Selective protein kinase Cθ (PKCθ) inhibitors for the treatment of autoimmune diseases.

Protein kinase Cθ (PKCθ) is a member of a large family of serine/threonine kinases that are involved in diverse cellular functions. PKCθ has roles in T-cell activation and survival, where the dependency of T-cell responses on this enzyme appears to be dictated by both the nature of the antigen and by the inflammatory environment. Studies in PKCθ-deficient mice have demonstrated that although anti-viral responses are PKCθ-independent, T-cell responses associated with autoimmune diseases are PKCθ-dependent. PKCθ-deficient mice are either resistant to or show markedly reduced symptoms in models of MS (multiple sclerosis), IBD (inflammatory bowel disease), arthritis and asthma. Thus potent and selective inhibition of PKCθ has the potential to block T-cell-mediated autoimmunity without compromising anti-viral responses. The present review describes the design and optimization of potent and selective PKCθ inhibitors and their efficacy in both in vitro and in vivo studies. First, our compounds confirm the critical role for PKCθ in T-cell activation and proliferation and secondly they help to demonstrate that murine and human memory T-cell function continues to be dependent on this enzyme. In addition, these inhibitors demonstrate impressive efficacy in treating established autoimmune disease in murine models of IBD and MS.

From inflammation to wound healing: using a simple model to understand the functional versatility of murine macrophages.

Macrophages are fundamental cells of the innate immune system. Their activation is essential for such distinct immune functions as inflammation (pathogen-killing) and tissue repair (wound healing). An open question has been the functional stability of an individual macrophage cell: whether it can change its functional profile between different immune responses such as between the repair pathway and the inflammatory pathway. We studied this question theoretically by constructing a rate equation model for the key substrate, enzymes and products of the pathways; we then tested the model experimentally. Both our model and experiments show that individual macrophages can switch from the repair pathway to the inflammation pathway but that the reverse switch does not occur.

Schistosoma mansoni arginase shares functional similarities with human orthologs but depends upon disulphide bridges for enzymatic activity.

Schistosome helminths constitute a major health risk for the human population in many tropical areas. We demonstrate for the first time that several developmental stages of the human parasite Schistosoma mansoni express arginase, which is responsible for the hydrolysis of l-arginine to l-ornithine and urea. Arginase activity by alternatively activated macrophages is an essential component of the mammalian host response in schistosomiasis. However, it has not been previously shown that the parasite also expresses arginase when it is in contact with the mammalian host. After cloning and sequencing the cDNA encoding the parasite enzyme, we found that many structural features of human arginase are well conserved in the parasite ortholog. Subsequently, we discovered that S. mansoni arginase shares many similar molecular, biochemical and functional properties with both human arginase isoforms. Nevertheless, our data also reveal striking differences between human and schistosome arginase. Particularly, we found evidence that schistosome arginase activity depends upon disulphide bonds by cysteines, in contrast to human arginase. In conclusion, we report that S. mansoni arginase is well adapted to the physiological conditions that exist in the human host.

Coordinated control of immunity to muscle stage Trichinella spiralis by IL-10, regulatory T cells, and TGF-beta.

We previously demonstrated that IL-10 is critical in the control of acute inflammation during development of Trichinella spiralis in the muscle. In this study, we use gene-targeted knockout mice, adoptive transfer of specific T cell populations, and in vivo Ab treatments to determine the mechanisms by which inflammation is controlled and effector T cell responses are moderated during muscle infection. We report that CD4(+)CD25(-) effector T cells, rather than CD4(+)CD25(+) regulatory T cells, suppress inflammation by an IL-10-dependent mechanism that limits IFN-gamma production and local inducible NO synthase induction. Conversely, we show that depletion of regulatory T cells during infection results in exaggerated Th2 responses. Finally, we provide evidence that, in the absence of IL-10, TGF-beta participates in control of local inflammation in infected muscle and promotes parasite survival.

Naturally occurring CD4+Foxp3+ regulatory T cells are an essential, IL-10-independent part of the immunoregulatory network in Schistosoma mansoni egg-induced inflammation.

In acute and chronic schistosomiasis, survival of the host requires a carefully balanced immune response against highly immunogenic parasite eggs. We characterized the phenotype, distribution, and functional role of CD4(+)Foxp3(+) naturally occurring regulatory T cells (naTregs) in schistosome egg-induced inflammation. In adoptive transfer experiments and by intracellular staining for Foxp3, we demonstrate significant frequencies of naTregs in hepatic granulomas and draining lymphoid tissues of mice infected with the trematode Schistosoma mansoni. Strikingly, egg-induced inflammation does not change the normal ratio between naTregs and effector CD4(+) T cells at the inflammatory site or in lymphoid organs in acute or chronic disease. However, increasing frequencies of CD103-expressing cells in the naTreg compartment indicate a change in phenotype for naTregs with disease progression. Because CD103 was described recently as an activation marker for naTregs, we speculate that naTregs in chronic schistosomiasis are potentially more suppressive. Furthermore, we found that most naTregs do not contribute to egg-induced IL-4 and IL-10 production. Importantly, depletion of CD25(+) naTregs strongly enhances the frequency of IL-4-producing effector T cells in acute egg-induced inflammation. It does not change clonal expansion of activated CD4(+) T cells. This regulation of egg-induced cytokine production does not require the presence of IL-10. These data demonstrate that naTregs limit egg-induced effector-cytokine production in our model. Our results identify naTregs as an important, IL-10-independent part of the regulatory network in schistosome egg-induced inflammation.

