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. Author manuscript; available in PMC: 2020 May 7.
Published in final edited form as: Cell Metab. 2019 Feb 14;29(5):1104–1118.e6. doi: 10.1016/j.cmet.2019.01.019

Calcium signaling controls pathogenic Th17 cell-mediated inflammation by regulating mitochondrial function

Ulrike Kaufmann 1,2,3, Sascha Kahlfuss 1,2, Jun Yang 1, Elitza Ivanova 1, Sergei B Koralov 1, Stefan Feske 1,4,5
PMCID: PMC6506368  NIHMSID: NIHMS1519779  PMID: 30773462

SUMMARY

Pathogenic Th17 cells play important roles in many autoimmune and inflammatory diseases. Their function depends on T cell receptor (TCR) signaling and cytokines that activate signal transducer and activator of transcription 3 (STAT3). TCR engagement activates stromal interaction molecule 1 (STIM1) and calcium (Ca2+) influx through Ca2+ release-activated Ca2+ (CRAC) channels. We here show that abolishing STIM1 and Ca2+ influx in T cells expressing a hyperactive form of STAT3 (STAT3C) attenuates pathogenic Th17 cell function and inflammation associated with STAT3C expression. Deletion of STIM1 in pathogenic Th17 cells impairs the expression of genes required for mitochondrial function and oxidative phosphorylation (OXPHOS), but enhances reactive oxygen species (ROS) production. STIM1 deletion or inhibition of OXPHOS is associated with a non-pathogenic Th17 gene expression signature and impaired pathogenic Th17 cell function. Our findings establish Ca2+ influx as a critical regulator of mitochondrial function and oxidative stress in pathogenic Th17 cell-mediated multiorgan inflammation.

Graphical abstract

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In Brief

XXX et al show an essential role of the store-operated calcium entry (SOCE) pathway in pathogenic Th17 cell function by regulating mitochondrial function, OXPHOS, and preventing oxidative stress. SOCE inhibition could be a potential therapeutic avenue in Th17 inflammatory diseases such as MS, colitis, psoriasis or steroid-resistant asthma.

INTRODUCTION

T helper 17 (Th17) cells are a subset of CD4+ T cells characterized by expression of the orphan nuclear receptor RORγt and production of interleukin (IL)-17 and IL-22 (Langrish et al., 2005; Zhou et al., 2007). Th17 cells play a dual role in immune responses to bacterial and fungal infections, as well as inflammation in a wide array of autoimmune and chronic inflammatory disorders (Korn et al., 2009). In humans, Th17 cells are present at the sites of autoimmune tissue inflammation in diseases such as multiple sclerosis (MS), inflammatory bowel disease (IBD) and psoriasis (Korn et al., 2009). Th17 cells also play a critical role in inflammatory airway diseases such as steroid-resistant asthma and chronic obstructive pulmonary disease (COPD) (Doe et al., 2010). The differentiation of Th17 cells is mediated by T cell receptor (TCR) signaling and cytokines including transforming growth factor-β (TGF-β) and IL-6. TGF-β activates Smad2/3 transcription factors, whereas IL-6 signals mediate STAT3 phosphorylation. Smad2/3 and STAT3, together with other transcription factors activated by TCR signaling, induce the expression of RORγt and Th17 differentiation. Besides IL-6, the cytokines IL-21 and IL-23 also signal via STAT3 and are critical for the differentiation of both murine and human Th17 cells (Korn et al., 2009). IL-23 in particular is required for the function of pathogenic Th17 cells and their ability to cause autoimmunity (Langrish et al., 2005). Furthermore, IL-1β receptor signaling regulates the expression of IRF4 and RORγt, thus promoting the differentiation of pathogenic Th17 cells (Chung et al., 2009). In vitro, pathogenic Th17 cells can be generated by a combination of IL-23, IL-1β and IL-6 (Ghoreschi et al., 2010) or TGF-β3 and IL-6 (Lee et al., 2012). Pathogenic Th17 cells can be distinguished from nonpathogenic Th17 cells by a transcriptional signature that includes cytokines (Il22, Il3, Csf2), transcription factors (HIF1α, Fosl2, Stat4, Rel) and other genes (Gpr65, Cd5l, Plzp) (Gaublomme et al., 2015; Lee et al., 2012).

STAT3 phosphorylation by cytokine receptor-associated janus kinases (JAK) causes its dimerization and translocation to the nucleus (Forbes et al., 2016). Engineered substitution of two cysteines (A661C and N663C) in its C terminus (STAT3C) facilitates disulfide linkage between two STAT3C monomers brought into proximity by Y705 phosphorylation and results in STAT3C dimer stabilization (Li and Shaw, 2006). Expression of such hyperactive STAT3C in CD4+ T cells strongly augments Th17 differentiation and spontaneous production of IL-17A. We generated Stat3CStopfl/fl Cd4-Cre mice (S3CD4) in which Cre-mediated deletion of an upstream floxed stop cassette results in T cell-specific expression of STAT3C (Fogli et al., 2013). Expression of STAT3C in T cells results in the expansion of Th17 cells, which preferentially home to the lungs, where they cause neutrophil infiltration and pulmonary inflammation (Fogli et al., 2013), and to the skin, triggering a psoriasis-like inflammation (Yang et al., 2018). Neutralization of IL-17 in S3CD4 mice greatly reduces lung inflammation and psoriatic disease (Fogli et al., 2013; Yang et al., 2018).

TCR signaling induces the production of the second messenger inositol-1,4,5-triphosphate (IP3), resulting in Ca2+ release from the endoplasmic reticulum (ER). The release of Ca2+ from the ER causes the activation of STIM1 and STIM2 that are localized in the ER membrane and function as Ca2+ sensors (Feske et al., 2012; Hogan et al., 2010). Activated STIM1 binds to and opens ORAI1, which is the pore-forming subunit of the CRAC channel and provides the bulk of Ca2+ influx (called store-operated Ca2+ entry, or SOCE) after TCR stimulation. SOCE activates several Ca2+ dependent enzymes and transcription factors including the phosphatase calcineurin and the nuclear factor of activated T cells (NFAT), which regulates the transcription of many cytokine genes including IL-17A, IL-21, IL-22 and IFNγ (Hermann-Kleiter and Baier, 2010). Inhibition of SOCE by genetic deletion of Orai1, Stim1 or Stim2 in murine CD4+ T cells impairs Th17 cell function and ameliorates the severity of CNS inflammation in the experimental autoimmune encephalomyelitis (EAE) model of MS and in IBD (Kaufmann et al., 2016; Kim et al., 2014; Ma et al., 2010; McCarl et al., 2010) in which Th17 cells play an important pathogenic role (Burkett et al., 2015).

The mechanisms by which SOCE regulates the development of pathogenic Th17 cells and enables them to cause autoimmune inflammation are poorly understood. To investigate the role of SOCE in the development and function of pathogenic Th17 cells, we generated mice whose T cells express hyperactive STAT3C but lack SOCE by crossing S3CCD4 mice with Stim1fl/fl Cd4-Cre (S1CD4) mice. T cells from the resulting S1–S3CCD4 mice lacked the spontaneous IL-17A expression of S3CCD4 Th17 cells and failed to induce severe lung inflammation, psoriasis-like skin disease and colitis. At the molecular level, STIM1-deficient Th17 cells showed a gene expression signature associated with non-pathogenic Th17 cells, had impaired mitochondrial function including oxidative phosphorylation (OXPHOS) and increased ROS production resulting in DNA damage and cell death. Inhibition of OXPHOS in pathogenic Th17 cells altered their gene expression profile to one resembling non-pathogenic Th17 cells. Our study demonstrates that SOCE is required for pathogenic Th17 cell function and multiorgan inflammation by regulating the mitochondrial function and antioxidant response of Th17 cells.

