Histopathological observation revealed the prevention of bone destruction in the infected mice. According to an analysis of splenic cytokine production pattern, production of IL-17A, TNF-α and IFN-γ were down-regulated and that of IL-4 and IL-10 was up-regulated. The real-time PCR analysis of inflamed paws showed the striking augmentation of the gene expression of bone-absorptive proinflammatory mediators (IL-1β, IL-6 and RANKL) observed in non-infected arthritic mice to be abrogated in infected mice. *S. mansoni* is a gonochoristic worm and forms a pair in the portal vein. Egg deposition in the infected host organs is the major stimulus of Th2 polarization (Grzych et al., 1991) and other immunomodulatory events such as Treg induction (Taylor et al., 2006). In our experiments, the severity of CIA correlated inversely with the numbers of worm pairs, theoretically proportional to the number of eggs produced (Osada et al., 2009). In addition, in a time-course experiment, the splenic cytokine modulation (including down-modulation of IL-17 and TNF-α) was observed from 6 to 8 week post-infection, which corresponds to the beginning of egg deposition (unpublished observation). However, repeated intra-peritoneal injections of soluble egg antigen (SEA) did not protect mice from CIA and viable eggs lost their ability to suppress IL-17 production by freeze-thawing and subsequent crushing treatment (Osada et al., 2010). Therefore, a viable infection, which supplies viable eggs continuously, may be essential for the schistosomeinduced anti-arthritic effects. By plotting the relationship between infection intensity and severity levels, we have found that a single pair of worms was enough to abrogate the augmentation of pro-inflammatory mediators and approximately 4 pairs were enough to

suppress the onset of arthritis (Osada et al., 2009).

2009).

Along with NOD mice, inducible T1D models have been used for diabetes research. One of them is streptozotocin (STZ)-induced diabetes. STZ is a glucosamine- nitrosourea compound specifically toxic to pancreatic islet β-cells (Yamamoto et al., 1981). Multiple lowdose administrations of STZ cause immune mechanism-mediated β-cell destruction and diabetes in mice (Kantwerk-Funke et al., 1991). In this T1D model, pathogenic roles of both IL-12/IFN-γ axis (Herold et al., 1996; Müller et al., 2002; Gysemans et al., 2005; Cetkovic-Cvrlje & Uckun, 2005) and IL-23/IL-17 axis (Miljkovic et al., 2005, Mensah-Brown et al., 2006) have been suggested, as in NOD mice.

↓:down-regulation, ↑:up-regulation

Table 2. Suppressive effects of parasitic helminths on experimental T1D.

Studies of the therapeutic effects of helminths on experimental T1D models are summarized in Table 2. As in the case of EAE, all three groups of helminths (nematodes, trematodes and cestodes) have preventive effects against experimental T1D models. As Treg cells play an important regulatory role against diabetogenesis (Brode et al., 2006; Ott et al., 2005), some authors suggest that they are also responsible for the anti-diabetogenic effects of helminths (Hübner et al., 2009; Zaccone et al., 2009). However, other reports do not support the importance of Treg cells (Liu et al., 2009; Espinoza-Jiménez et al., 2010). This inconsistency probably suggests the presence of distinct suppressive mechanisms in each parasite species. In our recent study, intestinal helminth infection protected mice from diabetes induced by multiple low-doses of STZ in a STAT6-independent manner (unpublished observation). This finding suggests that immune polarization from Th1 to Th2 induced by helminths cannot explain the anti-diabetogenic effect. Further studies using gene-targeted mice are needed to elucidate the anti-diabetogenic mechanisms of helminths.

#### **7.3 Experimental autoimmune arthritis**

There are various models of autoimmune arthritis in rodents. Collagen-induced arthritis (CIA) is one of the most widely used classical models of RA (Stuart et al., 1982). In this model, anti-collagen antibodies are responsible for the destruction of cartilage (Terato et al., 1992). Regarding cytokines, TNF-α, IL-1β and IL-6 are crucial to the inflammatory bone destruction in autoimmune arthritis (Ferraccioli et al., 2010; Möller & Villiger, 2006). However, the most important factor in the bone destruction is receptor activator of NFκB ligand (RANKL). This cytokine is induced to express on osteoblasts and synovial fibroblasts

