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CRAC (ORAI1, STIM1)

CRAC (ORAI1, STIM1)

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CRAC (ORAI1, STIM1)

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  1. CRAC Ca2+ channels are essential for CD4+ T cell function in inflammatory bowel disease Lymphocyte function is controlled by many ion channels Lymphocyte function is controlled by many ion channels Stefan Feske, MD , Department of Pathology New York University School of Medicine CRAC (ORAI1, STIM1) P2X TRP? Cav? Kv1.3 KCa3.1 TRPM7 MagT1 Ca2+ K+ Ca2+ TRPM4 Na+ Mg2+ (Ca2+) T cell ZIP, ZnT Zn2+ Cl– Disclosure CalciMedica: co-founder and consultant

  2. S. typhi. SFB B. fragilis DC MF Naïve CD4 Naïve CD4 Naïve CD4 IL-23 TGFb IL-12 IL-23R* Treg Foxp3 Th1 Th17 IL-21* IL-10 TGFb IL-17A IL-17F IL-22 CCR6* IFN-g*

  3. Store-Operated Ca2+ Entry (SOCE) S. typhi. SFB B. fragilis Antigen DC MF Naïve CD4 Ca2+ Naïve CD4 Naïve CD4 IL-23 TGFb IL-12 IL-23R Treg Foxp3 Th1 Th17 IL-21 IL-10 TGFb NFAT ERK NFkB IL-17A IL-17F IL-22 CCR6 IFN-g

  4. CRAC channel pore protein ORAI1 is required in CD4+ T cells to cause IBD in mice CD45RB T cell transfer model of colitis Weight loss Isolate naïve CD4+ T cells (CD45RBhi CD25neg) +/+ +/+ +/+ +/+ KI/KI KI/KI IBD WT or CRAC-deficient mice Rag2-/- mice (no T, B or Treg cells) Weeks after T cell transfer Orai1KI/KI WT WT Orai1KI/KI

  5. Ca2+ influx levels in CD4+ T cells determine severity of colitis CD4+ T cells WT [Ca2+]i Orai1KI Stim1fl/fl Weight loss after CD4+ T cell transfer min Stim2fl/fl Orai1fl/fl WT [Ca2+]i WT Stim1fl/fl Cd4Cre Cd4Cre min ER Weeks Nucleus WT Stim1fl/fl Cd4Cre [Ca2+]i WT Stim2fl/fl Cd4Cre min

  6. Ca2+ influx levels in CD4+ T cells determine severity of colitis WT [Ca2+]i Orai1KI Stim1fl/fl Weight loss after CD4+ T cell transfer min Stim2fl/fl Orai1fl/fl WT [Ca2+]i WT Stim1fl/fl Cd4Cre Cd4Cre min Ca2+ WT ER Weeks Stim1fl/fl Nucleus Stim2fl/fl Colitogenic Threshhold [Ca2+]i WT Orai1fl/fl Stim2fl/fl Cd4Cre time min

  7. Ca2+ signaling through CRAC channels is not required for Proliferation, survival or gut homing of CD4+ T cells MFI Lamina propria MFI • Normal numbers of CD4+ T cells (including Foxp3+Treg cells) • Normal expression of T cell homing molecules • Normal apoptosis and proliferation rates • in lamina propria (colon) and mesenteric lymph nodes

  8. SOCE levels determine IFNgand IL-17 production in lamina propria CD4+ T cells Lamina propria MFI Cytokine production by CD4+ T cells in lamina propria

  9. SOCE is required for Th17 cell function & differentiation IL-17A IL-17F IL-22 Th17 Naïve CD4+ T cell Th17 TGF-b IL-6 RORgt RORgt In vitro differentiatedTh17 cells

  10. SOCE is required for Th17 cell function & differentiation IL-17A IL-17F IL-22 Th17 Naïve CD4+ T cell Th17 TGF-b IL-6 RORgt RORgt CCR6 IL-23R Terminal differentiation IL-21R IL-21 IL-23 In vitro differentiatedTh17 cells (from DC) IL-21 IL-23R CCR6 % CCR6+ CD4+ * WT IL-23R Stim1fl/flCd4Cre Actin iso WT WT WT WT Stim1fl/flCd4Cre Stim1fl/fl Cd4Cre Stim1fl/fl Cd4Cre Stim1fl/flCd4Cre

  11. CRAC channel inhibition as anti-inflammatory treatment in IBD ? Inducible genetic Deletion using Stim1fl/fl ERT2Cre mice ERT2-Cre Tamoxifen Cre TAM nucleus Stim1fl/fl Stim1-/- – TAM + TAM

  12. Deletion of STIM1 (SOCE) in T cells after IBD development ameliorates intestinal inflammation Transfer of CD4+ CD45RB+ T cells from Stim1fl/fl ERT2Cre mice Vehicle (+ STIM1) Tamoxifen (– STIM1) Treg (Foxp3+) % CD4+ CD25+ Foxp3+ + STIM1 – STIM1

  13. Summary CRAC channels (ORAI1, STIM1, STIM2) in CD4+ T cells: Essential for Ca2+ influx Induction of IBD Th1 and Th17 function: production of proinflammatory cytokines (IL-17A, IL-17F, IL-22, IFNg, TNFa) in the gut Terminal Th17 differentiation Severity of IBD correlates with levels of Ca2+ influx and cytokine production Induced genetic deletion of STIM1 in T cells mimics CRAC inhibitors and leads to rapid recovery from IBD S. typhi. SFB B. fragilis DC MF Naïve CD4 Naïve CD4 Naïve CD4 IL-23 TGFb IL-12 IL-23R Treg Foxp3 Th1 Th17 Ca2+ IL-21 IL-10 IL-17A IL-17F IL-22 CCR6 Ca2+ IFN-g Ca2+ Adapted from: Arrieta Front Immunol (2012)

  14. Carl Weidinger Patrick Shaw Christie-Ann McCarl Sarah Khalil JianMa NIH/NIAID