RESULTS

Deletion of STIM1 inhibits STAT3-driven Th17 cell function and organ inflammation.

To investigate the role of SOCE in pathogenic Th17 cells and their ability to cause inflammation and autoimmunity, we used Stat3CStopfl/fl Cd4-Cre mice (S3CCD4) whose T cells express a hyperactive form of STAT3 (STAT3C). CD4+ T cells from these mice have augmented Th17 differentiation with increased levels of IL-17A and other Th17 cytokines and develop severe multiorgan inflammation (Fogli et al.; Yang et al., 2018). We crossed S3CCD4 to Stim1fl/fl (S1) mice to suppress SOCE in T cells (Figure 1A). Stimulation of T cells, by passive depletion of ER Ca2+ stores or by TCR crosslinking, from the resulting Stim1fl/flStat3CStopfl/fl Cd4-Cre (S1–S3CCD4) mice showed that S1–S3CCD4 T cells lacked SOCE compared to T cells from wild-type (WT) and S3CCD4 mice. The SOCE defect in S1–S3CCD4 T cells was similar to that in T cells from Stim1fl/fl Cd4-Cre (S1CD4) mice (Figure 1B, Figure S1A).

Figure 1. Deletion of STIM1 inhibits STAT3-driven pathogenic Th17 cell function and pulmonary inflammation.

Figure 1.

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(A) Generation of Stim1fl/flStat3CSTOPfl/fl Cd4-Cre (S1–S3CCD4) mice that lack STIM1 and express a hyperactive form of STAT3 in T cells. (B) SOCE in CD4+ T cells from WT, Stim1fl/fl Cd4-Cre (S1CD4), Stat3CSTOPfl/fl Cd4-Cre (S3CCD4) and S1–S3CCD4 mice that were loaded with Fura-2 and stimulated with thapsigargin (TG) in Ca2+ free Ringer solution followed by addition of 1 mM Ca2+. Bar graphs show means ± SEM of F340/380 emission at 460–800 sec from 3 mice per genotype. (C,D) IL-17A expression in CD4+ T cells (C) and frequencies of CD11b+ Gr-1+ neutrophils (D) isolated from the lungs of WT, S1CD4, S3CCD4 and S1–S3CCD4 mice. Representative flow cytometry plots (left) and bar graphs (right) of means ± SEM of 4 repeat experiments and 7–8 mice. (E) Representative lung histologies of 7–9 week old mice stained with H&E. (F) Lung function of 6–8 week old mice and analysis of airway resistance, compliance and elastance. Whisker plots represent 5–11 mice per group. *, p<0.05; **, p<0.01; ***, p<0.001. Statistical analysis in B, C and D by unpaired Student’s t test, in F by Mann-Whitney test. See also Figure S1.

Hyperactive STAT3C in T cells causes severe inflammation in various tissues including lung, joints and skin, and results in runting and premature death of S3CCD4 mice (Figure S1B) (Fogli et al., 2013; Yang et al., 2018). By contrast, S1–S3CCD4 mice had normal body weight and appeared phenotypically like WT littermates. S3CCD4 mice spontaneously developed severe dermatitis with fur loss, epidermal thickening and massive infiltration of inflammatory cells in the dermis (Figure S1C,D). This pronounced psoriatic-like phenotype was absent in S1–S3CCD4 mice and comparable to WT and S1CD4 mice. Protection was not due to increased numbers of immunosuppressive Foxp3+ Treg cells as Treg frequencies in S1–S3CCD4 mice were reduced compared to S3CCD4 mice (Figure S1E). Airway inflammation in S3CCD4 mice is characterized by infiltration of Th17 cells and neutrophils into the lungs of mice, increased mucus production, and decreased lung function resembling Th17-mediated asthma in humans (Fogli et al., 2013). To investigate if deletion of SOCE ameliorates Th17 cell-mediated lung inflammation, we first analyzed CD4+ T cells isolated from the lungs and spleens of WT, S1CD4, S3CCD4 and S1–S3CCD4 mice. Markedly increased frequencies of IL-17A expressing CD4+ T cells were detected in the spleen and lung of S3CCD4 compared to WT mice. Importantly, lack of SOCE in T cells isolated from the lungs of S1–S3CCD4 mice abolished IL-17A production (Figure 1C). IL-17A induces the recruitment of neutrophils through the stimulation of epithelial cells to secrete CXC chemokines (Laan et al., 1999). Whereas S3CCD4 mice showed markedly increased numbers of neutrophils in their lungs, S1–S3CCD4 mice had neutrophils numbers similar to those in WT and S1CD4 mice (Figure 1D). The histopathological analysis of lungs from S3CCD4 mice showed massive perivascular, peribronchiolar and interstitial inflammatory infiltrates with some germinal centers, overall resembling severe lymphocytic interstitial pneumonitis. S1–S3CCD4 mice, like WT and S1CD4 mice, showed no signs of inflammation (Figure 1E). To evaluate the effects of STIM1 deletion on lung function, we measured parameters of respiratory mechanics in mice using a forced-oscillation small animal ventilator. Compared to WT mice, airway resistance and elastance were significantly increased in S3CCD4 mice, whereas compliance was decreased, indicating that their pulmonary inflammation affects lung function (Figure 1F). By contrast, deletion of STIM1 almost completely normalized all three parameters of lung function to levels found in WT and S1CD4 mice. Taken together, deletion of STIM1 profoundly attenuates STAT3C-mediated Th17 cell responses and pulmonary and skin inflammation.

STIM1 regulates expression of genes associated with pathogenic Th17 cells.

To investigate how STIM1 regulates pathogenic Th17 cell function, we analyzed the effects of STIM1 deletion on STAT3-driven Th17 cell differentiation. In contrast to naive WT CD4+ T cells from WT mice, S3CCD4 T cells produced robust amounts of IL-17A without further stimulation when cultured in vitro under non-polarizing conditions. Spontaneous IL-17A production was abolished in S1–S3CCD4 T cells (Figure 2A). Furthermore, differentiation of CD4+ T cells into Th17 cells in the presence of IL-6 and TGF-β for 3 days resulted in strong expression of IL-17A by WT and S3CCD4 T cells, whereas S1CD4 and S1–S3CCD4 T cells almost completely lacked IL-17A, demonstrating an important role of SOCE in Th17 cell function and IL-17A expression. To understand how SOCE regulates the differentiation of CD4+ T cells into Th17 cells, we analyzed the expression of factors that control Th17 differentiation including the lineage-specific transcription factors RORγt and RORα, the transcription factors IRF4, BATF and hypoxia-inducible factor-1a (HIF1α), and receptors for the cytokines IL-1, IL-6, IL-23 (Korn et al., 2009). mRNA levels of RORγt, RORα, BATF, HIF1α as well as IL-1R1, IL-6R and IL-23R were not significantly different in CD4+ T cells from WT, S3CCD4 and S1–S3CCD4 mice that had been polarized into Th17 cells in vitro (Figure S2A). The exception was IRF4, whose mRNA levels were lower in S1–S3CCD4 compared to S3CCD4 Th17 cells, consistent with an important role of SOCE in IRF4 expression reported previously (Vaeth et al., 2017a). The normal expression of many factors required for Th17 differentiation in S1–S3CCD4 T cells was surprising given the strong attenuation of Th17 function in vitro and Th17 cell-mediated inflammation (Figure 1, Figure 2, Figure S1). These results suggested that STIM1 regulates additional factors that control the pathogenic function of Th17 cells.

