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1 Na+ Influx Via Orai1 Inhibits Intracellular ATP Induced Mtorc2 Signaling To 1 Na+ Influx via Orai1 Inhibits Intracellular ATP Induced mTORC2 Signaling to 2 Disrupt CD4 T Cell Gene Expression and Differentiation 3 4 Yong Miao, Jaya Bhushan, Adish Dani and Monika Vig* 5 Department of Pathology and Immunology 6 Washington University School of Medicine, St. Louis MO 63110, USA 7 *Correspondence to: [email protected] 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 23 Abstract: T cell effector functions require sustained calcium influx. However, the 24 signaling and phenotypic consequences of non-specific sodium permeation via calcium 25 channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its 26 depletion disrupts the functional assembly of Orai1 multimers. Here we show that α- 27 SNAP hypomorph, hydrocephalos with hopping gait, Napahyh/hyh mice harbor significant 28 defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. 29 Mechanistically, TCR stimulation induced rapid sodium influx in Napahyh/hyh CD4 T cells, 30 which reduced intracellular ATP, [ATP]i. Depletion of [ATP]i inhibited mTORC2 31 dependent NFκB activation in Napahyh/hyh cells but ablation of Orai1 restored it. 32 Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in 33 Napahyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific 34 sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and 35 may provide mechanistic insights into some divergent phenotypes associated with Orai1 36 function. 37 38 39 40 41 42 43 44 45 2 46 Introduction: 47 A sustained rise in cytosolic calcium levels is necessary for nuclear translocation 48 of calcium dependent transcription factors such as nuclear factor of activated T cell 49 (NFAT) (Crabtree, 1999, Parekh and Putney, 2005, Winslow et al., 2003, Macian, 2005, 50 Vig and Kinet, 2009, Vig and Kinet, 2007, Crabtree, 2001). NFAT proteins are essential 51 for the development of several tissues but have been found to be dispensable for thymic 52 development and function of Foxp3 regulatory T cells (Tregs) (Crabtree and Olson, 53 2002, Crabtree, 2001, Crabtree, 1999, Timmerman et al., 1997, Vaeth et al., 2012). The 54 role of proteins directly involved in sustained calcium influx, however, remains less well 55 established. Specifically, genetic ablation of ORAI1, the pore forming subunit of calcium 56 release activated calcium (CRAC) channels (Vig et al., 2006b, Vig et al., 2006a, Peinelt 57 et al., 2006), partially inhibits T cell effector cytokines in mice and does not affect Foxp3 58 Treg development (Vig et al., 2008, Vig and Kinet, 2009, Gwack et al., 2008, McCarl et 59 al., 2010). The role of ORAI2 as well as ORAI3, the two closely related homologs of 60 ORAI1 that are highly expressed in mouse T cells remains unestablished in mice and 61 humans although all ORAIs are capable of reconstituting CRAC currents in vitro (Mercer 62 et al., 2006, Lis et al., 2007, DeHaven et al., 2007). 63 STIM1 and STIM2, the ER resident calcium sensor proteins, are required for ER 64 calcium release and Orai1 activation and T lymphocyte effector functions (Oh-Hora et 65 al., 2008). However, ablation of STIMs, but not ORAIs, affects thymic development of 66 Tregs (Oh-Hora et al., 2013, McCarl et al., 2010) and ablation of STIMs, but not ORAI1, 67 resulting in multi-organ autoimmunity in mice and humans (Oh-Hora et al., 2008, McCarl 68 et al., 2010, Picard et al., 2009). Because STIMs perform several additional functions 3 69 such as regulation of calcium selectivity of ORAI1 channels (McNally et al., 2012) as 70 well as inhibition of voltage gated calcium channel Cav1.2 (Wang et al., 2010, Park et 71 al., 2010), role of sustained calcium influx or store-operated calcium entry (SOCE) in the 72 development of Tregs and autoimmunity remains correlative (Oh-Hora et al., 2008, Oh- 73 Hora et al., 2013). Likewise, the phenotypes of human patients harboring different Stim 74 and Orai mutations range from immunodeficiency to autoimmunity and cancer. Despite 75 this diversity, all phenotypes are currently correlated with reduced SOCE (Picard et al., 76 2009). 77 We have previously shown that α-soluble NSF-attachment protein (α-SNAP), a 78 cytosolic protein traditionally studied in the context of soluble NSF attachment protein 79 receptor (SNARE) complex disassembly and membrane trafficking (Clary et al., 1990), 80 directly binds Stim1 and Orai1 and is necessary for the functional assembly and ion 81 specificity of multimeric Orai1 channels (Miao et al., 2013, Li et al., 2016). In addition, α- 82 SNAP has been implicated in AMP kinase (AMPK) inhibition and zippering of SNAREs 83 in vitro (Park et al., 2014, Baur et al., 2007, Wang and Brautigan, 2013). SNAREs play a 84 direct role in exocytosis and are therefore required for cytotoxic T, natural killer and 85 mast cell degranulation (Baram et al., 2001, Puri et al., 2003, Hepp et al., 2005, Suzuki 86 and Verma, 2008). However, the role of α-SNAP is less clear in vivo, and remains 87 unexplored in the immune system. α-SNAP deletion is embryonic lethal in mice and a 88 hypomorphic missense mutation in α-SNAP, hydrocephalous with hopping gait, 89 (Napahyh/hyh) has been previously reported to cause neuro-developmental defects 90 (Bronson and Lane, 1990, Chae et al., 2004, Hong et al., 2004). 