Along with NOD mice, inducible T1D models have been used for diabetes research. One of them is streptozotocin (STZ)-induced diabetes. STZ is a glucosamine- nitrosourea compound specifically toxic to pancreatic islet β-cells (Yamamoto et al., 1981). Multiple lowdose administrations of STZ cause immune mechanism-mediated β-cell destruction and diabetes in mice (Kantwerk-Funke et al., 1991). In this T1D model, pathogenic roles of both IL-12/IFN-γ axis (Herold et al., 1996; Müller et al., 2002; Gysemans et al., 2005; Cetkovic-Cvrlje & Uckun, 2005) and IL-23/IL-17 axis (Miljkovic et al., 2005, Mensah-Brown et al.,

T1D model Helminth Treatment Proposed mechanism Refs

Table 2. Suppressive effects of parasitic helminths on experimental T1D.

elucidate the anti-diabetogenic mechanisms of helminths.

**7.3 Experimental autoimmune arthritis**

Spontaneous T1D in NOD mice *Litomosoides sigmodontis* Infection, Worm Ag IL-4↑, IL-5↑, Treg Hübner et al., 2009

Eggs, Egg Ag, Worm Ag i.p.

Studies of the therapeutic effects of helminths on experimental T1D models are summarized in Table 2. As in the case of EAE, all three groups of helminths (nematodes, trematodes and cestodes) have preventive effects against experimental T1D models. As Treg cells play an important regulatory role against diabetogenesis (Brode et al., 2006; Ott et al., 2005), some authors suggest that they are also responsible for the anti-diabetogenic effects of helminths (Hübner et al., 2009; Zaccone et al., 2009). However, other reports do not support the importance of Treg cells (Liu et al., 2009; Espinoza-Jiménez et al., 2010). This inconsistency probably suggests the presence of distinct suppressive mechanisms in each parasite species. In our recent study, intestinal helminth infection protected mice from diabetes induced by multiple low-doses of STZ in a STAT6-independent manner (unpublished observation). This finding suggests that immune polarization from Th1 to Th2 induced by helminths cannot explain the anti-diabetogenic effect. Further studies using gene-targeted mice are needed to

There are various models of autoimmune arthritis in rodents. Collagen-induced arthritis (CIA) is one of the most widely used classical models of RA (Stuart et al., 1982). In this model, anti-collagen antibodies are responsible for the destruction of cartilage (Terato et al., 1992). Regarding cytokines, TNF-α, IL-1β and IL-6 are crucial to the inflammatory bone destruction in autoimmune arthritis (Ferraccioli et al., 2010; Möller & Villiger, 2006). However, the most important factor in the bone destruction is receptor activator of NFκB ligand (RANKL). This cytokine is induced to express on osteoblasts and synovial fibroblasts

*Heligmosomoides polygyrus* Infection Independent of IL-10 and Treg Liu et al., 2009 *Trichinella spiralis* Infection IL-4↑, IL-10↑ Saunders et al., 2007 *Schistosoma mansoni* Infection / Egg i.p. Anti-insulin IgG↓ Cooke et al., 1999

*Schistosoma mansoni* Infection EL-Wakil et al., 2002

*Taenia crassiceps* Infection AAM Espinoza-Jiménez et al., 2010

NKT cells↑ Zaccone et al., 2003

Egg Ag i.p. Treg Zaccone et al., 2009

2006) have been suggested, as in NOD mice.

Streptozotocin-induced diabetes in mice

(Multiple low dose model)

↓:down-regulation, ↑:up-regulation

by IL-17 and acts on osteoclast precursors to stimulate their differentiation into multinucleated osteoclasts (Okamoto & Takayanagi, 2010). An essential role for IL-17 in the pathogenesis of CIA has been demonstrated directly by using IL-17-deficient mice (Nakae et al., 2003a). IL-17 is an essential pathological factor also in other models of arthritis; e.g. IL-1 receptor antagonist-deficient mice (Nakae et al., 2003b), Ag-induced arthritis (AIA) (Irmler et al., 2007) and glucose 6-phosphate isomerase (GPI)– induced arthritis (Iwanami et al., 2008). Thus, these models could be considered Th17-dependent. In contrast, IFN-γ is now considered an ameliorating factor in CIA (Kelchtermans et al., 2009; Chu et al., 2007) and in AIA (Irmler et al., 2007). However, in proteoglycan-induced arthritis, IFN-γ (not IL-17) is responsible for the pathogenesis (Doodes et al., 2008). Taken together, models of autoimmune arthritis are mainly dependent on Th17 but exceptions do exist. In human RA, the pathological importance of TNF-α, IL-1β, IL-6 and IL-17 has been demonstrated directly by the striking efficacy of biological drugs targeting those cytokines (Nixon et al., 2007; Jones & Ding, 2010; Genovese et al., 2010). In addition, an anti-RANKL monoclonal antibody has been approved for osteoporosis and is now under clinical development for RA (Pageau, 2009).