Figure 2. STIM1 controls expression of genes associated with pathogenic Th17 cells.

Figure 2.

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(A) CD4+ T cells from WT, S1CD4, S3CCD4 and S1–S3CCD4 mice were cultured under non-polarizing (Th0) conditions or differentiated into non-pathogenic Th17 cells (IL-6, TGFb) in vitro for 3 days. IL-17A expression in Th0 or Th17 cells after stimulation with PMA and ionomycin for 6h. Representative flow cytometry plots (left) and means ± SEM of 7–8 mice per genotype from 4 repeat experiments. (B-F) Gene expression analysis by RNA-Seq in pathogenic Th17 cells from S3CCD4 and S1–S3CCD4 mice cultured for 2 days with IL-1β, IL-6 and IL-23 in vitro. Data from 3 mice per cohort. (B) Volcano plot of 632 differentially expressed genes (DEG) using an adjusted p-value < 0.05 and fold change > 1.5 as cut off. (C) Ingenuity pathway analysis (IPA) of RNA-Seq data. Shown are the fraction of DEG in each pathway and their p-value. (D) Fold changed expression of DEG related to Th cell differentiation (from IPA analysis in C) in S1–S3CCD4 compared to S3CCD4 Th17 cells; adjusted p-values < 0.05 for all DEG. (E) Gene set enrichment analysis (GSEA) of S3CCD4 and S1–S3CCD4 Th17 cells identifies a non-pathogenic Th17 gene signature in S1–S3CCD4 cells (gene set M5615). (F) Heat map of DEG associated with non-pathogenic (left) and pathogenic (right) Th17 cells as defined by (Gaublomme et al., 2015; Lee et al., 2012). *, p<0.05; ***, p<0.001. Statistical analysis in A by unpaired Student’s t test; in B, D and F by Wald test and Benjamini-Hochberg method; in C by right-tailed Fisher’s exact test. See also Figure S2.

To identify these factors, we analyzed gene expression signatures in CD4+ T cells from S3CCD4 and S1–S3CCD4 mice that were differentiated into pathogenic Th17 cells in vitro in the presence of IL-1β, IL-6 and IL-23 (Ghoreschi et al., 2010). S3CCD4 T cells expressed IL-17A but not IFNγ, indicating their selective polarization into Th17 cells (Figure S2B,C). RNA sequencing of Th17 cells revealed the dysregulation of 632 genes by > 1.5-fold in S1–S3CCD4 compared to S3CCD4 T cells, of which 482 genes were up- and 150 genes downregulated in S1–S3CCD4 Th17 cells (Figure 2B). Among the most strongly downregulated genes were IL-17A and IL-17F, whereas IL-10 and IFNγ were upregulated (Figure 2B, Figure S2D). Ingenuity pathway analysis (IPA) demonstrated dysregulation of genes in S1–S3CCD4 compared to S3CCD4 T cells that are associated with T helper cell differentiation (Figure 2C). In particular, the expression of Tbx21, Gata3 and Foxp3, which regulate the differentiation of Th1, Th2 and Treg cells was upregulated in S1–S3CCD4 Th17 cells (Figure 2D). Gene set enrichment analyses (GSEA) showed a correlation of gene expression in S1–S3CCD4 Th17 cells with published signatures of non-pathogenic Th17 cells (Figure 2E, Figure S2E) (Lee et al., 2012), while gene expression in S3CCD4 Th17 cells correlated with published pathogenic Th17 cell signatures (Figure S2F). The expression of > 90% of all genes previously associated with non-pathogenic Th17 cells (Gaublomme et al., 2015; Lee et al., 2012) was significantly increased in S1–S3CCD4 compared to S3CCD4 Th17 cells including Maf, Ahr, Il10 and Ikzf3 (Figure 2F), suggesting that upon loss of SOCE, S3CCD4 Th17 cells acquire a non-pathogenic identity. Analysis of genes associated with pathogenic Th17 cells (Gaublomme et al., 2015; Lee et al., 2012) showed a more balanced effect with 63% of pathogenic Th17 signature genes upregulated in S1–S3CCD4 compared to S3CCD4 Th17 cells, and 37% of transcripts downregulated (Figure 2F). The net effect of STIM1 deletion in Th17 cells is a bias towards a dominant expression of genes associated with non-pathogenic Th17 cells, consistent with the attenuated severity of multiorgan inflammation in S1–S3CCD4 mice.

SOCE regulates expression of genes associated with cycle regulation.

IPA pathway and GSEA analyses revealed an altered expression of genes associated with several T cell signaling pathways and cell cycle regulation in S1–S3CCD4 compared to S3CCD4 Th17 cells. The latter included Ccne1 (Cyclin E1) and Cdk4 that play important roles in cell cycle regulation in T cells (Figure 3A,B)(Wells and Morawski, 2014). When we differentiated pathogenic Th17 cells in vitro and monitored cell numbers over a period of 7 days, we observed a significantly reduced expansion of S1–S3CCD4 compared to S3CCD4 Th17 cells as well as S1CD4 and WT Th17 cells (Figure 3C). To analyze the role of STIM1 in cell cycle progression of Th17 cells, we analyzed the levels of Ki-67, which is detected in all phases of the cell cycle except G0. While the vast majority of WT, S1CD4 and S3CCD4 Th17 cells were Ki-67 positive 3 days after stimulation and differentiation in vitro, less than half of S1–S3CCD4 Th17 cells had upregulated Ki-67 expression, suggesting that they did not enter the cell cycle (Figure 3D). Impaired cell cycle entry was also evident from strongly decreased Th17 cell proliferation analyzed by dilution of CellTrace violet dye after Th17 cell culture for 5 days. Whereas the majority of WT, S1CD4 and S3CCD4 Th17 cells proliferated vigorously in vitro, S1–S3CCD4 Th17 cells showed a pronounced block in proliferation (Figure 3E). These findings demonstrate that SOCE controls cell cycle entry and proliferation of pathogenic Th17 cells, thus contributing to their ability to cause inflammation.

Figure 3. STIM1 controls cell cycle gene expression.

Figure 3.

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(A) GSEA of RNA-Seq data (Figure 2) identifies DEG in S1–S3CCD4 Th17 cells associated with cell cycle regulation (gene set M16647). (B) Fold changed expression of DEG related to cell cycle regulation in S1–S3CCD4 compared to S3CCD4 Th17 cells. (C-E) Analysis of cell cycle, proliferation and viability of pathogenic Th17 cells from WT, S1CD4, S3CCD4 and S1–S3CCD4 mice cultured for 3–7 days in vitro with IL-1β, IL-6 and IL-23. (C) Total cell numbers during Th17 differentiation. (D) Ki-67 expression on day 3 of Th17 cell differentiation. Representative flow cytometry plots (left) and means ± SEM from 3–9 mice and 3 repeat experiments (right). (E) Dilution of CellTrace Violet dye in Th17 cell after 5 days in culture. Representative flow cytometry plots (left) and means ± SEM from 3–9 mice and 3 repeat experiments (right). ***, p<0.001. Statistical analysis in C-E by unpaired Student’s t test.

STIM1 is a critical regulator of mitochondrial function in pathogenic Th17 cells.