91 Here we show that reduced expression of α-SNAP causes unexpected defects in 4 92 CD4 T cell signaling, gene expression and Foxp3 Treg differentiation. Using RNAi 93 mediated ablation of Orai1 in Napahyh/hyh CD4 T cells and monensin treatment of 94 wildtype CD4 T cells, we demonstrate that Orai1 mediated sodium influx, but not hyh/hyh 95 reduced SOCE, depletes [ATP]i in T cell receptor (TCR) stimulated Napa CD4 T 96 cells. Furthermore, we find that depletion of [ATP]i levels disrupts mTORC2 activation 97 which, in turn, inhibits NFκB activation and in vivo as well as in vitro differentiation of 98 Foxp3 Tregs in Napahyh/hyh mice. Therefore, analysis of α-SNAP deficient mice reveals 99 that non-specific sodium permeation via Orai1 disrupts a novel signaling node and could 100 provide alternate mechanistic insights into the diversity of phenotypes observed in Stim 101 and Orai mutant human patients. 102 103 104 105 106 107 108 109 110 5 111 Results: 112 Napahyh/hyh mice harbor severe defects in the production of CD4 T cell 113 effector cytokines 114 Mice bearing Napahyh/hyh mutation on a mixed background have been 115 characterized previously in the context of neurodevelopmental disorders (Bronson and 116 Lane, 1990, Chae et al., 2004, Hong et al., 2004). We backcrossed Napahyh/hyh mice on 117 to C57BL/6 background and found that homozygous mutant Napahyh/hyh mice were 118 significantly smaller in size and died perinatally, within 2-3 weeks. To overcome the 119 issue of perinatal lethality, we generated fetal liver chimeras using irradiated CD45.1+ 120 congenic recipients reconstituted with CD45.2+ wildtype or Napahyh/hyh E15.5 embryos. 121 We analyzed fetal liver chimeras at 8-12 week post-transfer and found that the 122 reconstitution efficiency and total thymic (Figure 1A) and spleen cell numbers (Figure 123 1B) were comparable in wildtype (WT) and Napahyh/hyh chimeras. Relative abundance of 124 CD4 and CD8 T cells in the thymus (Figure 1C) and spleen (Figure 1D) was also normal 125 in Napahyh/hyh fetal liver chimeras. Therefore, we performed all the subsequent analysis 126 of wildtype and Napahyh/hyh CD4 T cells and Foxp3 Tregs using fetal liver chimeras, 127 unless otherwise specified. 128 α-SNAP null mice are embryonic lethal and, in accordance with previous reports, 129 Napahyh/hyh CD4 T cells showed ~40% depletion of α-SNAP levels (Figure 1E). Given 130 the role of α-SNAP in SNARE recycling (Clary et al., 1990), we first compared the levels 131 of cell surface receptors. Surprisingly, surface expression TCR and co-receptors was 132 found to be normal in Napahyh/hyh peripheral CD4 T cells (Figure 1F). Resting Napahyh/hyh 6 133 T lymphocytes showed largely normal surface expression of CD25, CD44 and CD69 134 and their up-regulation following receptor mediated stimulation was comparable to WT 135 (Figure 1G). 136 CRAC channel components, Orai1 and Stim1 are necessary for optimal 137 production and secretion of several T cell effector cytokines (Vig et al., 2008, Vig and 138 Kinet, 2009, Gwack et al., 2008, Oh-Hora et al., 2008). However, given a partial 139 depletion of α-SNAP in Napahyh/hyh mice, we first sought to determine whether 140 Napahyh/hyh CD4 T cells showed defects in the production of effector cytokines. 141 Surprisingly, we found significant defects in IL-2 (Figure 1H, 1J) and TNF-α production 142 by TCR stimulated Napahyh/hyh CD4 T cells (Figure 1I, 1J). Napahyh/hyh CD4 T cells 143 cultured under T helper 1 (Th1) polarizing conditions showed a minor defect in IFN-γ 144 production (Figure 1K & 1M), however, we observed a robust defect in IL-4 expression 145 in Th2 polarized Napahyh/hyh CD4 T cells (Figure 1L & 1M). Intracellular levels of T-bet or 146 Gata-3 did not appear to be significantly altered in Napahyh/hyh mice (Figure 1, figure 147 supplement 1). Furthermore, Napahyh/hyh CD4 T cells (Figure 1N-1O) and splenocytes 148 (Figure 1P) showed a partial defect in anti-CD3 induced proliferation. Taken together, 149 these data demonstrate that Napahyh/hyh CD4 T lymphocytes harbor a significant defect 150 in the production of several key effector cytokines, while exhibiting normal levels of cell 151 surface receptors.
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