As described in section 3, the global distribution and trends of RA do not match the hygiene hypothesis (Gabriel & Michaud, 2009; Shapira et al., 2010b). In addition, to our knowledge, there is no report of anti-RA effects of parasitic helminths in humans. Nonetheless, several investigators (including us) have found anti-arthritic effects of helminths or helminthderived products in rodents. Those studies are summarized in Table 3. We ourselves have reported suppressive effects of a blood fluke, *S. mansoni,* on mouse CIA (Osada et al., 2009). In that study, the *S. mansoni* infection reduced the severity of CIA as evaluated using scores of arthritis and numbers of arthritic paws. Histopathological observation revealed the prevention of bone destruction in the infected mice. According to an analysis of splenic cytokine production pattern, production of IL-17A, TNF-α and IFN-γ were down-regulated and that of IL-4 and IL-10 was up-regulated. The real-time PCR analysis of inflamed paws showed the striking augmentation of the gene expression of bone-absorptive proinflammatory mediators (IL-1β, IL-6 and RANKL) observed in non-infected arthritic mice to be abrogated in infected mice. *S. mansoni* is a gonochoristic worm and forms a pair in the portal vein. Egg deposition in the infected host organs is the major stimulus of Th2 polarization (Grzych et al., 1991) and other immunomodulatory events such as Treg induction (Taylor et al., 2006). In our experiments, the severity of CIA correlated inversely with the numbers of worm pairs, theoretically proportional to the number of eggs produced (Osada et al., 2009). In addition, in a time-course experiment, the splenic cytokine modulation (including down-modulation of IL-17 and TNF-α) was observed from 6 to 8 week post-infection, which corresponds to the beginning of egg deposition (unpublished observation). However, repeated intra-peritoneal injections of soluble egg antigen (SEA) did not protect mice from CIA and viable eggs lost their ability to suppress IL-17 production by freeze-thawing and subsequent crushing treatment (Osada et al., 2010). Therefore, a viable infection, which supplies viable eggs continuously, may be essential for the schistosomeinduced anti-arthritic effects. By plotting the relationship between infection intensity and severity levels, we have found that a single pair of worms was enough to abrogate the augmentation of pro-inflammatory mediators and approximately 4 pairs were enough to suppress the onset of arthritis (Osada et al., 2009).

Parasitic Helminths as Potential Therapeutic Agents Against Autoimmune Disorders 601

Increases in both IL-17 and IL-12 mRNA in CD and UC patients has been reported (Nielsen et al., 2003). The most striking evidence of the involvement of IL-23/Th17 axis is the finding of Th17-related genes (including IL-23R gene) as susceptibility genes for CD (Brand, 2009). In contrast, Th2-related cytokines are dominant in UC (Sarra et al., 2010). Likewise, models of colitis are also composed of diseases with a distinct pathogenesis (Strober et al., 2002). Experimental colitis induced by the intra-rectal injection of hapten, such as TNBS or DNBS, resembles human CD and its development seems independent of IFN-γR (Camoglio et al., 2000) and dependent on IL-17R signaling (Zhang et al., 2006). Colitis can be also induced by causing dysfunction in epithelial cell barrier. Supply of drinking water containing dextran sulfate sodium (DSS) induces this type of colitis. In this model, T and B cells are dispensable (Dieleman et al., 1994) and macrophages seem to play both a pathological role (Bauer et al., 2010) and a regulatory role (Qualls et al., 2006; Smith et al., 2007). Considering predominant expression of Th2-related cytokines in the chronic phase, DSS-induced colitis resembles UC (Alex et al., 2009). Interestingly, neutralization of IL-17 aggravates the model (Ogawa et al., 2004), suggesting that IL-17 plays a protective role against DSS-induced colitis. By contrast, another study demonstrated that the colitis was alleviated in IL-17A deficient mice (Ito et al., 2008). The reason for this discrepancy is not clear, but might be due to different mouse