Pathway analysis and GSEA in S1–S3CCD4 and S3CCD4 Th17 cells showed a marked deregulation of genes controlling mitochondrial function in the absence of STIM1 (Figure 4A,B). Most mitochondrial genes were downregulated in S1–S3CCD4 Th17 cells including some that play important roles in regulating the mitochondrial redox state such as thioredoxin 2 (Txn2) and Glycerol-3-Phosphate Dehydrogenase 2 (Gpd2)(Matsuzawa, 2017). The majority of downregulated genes, however, were subunits of electron transport chain (ETC) complexes that mediate mitochondrial respiration and oxidative phosphorylation (OXPHOS) with the strongest defects observed for genes encoding ETC complexes I, III, IV and V (Figure 4B,C). Overall, more than 60 genes encoding ETC complexes I-V were found to be reduced in S1–S3CCD4 compared to S3CCD4 Th17 cells. Significant reduction of complex I (NDUFB8), II (SDHB), III (UQCRC2) and IV (MTCO1) in S1–S3CCD4 Th17 cells was confirmed at the protein level (Figure 4D,E). These findings suggested that SOCE may be required for mitochondrial energy metabolism in Th17 cells.

Figure 4. STIM1 regulates mitochondrial gene expression and function in pathogenic Th17 cells.

Figure 4.

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(A-C) Analysis of RNA-Seq data from S3CCD4 and S1–S3CCD4 Th17 cells (Figure 2). (A) GSEA identifies DEG in S1–S3CCD4 Th17 cells associated with mitochondrial function (gene set M15112). (B) Fold changed expression of DEG related to mitochondrial function in S1–S3CCD4 compared to S3CCD4 Th17 cells. (C) Relative expression of electron transport chain (ETC) complex genes in S1–S3CCD4 compared to S3CCD4 Th17 cells. Heat map shows relative minimum (green) and maximum (red) values per row. (D,E) Expression of ETC complex proteins in CD4+ T cells from WT, S1CD4, S3CCD4, and S1–S3CCD4 mice differentiated into pathogenic Th17 cells in vitro for 3 days. (D) Representative Western blots of total cell lysates. (E) Quantification of ETC protein expression normalized to actin. Means ± SEM of 5 mice and 5 repeat experiments. (F,G) Mitochondrial respiration of pathogenic Th17 cells of WT, S1CD4, S3CCD4 and S1–S3CCD4 mice cultured into pathogenic Th17 cells for 3 days in vitro. (F) Oxygen consumption rates (OCR) measured by extracellular flux analysis. FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; Rot, rotenone; Anti, antimycin. (G) Means ± SEM of basal and maximal respiration and spare respiratory capacity from 5 repeat experiments and 7–10 mice per genotype. (H) Mitochondrial volume in pathogenic Th17 cells on day 3 in vitro measured using Mitotracker Deep Red. Representative flow cytometry plots (top) and means ± SEM of 9–10 mice and 4 repeat experiments (bottom). (I) Transmission electron microscopy images of pathogenic Th17 cells on day 3 in vitro. Left: Representative images at 7,100–15,000× magnification; boxes indicate areas enlarged in images on the right; scale bar 1 mm. Right: Means ± SEM of percentages of mitochondria with loose cristae structure from 2 mice per genotype and 218–285 mitochondria per genotype. *, p<0.05; **, p<0.01; ***, p<0.001. Statistical analysis in E by one-way analysis of variance (ANOVA); in G by unpaired Student’s t test; in I by Mann-Whitney test. See also Figures S3, S4 and S5.

To test the role of SOCE in mitochondrial function in pathogenic Th17 cells, we measured mitochondrial respiration in pathogenic Th17 cells in vitro. Oxygen consumption rates (OCR) as a readout of mitochondrial respiration were significantly reduced in S1–S3CCD4 compared to S3CCD4 Th17 cells. This included decreased basal respiration as well as maximal respiration, spare respiratory capacity (SRC) and ATP production in S1–S3CCD4 Th17 cells after uncoupling of mitochondria with FCCP (Figure 4F,G; Figure S3A). It is noteworthy that these differences in mitochondrial respiration were specific to pathogenic effector Th17 cells because freshly isolated naïve CD4+ T cells from S1–S3CCD4 mice showed comparable basal and maximal OCR, SRC and ATP production compared to naive WT, S1CD4 and S3CCD4 T cells (Figure S3B). Despite the defects in ETC complex expression and mitochondrial respiration in S1–S3CCD4 Th17 effector cells, their mitochondrial membrane potential (MMP) was comparable to that of pathogenic S3CCD4 Th17 cells (Figure S3C). This finding is likely explained by the reduced proton leak of mitochondria in S1–S3CCD4 compared to S3CCD4 Th17 cells (Figure S3D). Impaired mitochondrial respiration in S1–S3CCD4 compared to S3CCD4 Th17 cells was not due to decreased numbers of mitochondria (Figure S3E) or reduced expression of regulators of mitochondrial gene expression, genome replication and biogenesis such as TFAM, PGC-1b and PPRC (Figure S3F)(Scarpulla, 2011). Although mitochondrial numbers were normal in S1–S3CCD4 Th17 cells, mitochondrial mass was increased compared to S3CCD4 (and WT or S1CD4) Th17 cells (Figure 4H). The shape of mitochondria and their cristae structure affects mitochondrial function. Fission of mitochondria was shown to lead to cristae expansion and reduced ETC efficiency, whereas mitochondrial fusion promotes ETC function and OXPHOS in memory T cells (Buck et al., 2016), e.g. by affecting the formation of respiratory chain supercomplexes (Cogliati et al., 2013). Examination of mitochondrial morphology in pathogenic Th17 cells by transmission electron microscopy showed that the mitochondria in S3CCD4 Th17 cells had a dense cristae structure, whereas the majority of mitochondria in S1–S3CCD4 Th17 cells had loose cristae and appeared swollen (Figure 4I, Figure S4), which may explain their increased size. The altered inner mitochondrial membrane (cristae) architecture of S1–S3CCD4 Th17 cells is consistent with reduced ETC function and OXPHOS. Together, these findings demonstrate that deletion of STIM1 in pathogenic Th17 cells compromises mitochondrial gene expression, structure and function.

It is noteworthy that glycolysis and mTOR signaling were largely normal in pathogenic S1–S3CCD4 Th17 cells (Figure S5). Th17 cell differentiation depends on the AKT-mTOR signaling pathway and aerobic glycolysis (Dang et al., 2011; Michalek et al., 2011; Shi et al., 2011) and we recently showed that complete inhibition of SOCE by deletion of STIM1 and STIM2 severely impairs glycolysis and proliferation after TCR stimulation (Vaeth et al., 2017a). By contrast, we found that expression of glycolysis-associated genes was not altered in pathogenic Th17 cells from S1–S3CCD4 compared to S3CCD4 mice after differentiation for 3 days in vitro (Figure S5A). This finding was consistent with comparable glycolysis (determined by measurements of the extracellular acidification rate, ECAR) (Figure S5B), phosphorylation of AKT, mTOR and S6 (Figure S5D) and glucose uptake (Figure S5E) in T cells of S1–S3CCD4, S3CCD4, S1CD4 and WT mice 3 days after differentiation into pathogenic Th17 cells in vitro. A moderate reduction in mTOR and S6 phosphorylation and glucose uptake was observed in S1–S3CCD4 Th17 cells 1 day after TCR stimulation (Figure S5C, E), consistent with a role of SOCE in the metabolic switch of naive T cells to glycolysis early after TCR stimulation (Vaeth et al., 2017a). Th17 cell differentiation and the pathogenic function of Th17 cells in EAE was shown to require glutamine uptake via the glutamine transporters ASCT2 (Slc1a5) and LAT1 (Slc7a5) (Nakaya et al.; Sinclair et al.). Both genes, as well as those of other glutamine transporters (Slc38a1, Slc38a2 and Slc3a2), were expressed at similar levels in S1–S3CCD4 and S3CCD4 Th17 cells (Figure S5F). The expression of enzymes mediating glutamine metabolism and its conversion into α-ketoglutarate was normal (Gls, Gpt) or only moderately reduced (Gls2, Glud1) in S1–S3CCD4 compared to S3CCD4 Th17 cells (Figure S5G). Collectively, these data show that the pathogenic function of Th17 cells does not depend on the regulation of glucose or glutamine metabolism by STIM1 and SOCE.