IL-13 activates a mechanism of tissue fibrosis that is completely TGF-beta independent.

Fibrosis is a characteristic feature in the pathogenesis of a wide spectrum of diseases. Recently, it was suggested that IL-13-dependent fibrosis develops through a TGF-beta1 and matrix metalloproteinase-9-dependent (MMP-9) mechanism. However, the significance of this pathway in a natural disorder of fibrosis was not investigated. In this study, we examined the role of TGF-beta in IL-13-dependent liver fibrosis caused by Schistosoma mansoni infection. Infected IL-13-/- mice showed an almost complete abrogation of fibrosis despite continued and undiminished production of TGF-beta1. Although MMP-9 activity was implicated in the IL-13 pathway, MMP-9-/- mice displayed no reduction in fibrosis, even when chronically infected. To directly test the requirement for TGF-beta, studies were also performed with neutralizing anti-TGF-beta Abs, soluble antagonists (soluble TGF-betaR-Fc), and Tg mice (Smad3-/- and TGF-betaRII-Fc Tg) that have disruptions in all or part of the TGF-beta signaling cascade. In all cases, fibrosis developed normally and with kinetics similar to wild-type mice. Production of IL-13 was also unaffected. Finally, several genes, including interstitial collagens, several MMPs, and tissue inhibitors of metalloprotease-1 were up-regulated in TGF-beta1-/- mice by IL-13, demonstrating that IL-13 activates the fibrogenic machinery directly. Together, these studies provide unequivocal evidence of a pathway of fibrogenesis that is IL-13 dependent but TGF-beta1 independent, illustrating the importance of targeting IL-13 directly in the treatment of infection-induced fibrosis.

The pathogenesis of schistosomiasis is controlled by cooperating IL-10-producing innate effector and regulatory T cells.

IL-10 reduces immunopathology in many persistent infections, yet the contribution of IL-10 from distinct cellular sources remains poorly defined. We generated IL-10/recombination-activating gene (RAG)2-deficient mice and dissected the role of T cell- and non-T cell-derived IL-10 in schistosomiasis by performing adoptive transfers. In this study, we show that IL-10 is generated by both the innate and adaptive immune response following infection, with both sources regulating the development of type-2 immunity, immune-mediated pathology, and survival of the infected host. Importantly, most of the CD4(+) T cell-produced IL-10 was confined to a subset of T cells expressing CD25. These cells were isolated from egg-induced granulomas and exhibited potent suppressive activity in vitro. Nevertheless, when naive, naturally occurring CD4(+)CD25(+) cells were depleted in adoptive transfers, recipient IL-10/RAG2-deficient animals were more susceptible than RAG2-deficient mice, confirming an additional host-protective role for non-T cell-derived IL-10. Thus, innate effectors and regulatory T cells producing IL-10 cooperate to reduce morbidity and prolong survival in schistosomiasis.

P-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon gamma and the IL-13 decoy receptor.

The selectin family of cell adhesion molecules is widely thought to promote inflammatory reactions by facilitating leukocyte recruitment. However, it was unexpectedly found that mice with targeted deletion of the P-selectin gene (PsKO mice) developed unpolarized type 1/type 2 cytokine responses and severely aggravated liver pathology following infection with the type 2-promoting pathogen Schistosoma mansoni. In fact, liver fibrosis, which is dependent on interleukin 13 (IL-13), increased by a factor of more than 6, despite simultaneous induction of the antifibrotic cytokine interferon gamma (IFN-gamma). Inflammation, as measured by granuloma size, also increased significantly in the absence of P-selectin. When infected PsKO mice were treated with neutralizing anti-IFN-gamma monoclonal antibodies, however, granuloma size was restored to wild-type levels; this finding revealed the potent proinflammatory role of IFN-gamma when expressed concomitantly with IL-13. Untreated PsKO mice also exhibited a significant (sixfold) reduction in decoy IL-13 receptor (IL-13 receptor alpha-2) expression when compared with infected wild-type animals. It is noteworthy, however, that when decoy receptor activity was restored in PsKO mice by treatment with soluble IL-13 receptor alpha-2-Fc, the exacerbated fibrotic response was completely inhibited. Thus, reduced expression of the decoy IL-13 receptor mediated by the elevated type 1 cytokine response probably accounts for the enhanced activity of IL-13 in PsKO mice and for the resultant increase in collagen deposition. In conclusion, the current study has revealed the critical role of P-selectin in the progression of chronic liver disease caused by schistosome parasites. By suppressing IFN-gamma and up-regulating the decoy IL-13 receptor, P-selectin dramatically inhibits the pathologic tissue remodeling that results from chronic type 2 cytokine-mediated inflammation.