Using these models of colitis, a number of studies on helminth effects have been conducted (Table 4). Generally, helminths seem to ameliorate TNBS/DNBS- induced colitis. It is worth noting that even worms that usually cause intestinal pathology by laying a large number of eggs in the mesenteric vein (i.e. *Schistosoma* spp.) have anti-colitic effects (Moreels et al., 2004; Ruyssers et al., 2009; Elliott et al., 2003; Mo et al., 2007; Zhao et al., 2009; Bodammer et al., 2011; Smith et al., 2007). A down-regulation of Th1/Th17-related cytokines and upregulation of anti-inflammatory cytokines are observed in most studies. However, only a few papers have provided direct evidence of the involvement of certain cells or cytokines. For instance, anti-colitic effects of *S.mansoni* (Elliott et al., 2003) and *H.diminuta* (Hunter et al., 2005) were demonstrated to be STAT6-dependent. The latter authors also a showed the anti-colitic effect of the worms to be dependent on IL-10 and the presence of macrophages (Hunter et al., 2010). In contrast to the regularly observed helminths' ameliorating effects on Th1/Th17 dominant (TNBS/DNBS- induced) models, macrophage-mediated or Th2 dominant (DSS-induced or oxazolone- induced) colitis was not alleviated or rather worsened by some helminth infections (Table 4). This exacerbation seems due to the robust Th2-polarization by the helminths; e.g. *H. diminuta* infection worsened oxazolone-induced colitis via IL-5 induction and consequently caused eosinophila (Wang et al., 2010), demonstrated by a loss of exacerbation in eotaxin-deficient mice or anti-IL-5 antibodytreated mice. The finding that anti-colitic effects of schistosome male worms were lost in DSS-induced colitis when mice were infected with worms of mixed sex (Smith et al., 2007) may support the speculation, because the Th2-polarizing ability of schsitosome eggs (produced in mixed sex infection) is more potent than that of adult worms (Grzych et al., 1991). Weinstock's group has been studying anti-colitic mechanisms of the intestinal helminth *H.polygyrus* by using colitis in IL-10-deficient or TGF-βRII dominant negative (DN) mice (Elliott et al., 2004, 2008; Ince et al., 2009). According to the studies, the helminth's anticolitic effect depends not on IL-10 but on TGF-β signaling. This finding does not mean that host-derived TGF-β is necessary, because it has been recently shown that *H. polygyrus*derived "TGF-β-like molecules" mobilize host TGF-β signaling and induce subsequent Treg

strains used in their experiments as indicated by Ito et al.

cell development (McSorley et al., 2010; Grainger et al., 2010).

↓:down-regulation, ↑:up-regulation

Table 3. Suppressive effects of parasitic helminths on experimental arthritis.

In addition to the down-regulation of pro-inflammatory cytokines, pathogenic anti-collagen IgG levels were lowered in the infected mice. Since our publication, other investigators also have demonstrated therapeutic effects of schistosomes on arthritis models. He et al. (2010) showed that *S.japonicum* suppressed CIA. However, infection at 2 weeks before CIA induction resulted in an exacerbation of the disease, whereas infection at 7 weeks before induction prevented the disease. This is in contrast to our study in which infection at 2 weeks before induction reduced the severity of CIA. The reason of this discrepancy is not clear, but as speculated by He et al., the difference in parasite species (*S.mansoni* and *S.japonicum*) might have affected the outcome. Sun et al. (2010) demonstrated that a recombinant protein of *S. japonicum* (rSj16) ameliorated adjuvant-induced arthritis in rats. The authors observed a suppression of IL-12 production and augmentation of IL-10 production in rSj16-treated bone marrow-derived dendritic cells (BMDCs). A filarial nematode-derived phosphorylcholine- containing glycoprotein, ES-62, was proven to suppress ongoing CIA (McInnes et al., 2003). In vitro, ES-62 suppressed LPS-induced production of TNF-α and IL-6 from RA synovial cells. Moreover, ES-62 also inhibited TNF-α production in human T cells and macrophages. Regarding the involvement of regulatory cytokines and cells, Shi et al. (2011) reported that IL-10 from CD4+ cells of infected mice was important for anti-arthritic effects of the rat tapeworm *Hymenolepis diminuta*. They also found that the absence of IL-4Rα chain signaling in mice cancelled the anti-arthritic effects of the worm. These findings seem to suggest the importance of Th2-polarization by the parasite, however, we recently found that the Th2-polarizing intestinal parasite *H. polygyrus* did not protect mice from CIA but rather, exacerbated the disease (unpublished observation). The anti-arthritic mechanisms of helminths seem complicated, probably because of the heterogeneity of both experimental models and parasites.