STIM1 controls mitochondrial ROS levels in pathogenic Th17 cells.

An important role of mitochondrial function besides ATP production is the generation of mitochondrial ROS (mROS). Because mitochondrial respiration and ETC complex expression were reduced in S1–S3CCD4 Th17 cells, we expected to find reduced mROS levels, which might account for impaired pathogenic Th17 cell function given the role of ROS in redox signaling and T cell function including activation of NFAT (Mak et al., 2017; Sena et al., 2013). However, basal and oligomycin-induced mROS levels of in vitro differentiated S1–S3CCD4 Th17 cells were significantly increased compared to WT, S1CD4 and S3CCD4 Th17 cells (Figure 5A). At high levels, ROS causes T cell apoptosis (Belikov et al., 2015) and we observed reduced numbers of live PI– Annexin V– S1–S3CCD4 Th17 cells compared to WT, S1CD4 and S3CCD4 Th17 cells (Figure 5B). Reduced viability of S1–S3CCD4 Th17 cells did not correlate with altered expression of pro- or anti-apoptotic genes in the RNA-Seq data.

Figure 5. STIM1 controls mitochondrial ROS levels in pathogenic Th17 cells.

Figure 5.

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(A-F) Analysis of pathogenic Th17 cells from WT, S1CD4, S3CCD4 and S1–S3CCD4 mice cultured with IL-1β, IL-6 and IL-23 for 3 days in vitro. (A) mROS levels. Th17 cells were loaded with MitoSOX, treated with 1 mM oligomycin and analyzed for basal (0 min, right) and oligomycin-induced (0–120 min, left) MitoSOX fluorescence. Means ± SEM of the mean fluorescence intensity (MFI) of S1CD4, S3CCD4 and S1–S3CCD4 cells normalized to the MFI in WT cells at 0 min (DMFI). Data from 5–9 mice and 4 repeat experiments. (B) Apoptosis measured by propidium iodide (PI) and Annexin V staining of Th17 cells after 5 days in vitro. Representative flow cytometry plots (left) and means ± SEM of 3–6 mice and 3 repeat experiments (right). Live cells defined as PI– Annexin V–. (C) Relative expression of Sod2, Gpx1, Gpx4, Bcl2 and Nfe2l2 in Th17 cells determined by RNA-Seq (Figure 2). (D) Protein expression of antioxidants in pathogenic Th17 cells from WT, S1CD4, S3CCD4 and S1S3CCD4 mice cultured for 3 days in vitro. Representative Western blots (left) and means ± SEM of 3 (SOD2) and 6 (NRF2) mice. Expression was normalized to actin. (E) Glutathione levels. Pathogenic Th17 cells from S3CCD4 and S1–S3CCD4 mice cultured for 24h in vitro and analyzed for the ratio of total glutathione (GSH) to oxidized glutathione (GSSG). Means ± SEM of 5 mice per genotype from 2 independent experiments. (F-H) Analysis of pathogenic Th17 cells cultured for 3 days in vitro. Th17 cells were treated or not with NAC for 24h before analysis. (F) Basal mROS production measured by MitoSOX. (G) DNA damage analyzed by H2A.X phosphorylation on serine (Ser) 139. (H) Viability of Th17 cells analyzed using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit. Panels B and D-F show representative flow cytometry plots (left) and means ± SEM of 5–7 mice from at least 2 repeat experiments (right). *, p<0.05; **, p<0.01; ***, p<0.001. Statistical analysis in A-C and E-H by unpaired Student’s t test; in D by one-way analysis of variance (ANOVA). See also Figure S6.

ROS levels in cells are regulated by anti-oxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GPX), which catalyze the conversion of O2− into H2O2 and H2O2 into H2O, respectively (Belikov et al., 2015). mRNA levels of Sod2, Gpx1 and Gpx4 were significantly reduced in S1–S3CCD4 compared to S3CCD4 Th17 cells (Figure 5C). In addition, mRNA levels of the transcription factor Nrf2, which regulates antioxidant gene expression (Motohashi and Yamamoto, 2004), and Bcl2, whose antioxidant function protects cells from oxidative death, were reduced in S1–S3CCD4 Th17 cells. mRNA levels of Nrf1, the NRF2 inhibitor Keap1 and Gclc, which encodes the catalytic subunit of glutamate-cysteine ligase required for glutathione production, were not altered in S1–S3CCD4 compared to S3CCD4 Th17 cells (Figure S6A). Whereas protein levels of GPX1/2, GPX4 and GCLC were not altered in S1–S3CCD4 Th17 cells (Figure S6B), those of NRF2 and its transcriptional target SOD2 were significantly increased in S1–S3CCD4 relative to S3CCD4, S1CD4 and WT Th17 cells (Figure 5D). Elevated NRF2 protein levels are likely secondary to increased mROS in S1–S3CCD4 Th17 cells as ROS is known to oxidize KEAP1, which results in the dissociation of KEAP1 from NRF2 and stabilization of NRF2 protein (Motohashi and Yamamoto, 2004). Collectively, our data suggest that SOCE has a dual effect on the antioxidant response in pathogenic Th17 cells by promoting the transcription of antioxidant genes and by regulating NRF2 protein stability. The antioxidant glutathione (GSH) is essential to prevent ROS-mediated damage. GSH-deficient murine T cells have increased ROS levels, lack IL-17A and fail to cause EAE, suggesting that GSH plays an important role in pathogenic Th17 cell function (Mak et al., 2017). We found that levels of the reduced form of GSH were decreased in pathogenic Th17 cells of S1–S3CCD4 compared to S3CCD4 mice at 24h and 48h after TCR stimulation (Figure S6C). In addition, we measured the ratio of total glutathione (GSH plus GSSG) to oxidized glutathione disulfide (GSSG) to assess glutathione turnover. S1–S3CCD4 Th17 cells showed a reduced ratio of total to oxidized glutathione compared to S3CCD4 Th17 cells (Figure 5E). These data suggest that the consumption of GSH is increased consistent with increased ROS levels and oxidative stress in the absence of SOCE.

