PI3Kδ drives the pathogenesis of experimental autoimmune encephalomyelitis by inhibiting effector T cell apoptosis and promoting Th17 differentiation

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Abstract

The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.

Introduction

MS is a chronic, demyelinating disease of the central nervous system (CNS) and multiple lines of evidence indicate that it is, at least in part, an autoimmune disease [1], [2]. Following breakdown of immunological tolerance to CNS antigens by an as yet unknown mechanism, T and B cells invade the CNS leading to an accumulation of innate immune effector cells within the brain and spinal cord. Activation of inflammatory signaling results in CNS demyelination caused by oligodendrocyte loss and cytotoxic effects on myelinated axons. The resulting paralysis and other neurological symptoms seen in MS patients are unpredictable, difficult to manage and treat effectively and are a major health and socio-economic burden in many developed countries. The autoimmune aspects of MS are well modelled in the rodent model experimental autoimmune encephalomyelitis (EAE). In EAE, CNS-autoreactive CD4+ T cells are activated or introduced, then invade the CNS and the resulting inflammatory cascade resembles human MS in many aspects of clinical and cellular pathogenesis. Until recently, MS and EAE were both considered driven by autoreactive Th1 cells. However, recent evidence has revealed that the T cell lineage most likely to be driving EAE pathogenesis are Th17 cells, a pro-inflammatory helper T cell type characterised by expression of the cytokine IL-17 [3], [4]. Th17 cells differentiate down a separate lineage commitment pathway from Th1 and Th2 cells, dependent upon the cytokines IL-6, TGFβ, IL-23 and IL-1β [5], [6], [7]. Numerous studies in mice have now shown that CD4+ T cells which produce IL-17 drive organ-specific autoimmune inflammation and IL-17 has been strongly implicated in human autoimmune disease (reviewed in Weaver et al. [4] and Harrington et al. [3]). Therefore, understanding the mechanisms controlling their generation is an important objective.

Class IA PI3Ks are a family of lipid and protein kinases that play a critical role in an array of intracellular signaling cascades involved in both physiological and pathological processes. The three class IA PI3Ks each function as a heterodimeric complex consisting of a catalytic subunit (p110α, p110β, p110δ) and one of seven adaptor subunits that arise following alternative splicing of the genes encoding p85α, p85β and p55γ [8]. Whereas the p110α and p110β catalytic subunits are expressed ubiquitously and play non-redundant roles in embryonic development [9], [10], p110δ is mainly expressed in leukocytes and mice lacking p110δ expression or activity do not show adverse developmental phenotypes [11]. Despite this, recent evidence suggests that PI3Kδ plays a diverse and critical role in many aspects of leukocyte activation and migration; PI3Kδ is required for B cell and NK cell trafficking, maintenance of directed neutrophil migration [12], [13], [14], [15], [16], [17], [18] and mast cells require PI3Kδ for efficient proliferation, adhesion, migration and degranulation [19], [20]. In addition, neutrophil and NK cell development, activation and cytokine secretion are also affected by inactivation of PI3Kδ [15], [21], [22], [23], [24]. In B cells, activation, T-cell dependent and independent IgM and IgG production, B-cell receptor-induced proliferation and IL-4-induced survival are markedly reduced when PI3Kδ kinase activity is absent [25], [26], [27], [28]. While p110δ has been shown to play a limited role in T cell trafficking, it is required for Th1, Th2 and Treg differentiation, activation and regulation [14], [17], [28], [29], [30], [31], most likely due to demonstrated defects in TCR as well as cytokine receptor signaling. These combined processes to which PI3Kδ contributes have led to proposals to target this kinase with small molecule isoform-selective inhibitors in human autoimmune diseases where multiple detrimental inflammatory signals are involved.

While it is clear that PI3Kδ plays an important role in numerous signaling pathways involved in leukocyte activation or migration that could impact on the development of autoimmune diseases such as MS, it is not currently known whether PI3Kδ plays a role in the development of pathogenic Th17 responses. Moreover, while the results of previous studies indicate that the reduced level of T lymphocyte activation observed in p110δ-deficient mice is attributable, at least in part, to an intrinsic defect in T cell signaling [14], [17], [28], [29], [30], [31], the extent to which PI3Kδ contributes to dendritic cell migration and function or lymphocyte apoptosis in the context of activation of an autoimmune response is not known. In the present study, we have addressed these issues and the role of PI3Kδ in CNS autoimmunity using mice in which p110δ has been genetically-inactivated (p110δD910A/D910A mice) and using a highly selective PI3Kδ inhibitor.

Section snippets

Animals and pharmacological inhibitors

p110δD910A/D910A mice on a C57Bl/6 background [28] were obtained from Dr. Margaret Hibbs, Ludwig Institute for Cancer Research, Melbourne, and bred at the University of Adelaide Animal House. Age and sex-matched C57BL/6 mice were purchased from The University of Adelaide Animal House, South Australia or the Australian Research Council and housed at the University of Adelaide Animal House. OT-II mice were provided by Dr. Christian Engwerda at the Queensland Institute of Medical Research.

Lack of p110δ catalytic activity limits the pathogenesis of EAE

To study the role of p110δ in CNS autoimmune disease, WT and p110δD910A/D910A mice were monitored for signs of clinical disease following EAE induction (Fig. 1A and B and Table 1). EAE severity was reduced in p110δD910A/D910A mice, when compared with WT mice, from day 15 post-immunization. There were no significant differences between WT and p110δD910A/D910A mice with respect to incidence of disease, the day of clinical onset or the day of peak clinical disease (Table 1). However, the peak

Discussion

This study has led to a number of novel observations with respect to the role of PI3Kδ in immunity. First, we have shown that it plays an important role in autoimmunity and deficiency in p110δ inhibits EAE, a model that mimics the autoimmune aspects of MS. Second, we have demonstrated that the reduction in EAE pathogenesis is not only due to a decrease in T cell activation, but it is also attributable to higher levels of activation-induced cell death in T cells lacking functional p110δ. Third,

Conclusion

This study has provided several novel insights into the role of PI3Kδ in CNS autoimmune disease and particularly, in regard to T helper cell function. PI3Kδ activity is required for efficient Th17 differentiation and function both in vitro and in vivo and protects CD4+ T cells from undergoing apoptosis during an autoimmune response. These effects on T cells are apparently independent of DC function. Cumulatively, these alterations in the T cell compartment ultimately result in impaired

Acknowledgements

The authors would like to thank Dr. Margaret Hibbs at the Ludwig Institute for Cancer Research, Melbourne, for the p110δD910A/D910A mice, Dr. Christian Engwerda at the Queensland Institute of Medical Research for OT-II mice, Calistoga Pharmaceuticals for the IC87114 inhibitor and laboratory animal services at the University of Adelaide for assistance with animal husbandry and maintenance. This work was supported by funds from the US Multiple Sclerosis Society and Multiple Sclerosis Research

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    Present address: Department of Clinical Neurosciences, University of Calgary, Hotchkiss Brain Institute, AB, Canada.

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    Present address: Department of Hematology, Hanson Institute, IMVS, SA, Australia.

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    These authors contributed equally to this work.

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