#### **7.4 Experimental colitis as a model of IBD**

Human IBD includes heterogeneous inflammatory diseases of the intestines. For instance, in terms of T cell subset, CD had been considered a "Th1 disease", but at present, the importance of the Th17 subset in the pathogenesis of CD is emphasized (Sarra et al., 2010).

*Acanthocheilonema viteae* ES-62 i.p. IFN-↓, TNF-↓, IL-6↓, Anti-

*Schistosoma mansoni* Infection IL-17↓, TNF-↓, IL-6↓, RANKL

*Ascaris suum* Worm Ag i.p. Rocha et al., 2008

*Schistosoma japonicum* Infection IL-4↑, Anti-collagen IgG↓ He et al., 2010

*Ascaris suum* Worm Ag i.p./p.o. NO↓, IL-1↓ Rocha et al., 2008

*Schistosoma japonicum* rSj16 i.p. TNF-↓, IL-1↓,NO↓, IL-10↑ Sun et al., 2010

*Hymenolepis diminuta* Infection IL-10 from CD4+ cells Shi et al., 2011

In addition to the down-regulation of pro-inflammatory cytokines, pathogenic anti-collagen IgG levels were lowered in the infected mice. Since our publication, other investigators also have demonstrated therapeutic effects of schistosomes on arthritis models. He et al. (2010) showed that *S.japonicum* suppressed CIA. However, infection at 2 weeks before CIA induction resulted in an exacerbation of the disease, whereas infection at 7 weeks before induction prevented the disease. This is in contrast to our study in which infection at 2 weeks before induction reduced the severity of CIA. The reason of this discrepancy is not clear, but as speculated by He et al., the difference in parasite species (*S.mansoni* and *S.japonicum*) might have affected the outcome. Sun et al. (2010) demonstrated that a recombinant protein of *S. japonicum* (rSj16) ameliorated adjuvant-induced arthritis in rats. The authors observed a suppression of IL-12 production and augmentation of IL-10 production in rSj16-treated bone marrow-derived dendritic cells (BMDCs). A filarial nematode-derived phosphorylcholine- containing glycoprotein, ES-62, was proven to suppress ongoing CIA (McInnes et al., 2003). In vitro, ES-62 suppressed LPS-induced production of TNF-α and IL-6 from RA synovial cells. Moreover, ES-62 also inhibited TNF-α production in human T cells and macrophages. Regarding the involvement of regulatory cytokines and cells, Shi et al. (2011) reported that IL-10 from CD4+ cells of infected mice was important for anti-arthritic effects of the rat tapeworm *Hymenolepis diminuta*. They also found that the absence of IL-4Rα chain signaling in mice cancelled the anti-arthritic effects of the worm. These findings seem to suggest the importance of Th2-polarization by the parasite, however, we recently found that the Th2-polarizing intestinal parasite *H. polygyrus* did not protect mice from CIA but rather, exacerbated the disease (unpublished observation). The anti-arthritic mechanisms of helminths seem complicated, probably

collagen IgG↓

*Nippostrongylus brasiliensis* Infection Salinas-Carmona et al., 2009

↓, Anti-collagen IgG↓

McInnes et al., 2003

Osada et al., 2009

Arthritis model Helminth Treatment Proposed mechanism Refs

Table 3. Suppressive effects of parasitic helminths on experimental arthritis.

because of the heterogeneity of both experimental models and parasites.

Human IBD includes heterogeneous inflammatory diseases of the intestines. For instance, in terms of T cell subset, CD had been considered a "Th1 disease", but at present, the importance of the Th17 subset in the pathogenesis of CD is emphasized (Sarra et al., 2010).