To test the effects of elevated mROS in STIM1-deficient pathogenic Th17 cells, we treated S1–S3CCD4 and S3CCD4 Th17 cells with the ROS scavenger N-acetylcysteine (NAC), which resulted in a significant reduction of mROS in S1–S3CCD4 Th17 cells to levels comparable in untreated S3CCD4 Th17 cells (Figure 5F). Since high ROS levels cause DNA damage and cell death (Belikov et al., 2015), we measured the phosphorylation of histone H2A.X (pH2A.X) on serine 139 (S139), which is an early marker of DNA damage. S1–S3CCD4 Th17 cells had significantly increased levels of H2A.X phosphorylation compared to S3CCD4 Th17 cells (Figure 5G). Treatment of S1–S3CCD4 Th17 cells with NAC significantly reduced H2A.X phosphorylation to levels found in untreated S3CCD4 Th17 cells. Importantly, treatment of S1–S3CCD4 Th17 cells with NAC significantly decreased the number of dead cells to levels found in untreated S3CCD4 Th17 cells (Figure 5H). It is noteworthy that addition of NAC during Th17 cell differentiation in vitro had no effect on the reduced expression of ETC proteins in S1–S3CCD4 compared to S3CCD4, S1CD4 and WT Th17 cells (Figure S6D,E), demonstrating that reduced ETC gene expression is not secondary to increased mROS or cell death in the absence of STIM1 in pathogenic Th17 cells. Taken together, our findings indicate that depletion of SOCE in pathogenic Th17 cells results in increased mROS production, DNA damage and decreased viability of Th17 cells.

Mitochondrial function and OXPHOS are critical for pathogenic Th17 cell function.

Increased DNA damage and cell death were not limited to S1–S3CCD4 Th17 cells in vitro. Pathogenic Th17 cells isolated from the lungs of S1–S3CCD4 mice also showed strongly enhanced H2A.X phosphorylation and cell death compared to S3CCD4 mice (Figure 6A,B). To prove that mitochondrial function and OXPHOS are critical for the pathogenicity of Th17 cells, we treated Th17 cells isolated from the lungs of S3CCD4 mice with oligomycin to block ATP synthase and OXPHOS. IL-17A expression was significantly reduced in oligomycin-treated pathogenic Th17 cells (Figure 6C). An even more striking suppression of IL-17A levels was observed when CD4+ splenocytes of S3CCD4 mice were differentiated into pathogenic Th17 cells in vitro and treated with oligomycin (Figure 6D). It is noteworthy that reduction of IL-17A expression by oligomycin in Th17 cells of S3CCD4 mice was not due to decreased SOCE (Figure S7A). Next, we investigated the expression of genes associated with pathogenic and non-pathogenic Th17 cells. Oligomycin treatment significantly reduced the expression of Rorc, Il17a and Il3 expressed by pathogenic Th17 cells, whereas mRNA levels of genes associated with a non-pathogenic Th17 cell signature such as Ikzf3, Ahr and Maf were increased following oligomycin treatment (Figure 6E and 2F), suggesting that OXPHOS regulates the expression of genes associated with pathogenic Th17 cells.

Figure 6. OXPHOS is critical for pathogenic Th17 cell function.

Figure 6.

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(A-B) Analysis of CD4+ T cells isolated from the lungs of 6–9 week old S3CCD4 and S1–S3CCD4 mice. (A) DNA damage in T cells analyzed by H2A.X phosphorylation. (B) Viability of T cells analyzed using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit. (C) IL-17A expression by pathogenic Th17 cells isolated from the lungs of 6–9 week old S3CCD4 mice and treated with 1 μM oligomycin or DMSO for 1h before and during the 6h stimulation with PMA and Ionomycin (P+I). (D) IL-17A expression by pathogenic Th17 cells of S3CCD4 mice cultured for 3 days in vitro with IL-1β, IL-6 and IL-23. Cells were treated with DMSO or oligomycin for 2 days before and during stimulation with P+I for 6h. Panels AD show representative flow cytometry plots (left) and means ± SEM from 6–7 mice and 2 repeat experiments (right). (E) Oligomycin-dependent expression of pathogenic and non-pathogenic Th17 cell signature genes. Pathogenic Th17 cells from S3CCD4 mice cultured as in D were treated for 2 days with DMSO or oligomycin before realtime PCR analysis of gene expression. Means ± SEM of 6 mice and 2 repeat experiments. *, p<0.05; **, p<0.01; ***, p<0.001. Statistical analysis in A and B by unpaired Student’s t test; in C, D and E by paired Student’s t test.

To test the effects of OXPHOS on pathogenic Th17 cells in vivo, we transferred CD25low CD4+ T cells from S3CCD4 mice into Rag1−/− mice to induce Th17 cell-mediated inflammation. Recipient mice lost >10% of their body weight by 5 weeks after T cell transfer, consistent with the expected colitis in these mice (Figure S7B). Recipient mice also developed severe pulmonary inflammation with peribronchiolar and perivascular leukocyte infiltration (Figure 7A). Treatment of mice with oligomycin to suppress pathogenic Th17 cell function in vivo was not feasible due to the toxic effects of prolonged oligomycin application. We therefore analyzed the effects of OXPHOS inhibition on pathogenic Th17 cells isolated from the lungs and colon lamina propria (LP) of recipient Rag1−/− mice. Oligomycin treatment of pulmonary and LP Th17 cells significantly attenuated the expression of IL-17A whereas it had no effect on IFNγ production (Figure 7B,C). Inhibition of glycolysis with 2-DG, which was shown to be essential for Th17 cell function (Dang et al., 2011; Michalek et al., 2011; Shi et al., 2011), significantly reduced the expression of IL-17A (but not IFNγ) in pathogenic Th17 cells isolated from the lung, whereas IL-17A expression in LP Th17 cells was normal (Figure 7B,C). Simultaneous inhibition of OXPHOS and glycolysis almost completely suppressed IL-17A production in both tissues. Similar results were obtained with Th17 cells isolated from the lungs of S3CCD4 mice that were treated with oligomycin, 2-DG or both (Figure S7C). Collectively, these findings show that OXPHOS is critical for the function of pathogenic Th17 cells. To investigate the T cell-intrinsic role of SOCE in the pathogenic function of STAT3-dependent Th17 cells, we adoptively transferred T cells from S1–S3CCD4 and S3CCD4 mice to Rag1−/− hosts to induce colitis. Transfer of naïve CD4+ T cells from S3CCD4 mice resulted in severe weight loss and transmural colonic inflammation, pronounced patch inflammation and mucine depletion resembling severe lymphocytic cryptitis in Rag1−/− hosts (Figure 7D–F). By contrast, STIM1-deficient T cells from S1–S3CCD4 mice induced only moderate and delayed weight loss with attenuated severity of colitis. The differences in weight loss and colitis severity correlated with reduced levels of proinflammatory cytokines IL-17A, IFNγ and TNFα in S1–S3CCD4 compared to S3CCD4 CD4+ T cells isolated from mesenteric lymph nodes of Rag1−/− mice (Figure 7G). Collectively these findings demonstrate that the function of pathogenic Th17 cells and the resulting multiorgan inflammation are dependent on SOCE.

Figure 7. STIM1 and OXPHOS are critical for pathogenic Th17 cell function.

Figure 7.

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(A-C) Analysis of pathogenic Th17 cells 5 weeks after i.v. injection of 1×106 CD4+CD25low T cells from S3CCD4 mice into Rag1−/− mice. (A) Lung histologies and H&E stains of Rag1−/− mice. Images are representative of 9 mice per cohort. (B) IL-17A and IFNγ production by T cells isolated from the lung and colon lamina propria (LP) of Rag1−/− mice. Isolated T cells were treated with DMSO, 1 μM oligomycin, 2 mM 2-DG or oligomycin plus 2-DG for 1 h before and during 6h stimulation with PMA and ionomycin. (C) Means ± SEM of IL-17A and IFNγ production normalized to DMSO from 2 repeat experiments and 9 mice per group. (D-G) STIM1 deletion in pathogenic Th17 cells attenuates colitis. 1×106 naïve CD4+ T cells from WT, S1CD4, S3CCD4 and S1–S3CCD4 mice were injected into Rag1−/− mice, which were analyzed 10 weeks later. (D) Body weight change relative to the starting weight. (E) Representative colon histologies of mice stained with H&E. (F) Colitis disease score based on colon histologies of 14–16 mice per cohort. (G) Flow cytometry analysis of IL-17A, IFN-g and TNFα expression by CD4+ T cells isolated from mesenteric LNs and stimulated ex vivo with PMA and ionomycin for 6h. Means ± SEM of 5–8 mice. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001. Statistical analysis in C by paired Student’s t test; in D, F and G by unpaired Student’s t test. See also Figure S7.

DISCUSSION

Pathogenic Th17 cells cause inflammation in a variety of autoimmune diseases as well as steroid-resistant asthma and COPD (Doe et al., 2010; Korn et al.). We and others had shown that SOCE mediated by STIM1 is essential for Th17 cell function and autoimmunity in mouse models of MS and IBD (Kaufmann et al., 2016; Kim et al., 2014; Ma et al., 2010; McCarl et al., 2010; Vaeth et al., 2017b), but the mechanisms by which SOCE regulates the function of pathogenic Th17 cells remained poorly understood. To investigate the role of SOCE specifically in pathogenic, proinflammatory Th17 cells, we deleted STIM1 in S3CCD4 mice. Enhanced STAT3 signaling in CD4+ T cells of S3CCD4 mice results in spontaneous Th17 differentiation and Th17-mediated multiorgan inflammation (Fogli et al., 2013; Yang et al., 2018), making these mice an excellent model to investigate the pathways involved in pathogenic Th17 cell function. We found that SOCE is essential for pathogenic Th17 cell function because STIM1 deletion resulted in (i) suppressed IL-17A production, (ii) prevented severe pulmonary, skin and intestinal inflammation and (iii) a shift towards the expression of genes associated with a non-pathogenic Th17 cell signature. Attenuated Th17 cell function was associated with decreased expression of genes associated with mitochondrial function, in particular ETC complexes, impaired OXPHOS and altered mitochondrial morphology, suggesting that STIM1-dependent gene expression is necessary for mitochondrial function and structural intergrity. Despite impaired mitochondrial respiration, mROS levels were elevated in SOCE-deficient S1–S3CCD4 Th17 cells resulting in enhanced DNA damage and death of Th17 cells, which likely contributes to the protection of S1–S3CCD4 mice from Th17-mediated multiorgan inflammation. Direct inhibition of OXPHOS in pathogenic Th17 cells from S3CCD4 mice reduced IL-17A production but enhanced expression of genes associated with non-pathogenic Th17 cells, suggesting that OXPHOS is required for pathogenic Th17 cell function. Taken together, our data demonstrate that SOCE is required for the pathogenic function of Th17 cells and their ability to cause inflammation by regulating mitochondrial function, especially OXPHOS, and preventing oxidative stress in Th17 cells.

Our findings indicate that the SOCE and STAT3 pathways synergize to mediate pathogenic Th17 cell function. The promoters of several genes required for the differentiation and function of Th17 cells have binding sites for both STAT3 and the transcription factor NFAT including Rorc, Il17a and Il21 (Hermann-Kleiter and Baier, 2010). NFAT, which is activated by the Ca2+-regulated phosphatase calcineurin, is known to cooperatively bind with other transcription factors such as IRF4, AP-1 and Runx1 to the promotors of Rorc and Il17 in Th17 cells (Hermann-Kleiter and Baier, 2010). NFATc1 was also shown to cooperatively bind to DNA together with STAT3 in pancreatic cancer cells thereby promoting cancer initiation and progression (Baumgart et al., 2014). A similar direct cooperation between NFAT and STAT3 in Th17 cells has not been reported. NFAT and STAT3 may also cooperate indirectly as IL-6R signaling via STAT3 enhances the expression of NFATc2 and thereby increases NFATc2 function following TCR stimulation (Diehl et al., 2002).

It is noteworthy that the expression of transcription factors (RORγt, RORα, HIF1α, BATF) and cytokine receptors (IL-1R, IL-6R, IL-23R) required for Th17 differentiation was not altered in S1–S3CCD4 Th17 cells despite their abolished pathogenic function. The only exception was IRF4, whose expression in S1–S3CCD4 Th17 cells was reduced consistent with the known role of IRF4 in Th17 differentiation (Ciofani et al., 2012) and our previous observation that IRF4 expression in CD4+ T cells depends on SOCE (Vaeth et al., 2017a). These data suggest that expression of RORγt, RORα, HIF1α, BATF, IL-1R, IL-6R and IL-23R in S1–S3CCD4 Th17 cells is not sufficient to render Th17 cells pathogenic given the strongly attenuated multiorgan inflammation in S1–S3CCD4 mice. A potential explanation for this attenuation is the relative upregulation of genes in S1–S3CCD4 Th17 cells that were reported to be highly expressed in non-pathogenic Th17 cells including Maf, Ikzf3, Ahr, Socs2 and Ctlab2. Overall, S1–S3CCD4 Th17 cells had a gene expression signature that resembled nonpathogenic rather than pathogenic Th17 cells.

We here show that SOCE is essential for mitochondrial gene expression, function and structural integrity in pathogenic Th17 cells. In the absence of STIM1, the expression of nuclear encoded ETC genes was reduced resulting in strongly impaired mitochondrial respiration and OXPHOS. Moreover, we show that OXPHOS is required for the function of pathogenic Th17 cells as OXPHOS inhibition suppressed IL-17A expression and biased their gene expression profile toward that of non-pathogenic Th17 cells. The reliance of pathogenic Th17 cells on mitochondrial respiration is consistent with a recent study showing that in vivo differentiated Th17 cells require OXPHOS as oligomycin impaired their effector function and ameliorated TNBS-induced colitis in vivo (Franchi et al., 2017). The role of SOCE in regulating OXPHOS is consistent with our recent reports that the expression of ETC genes in effector T cells depends on SOCE (Vaeth et al., 2017a) and that fatty acid oxidation (FAO) in fibroblasts requires SOCE (Maus et al., 2017). How SOCE regulates ETC gene expression is not known, but preliminary in silico analyses revealed binding of NFAT to the promoters of several ETC genes (including Ndufs3, Ndufs6 and Uqcrc1) that were downregulated in S1–S3CCD4 Th17 cells. The reliance of pathogenic Th17 cells on OXPHOS is similar to long-lived memory CD8+ T cells which depend on FAO and OXPHOS for survival and function (van der Windt et al., 2012). Given the long-lived nature and stem cell-like molecular signature reported for Th17 cells (Muranski et al., 2011), it is possible that OXPHOS allows pathogenic Th17 cells to persist as effector cells, self-renew and generate effector progeny.

Surprisingly, we did not observe a defect in glycolysis in STIM1-deficient Th17 cells that could account for their abolished pathogenicity. The differentiation of Th17 cells, like that of other proinflammatory effector CD4+ T cell subsets, depends on glycolysis (Michalek et al., 2011; Pearce et al., 2013; Wang and Solt, 2016). However, we found that the expression of glycolytic enzymes and transcription factors such as HIF-1a, c-Myc and IRF4 that regulate glycolysis (Dang et al., 2011; Mahnke et al., 2016; Shi et al., 2011) was unaltered in S1–S3CCD4 compared to S3CCD4 Th17 cells. Glycolysis (ECAR), glucose uptake and the phosphorylation of AKT, mTOR and S6, which regulate glycolytic gene expression, were also normal. These findings were surprising because we had recently shown an important role of SOCE in the metabolic switch of naive T cells to aerobic glycolysis after TCR stimulation, which allows T cells to enter the cell cycle and proliferate (Vaeth et al., 2017a). Indeed, we observed a moderate defect in mTOR signaling and glucose uptake of STIM1-deficient pathogenic Th17 cells one day after T cell stimulation, which was no longer observable after 3 days, suggesting that SOCE is required for the induction of glycolysis early after TCR stimulation but is dispensable for maintaining glycolysis during the differentiation of pathogenic Th17 cells. Our findings do not contradict the important role of mTOR signaling or glycolysis in Th17 cell function; they demonstrate however that STIM1 deletion interferes with pathogenic Th17 cell function in a manner independent of glycolysis.