**7.4 Experimental colitis as a model of IBD**

*Heligmosomoides polygyrus* Infection

Collagen-induced arthritis (CIA) in mice

Zymosan-induced arthritis (ZYA) in mice/rats

Adjuvant-induced arthritis (AIA) in rats

Spontaneous arthritis in MRL/lpr mice

↓:down-regulation, ↑:up-regulation

FCA-induced monoarthritis in rats Increases in both IL-17 and IL-12 mRNA in CD and UC patients has been reported (Nielsen et al., 2003). The most striking evidence of the involvement of IL-23/Th17 axis is the finding of Th17-related genes (including IL-23R gene) as susceptibility genes for CD (Brand, 2009). In contrast, Th2-related cytokines are dominant in UC (Sarra et al., 2010). Likewise, models of colitis are also composed of diseases with a distinct pathogenesis (Strober et al., 2002). Experimental colitis induced by the intra-rectal injection of hapten, such as TNBS or DNBS, resembles human CD and its development seems independent of IFN-γR (Camoglio et al., 2000) and dependent on IL-17R signaling (Zhang et al., 2006). Colitis can be also induced by causing dysfunction in epithelial cell barrier. Supply of drinking water containing dextran sulfate sodium (DSS) induces this type of colitis. In this model, T and B cells are dispensable (Dieleman et al., 1994) and macrophages seem to play both a pathological role (Bauer et al., 2010) and a regulatory role (Qualls et al., 2006; Smith et al., 2007). Considering predominant expression of Th2-related cytokines in the chronic phase, DSS-induced colitis resembles UC (Alex et al., 2009). Interestingly, neutralization of IL-17 aggravates the model (Ogawa et al., 2004), suggesting that IL-17 plays a protective role against DSS-induced colitis. By contrast, another study demonstrated that the colitis was alleviated in IL-17A deficient mice (Ito et al., 2008). The reason for this discrepancy is not clear, but might be due to different mouse strains used in their experiments as indicated by Ito et al.

Using these models of colitis, a number of studies on helminth effects have been conducted (Table 4). Generally, helminths seem to ameliorate TNBS/DNBS- induced colitis. It is worth noting that even worms that usually cause intestinal pathology by laying a large number of eggs in the mesenteric vein (i.e. *Schistosoma* spp.) have anti-colitic effects (Moreels et al., 2004; Ruyssers et al., 2009; Elliott et al., 2003; Mo et al., 2007; Zhao et al., 2009; Bodammer et al., 2011; Smith et al., 2007). A down-regulation of Th1/Th17-related cytokines and upregulation of anti-inflammatory cytokines are observed in most studies. However, only a few papers have provided direct evidence of the involvement of certain cells or cytokines. For instance, anti-colitic effects of *S.mansoni* (Elliott et al., 2003) and *H.diminuta* (Hunter et al., 2005) were demonstrated to be STAT6-dependent. The latter authors also a showed the anti-colitic effect of the worms to be dependent on IL-10 and the presence of macrophages (Hunter et al., 2010). In contrast to the regularly observed helminths' ameliorating effects on Th1/Th17 dominant (TNBS/DNBS- induced) models, macrophage-mediated or Th2 dominant (DSS-induced or oxazolone- induced) colitis was not alleviated or rather worsened by some helminth infections (Table 4). This exacerbation seems due to the robust Th2-polarization by the helminths; e.g. *H. diminuta* infection worsened oxazolone-induced colitis via IL-5 induction and consequently caused eosinophila (Wang et al., 2010), demonstrated by a loss of exacerbation in eotaxin-deficient mice or anti-IL-5 antibodytreated mice. The finding that anti-colitic effects of schistosome male worms were lost in DSS-induced colitis when mice were infected with worms of mixed sex (Smith et al., 2007) may support the speculation, because the Th2-polarizing ability of schsitosome eggs (produced in mixed sex infection) is more potent than that of adult worms (Grzych et al., 1991). Weinstock's group has been studying anti-colitic mechanisms of the intestinal helminth *H.polygyrus* by using colitis in IL-10-deficient or TGF-βRII dominant negative (DN) mice (Elliott et al., 2004, 2008; Ince et al., 2009). According to the studies, the helminth's anticolitic effect depends not on IL-10 but on TGF-β signaling. This finding does not mean that host-derived TGF-β is necessary, because it has been recently shown that *H. polygyrus*derived "TGF-β-like molecules" mobilize host TGF-β signaling and induce subsequent Treg cell development (McSorley et al., 2010; Grainger et al., 2010).

Parasitic Helminths as Potential Therapeutic Agents Against Autoimmune Disorders 603

There are two ways of developing parasite-based biomedicines for clinical use. One approach is the direct applications of non-pathogenic/hypo-virulent viable helminths to patients, as introduced in section 8. In addition to TSO and hookworms, other hypo-virulent helminths could be considered for human application. However, before clinical trials, sufficient accumulation of epidemiological and experimental evidence of their therapeutic efficacy is required. Hypo-virulent intestinal nematodes (e.g. *Trichostrongylus* spp.), intestinal trematodes (e.g. *Metagonimus* sp.) and intestinal tapeworms (e.g. *Hymenolepis diminuta*) may become candidates for such studies in the future. It is also essential that the parasites can be maintained in domestic or experimental animals. This is because parasites that infect only humans cannot be maintained and expanded efficiently for clinical use. Another way of developing parasite-based biomedicines comes from the identification of effector molecules of parasites. Considerable numbers of immunomodulatory molecules have been identified from helminths (Harnett W & Harnett MM, 2010). The majority have shown therapeutic effects on experimental autoimmunity or allergy. Some investigators reported that viable parasites were superior to administration of the antigens of parasites (Hunter et al., 2010; Bodammer et al., 2011; Osada et al., 2010) in therapeutic efficacy. In addition, there is still considerable controversy over the roles of regulatory cells (e.g. Treg, Breg or AAMΦ) and regulatory cytokines (e.g. IL-4, IL-10, TGF-β) in helminth-induced immunomodulation. Therefore, further investigation is needed to elucidate the immunomodulatory mechanisms of viable parasite infections, and new findings obtained there should help to establish an optimal screening system for anti-autoimmune/anti-

Our studies introduced in this chapter were supported by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (nos. 20590435, 22590386) and Health and Labour Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan (H22-Kokui-Shitei-004). I am grateful to Prof. Tamotsu Kanazawa and other laboratory members for their valuable scientific advice. I also acknowledge the technical assistance of Ms. Yukari Horie and the clerical assistance of Ms. Tomoko Fujitomi.

Alex, P., Zachos, N.C., Nguyen, T., Gonzales, L., Chen, T.E., Conklin, L.S., Centola, M. & Li,

Aoyama, H., Hirata, T., Sakugawa, H., Watanabe, T., Miyagi, S., Maeshiro, T., Chinen, T.,

Ascherio, A. & Munger, K. L. (2010). 99th Dahlem conference on infection, inflammation and

DSS and TNBS-induced colitis. *Inflamm Bowel Dis.* 15 (3):341-352.

epidemiological evidence. *Clin Exp Immunol*. 160 (1):120-124.

infection. *Am J Trop Med Hyg.* 76 (5):972-976.

X. (2009). Distinct cytokine patterns identified from multiplex profiles of murine

Kawane, M., Zaha, O., Nakayoshi, T., Kinjo, F. & Fujita, J. (2007). An inverse relationship between autoimmune liver diseases and *Strongyloides stercoralis*

chronic inflammatory disorders: Epstein-Barr virus and multiple sclerosis:

**9. Concluding remarks**

allergic substances from parasitic helminths.

**10. Acknowledgements**

**11. References**

Table 4. Effects of parasitic helminths on experimental colitis.

#### **8. Clinical trials of parasitic helminths against immunological disorders**

The administration of non-pathogenic or hypo-virulent parasitic worms could be considered for the treatment of immunological disorders. Several clinical trials using parasitic worms have been and are currently being conducted. Significant therapeutic effects have been confirmed in some of these studies. Weinstock's group conducted trials with *Trichuris suis* (porcine whipworm) ova (TSO) against CD (Summers et al., 2005a) and UC (Summers et al., 2005b), and demonstrated significant efficacy. TSO is also being tested for MS and promising results have been obtained in a phase I trial (Fleming et al., 2011). Regarding allergic disorders, Bager et al. (2010) found no therapeutic effect of TSO on allergic rhinitis. However, Summers et al. (2010) critically commented on the report that it was premature to conclude that TSO is ineffective on allergic rhinitis because the TSO treatment was too late and not sufficient. *Necator americanus* (Hookworm) is also under clinical trials for asthma (Feary et al., 2010) and CD (Croese et al., 2006). The advantage of this worm is its long life in the host (at least 6 years) and no need for repeated inoculation (Elliott and Weinstock, 2009). The parasite was well-tolerated without severe adverse effects on asthmatic patients, but a safe dosage of the parasites (10 infective larvae) did not show significant therapeutic efficacy (Feary et al., 2010).