We here show that SOCE regulates mROS levels in pathogenic Th17 cells. ROS has multiple functions in T cells. Low ROS concentrations mediate redox signaling that promotes T cell activation and cytokine production (Belikov et al., 2015). Deletion of the ETC complex III gene Uqcrfs1 in murine T cells reduced mROS levels and impaired IL-2 production and T cell proliferation, which were associated with decreased SOCE and NFAT activation (Sena et al., 2013). At high levels, ROS causes T cell apoptosis (Belikov et al., 2015). We found that mROS was increased in S1–S3CCD4 compared to S3CCD4 Th17 cells resulting in enhanced DNA damage and death of Th17 cells, likely accounting for some of the protection from multiorgan inflammation in S1–S3CCD4 mice. mROS levels were increased in STIM1-deficient Th17 cells despite reduced expression of ETC genes and mitochondrial respiration. The most likely explanation for this finding is the abnormal mitochondrial morphology in S1–S3CCD4 Th17 cells characterized by loose cristae packing. The efficient transfer of electrons through the ETC requires the assembly of ETC supercomplexes consisting of CI, CIII and CIV (Mannella, 2008; Winge, 2012). The supramolecular organization of ETC supercomplexes is not only necessary to enhance the catalytic activity of the ETC and its ability to transfer electrons with high efficiency, it also reduces the electron leak and ROS production by mitochondria. In fact, superoxide production from complex I was strongly increased when the association of complex I and III was disrupted (Maranzana et al., 2013). Reduced expression of ETC genes in STIM1-deficient Th17 cells and altered inner mitochondrial membrane architecture are likely to interfere with the assembly of ETC supercomplexes, thereby increasing the electron leak and ROS production.

An additional explanation for increased mROS levels in S1–S3CCD4 Th17 cells is their deregulated antioxidant response. We found that mRNA levels of antioxidant genes (Sod2, Gpx1 Gpx4) and the transcription factor Nrf2, which regulates the expression of Sod2 and genes involved in glutathione synthesis (Belikov et al., 2015), are reduced in STIM1-deficient pathogenic Th17 cells. Protein levels of NRF2 and SOD2, however, were elevated in S1–S3CCD4 Th17 cells. The discrepancy between NRF2 mRNA and protein levels suggests a dual role of SOCE in the regulation of the antioxidant response. At the transcriptional level, SOCE promotes the expression of Nrf2 and its target genes Sod2, Nqo1 and Slc7a11. At the protein level, increased mROS due to increased electron leak from the ETC in STIM1-deficient Th17 cells likely enhances NRF2 protein stability via oxidation of the adaptor protein KEAP1, which results in dissociation of KEAP1 from NRF2 and prevents proteasomal degradtion of NRF2 (Motohashi and Yamamoto, 2004). Despite increased NRF2 protein levels in S1–S3CCD4 Th17 cells, the antioxidant response is insufficient to reduce ROS levels and to prevent DNA damage and apoptosis of Th17 cells. An important antioxidant and ROS scavenger, which regulates redox signaling in T cells and prevents cell death, is glutathione (GSH) (Belikov et al., 2015). We observed that the ratio of total GSH to its oxidized form GSSG was reduced in S1–S3CCD4 Th17 cells after TCR stimulation, demonstrating that glutathione consumption is increased in STIM1-deficient Th17 cells, which is consistent with increased ROS levels and oxidative stress in the absence of SOCE. GSH was recently shown to be essential for T cell function as deletion of glutamate cysteine ligase (GCLC) in T cells prevented GSH production and increased ROS levels (Mak et al., 2017). GCLC-deficient T cells lacked IL-17A and IFNγ production and failed to cause EAE, suggesting that GSH plays an important role in pathogenic Th17 cells.

Our study identifies an important role of SOCE in pathogenic Th17 cells. Inhibition of SOCE may represent a novel therapeutic approach to attenuate Th17 cell-mediated inflammation in the context of autoimmune and inflammatory diseases such as MS, colitis, psoriasis or steroid-resistant asthma.

Limitations of Study

The pathogenic Th17 cells we investigate develop in response to a hyperactive form of STAT3. While several cytokines (including IL-6, IL-23) that drive the differentiation of pathogenic Th17 cells signal through STAT3, the strong STAT3C signal in our model may overemphasize the role of STAT3 in pathogenic Th17 cells at the expense of other signals. The reasons we chose the STAT3C model to analyze the role of SOCE in pathogenic Th17 cells are the following: (a) T cell-specific induction of STAT3C results in selective differentiation of pathogenic, proinflammatory Th17 cells in vivo, with no obvious impact on other Th subsets; (b) there are currently no molecular markers for pathogenic Th17 cells in vivo that can be used to differentiate them from non-pathogenic Th17 cells and to conduct a molecular and functional analysis in vitro. Future studies should analyze (i) how SOCE regulates the function of naturally developing pathogenic Th17 cells in vivo once reliable markes become available and (ii) the metabolic requirements of pathogenic and nonpathogenic Th17 cells and the role of SOCE in these metabolic pathways.

Supplementary Material

1

Highlights.

  • STIM1-mediated calcium flux controls ETC expression in pathogenic Th17 cells

  • STIM1 deficiency impairs OXPHOS and increases mROS in Th17 cells

  • Loss of STIM1 and OXPHOS inhibition result in a non-pathogenic Th17 gene signature

  • Lack of STIM1 protects from Th17 cell-mediated lung, intestinal and skin inflammation

ACKNOWLEDGEMENTS

We thank Dr. C.Z. Liu for histological analysis, Dr. A. Liang, C. Petzold and J. Sall for assistance with electron microscopy, the Genome Technology Center (GTC) for RNA sequencing, T. Lhakhang (Applied Bioinformatics Laboratory) for bioinformatics support and Dr. T. Papagiannakopoulos as well as members of the Feske lab for helpful discussions. Bioinformatics analysis used computing resources at the High Performance Computing Facility (HPCF) at NYU Langone Medical Center. This work was supported by National Institutes of Health (NIH) grants AI097302 and AI130143 to S.F. and HL125816 to S.B.K., postdoctoral fellowships by the Deutsche Forschungsgemeinschaft (DFG) KA 4083/2–1 to U.K. and KA 4514/1–1 to S.K. and a postdoctoral fellowship from the National Multiple Sclerosis Society (NMSS) FG-1608–25544 to U.K.. The GTC and the Experimental Pathology core are supported by Cancer Center Support Grant P30CA016087 (Laura and Isaac Perlmutter Cancer Center). The Seahorse XFe24 instrument used for this study was purchased with funding from NIH grant 1S10OD016304-01.

Footnotes

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Declaration of Interests: S.F. is a scientific cofounder of Calcimedica. The other authors declare no conflict of interest.

Data and materials availability: The mouse lines described in this study are available from our laboratory and require a Material Transfer Agreement (MTA). The GEO accession number for RNA sequencing data is GSE125204.

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