IL-23R–activated STAT3/STAT4 is essential for Th1/Th17-mediated CNS autoimmunity
Priscilla W. Lee, … , Michael K. Racke, Amy E. Lovett-Racke
Priscilla W. Lee, … , Michael K. Racke, Amy E. Lovett-Racke
Published September 7, 2017
Citation Information: JCI Insight. 2017;2(17):e91663. https://doi.org/10.1172/jci.insight.91663.
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Research Article Immunology

IL-23R–activated STAT3/STAT4 is essential for Th1/Th17-mediated CNS autoimmunity

Abstract

The factors that promote the differentiation of pathogenic T cells in autoimmune diseases are poorly defined. Use of genetically modified mice has provided insight into molecules necessary for the development of autoimmunity, but the sum of the data has led to contradictory observations based on what is currently known about specific molecules in specific signaling pathways. To define the minimum signals required for development of encephalitogenic T cells that cause CNS autoimmunity, myelin-specific T cells were differentiated with various cytokine cocktails, and pathogenicity was determined by transfer into mice. IL-6+IL-23 or IL-12+IL-23 generated encephalitogenic T cells and recapitulated the essential cytokine signals provided by antigen-presenting cells, and both IL-6 and IL-12 induced IL-23 receptor expression on both mouse and human naive T cells. IL-23 signaled through both STAT3 and STAT4, and disruption in STAT4 signaling impaired CNS autoimmunity independent of IL-12. These data explain why IL-12–deficient mice develop CNS autoimmunity, while STAT4-deficient mice are resistant. CD4+ memory T cells from multiple sclerosis patients had significantly higher levels of p-STAT3/p-STAT4, and p-STAT3/p-STAT4 heterodimers were observed upon IL-23 signaling, suggesting that p-STAT3/p-STAT4 induced by IL-23 signaling orchestrate the generation of pathogenic T cells in CNS autoimmunity, regardless of Th1 or Th17 phenotype.

Authors

Priscilla W. Lee, Alan J. Smith, Yuhong Yang, Amanda J. Selhorst, Yue Liu, Michael K. Racke, Amy E. Lovett-Racke

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Figure 5

IL-23 induces the formation of p-STAT3/p-STAT4 heterodimers.

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IL-23 induces the formation of p-STAT3/p-STAT4 heterodimers. Splenocytes...
Splenocytes from naive Vα2.3/Vβ8.2 TCR Tg mice were transfected with either si-NS or si-Stat4 for 18 hours. (A) Knockdown of STAT4 was determined by Western blot of si-NS and si-STAT4 transfected cells using tubulin as the control protein. (B) The si-STAT4 or si-NS transfected cells were activated with anti-CD3/CD28 plus IL-6+IL-23 for 60 hours and then adoptively transferred into naive B10.PL mice (5 × 106 cells per mouse). The number of mice with clinical signs/total number of mice in each group is shown as follows: si-NS (7/10) and si-STAT4 (4/10). (C) Supernatants from anti-CD3/CD28 plus IL-6+IL-23 culture condition were analyzed by ELISA for IFN-γ, IL-17, and GM-CSF. *P < 0.05 (Mann-Whitney U test). (D) Purified CD4+ T cells from naive B10.PL mice were activated in vitro with anti-CD3/CD28 in the presence of the combinations of IL-6+IL-23 or IL-12+IL-23 for 64 hours. CD4+ T cells from naive mice served as a negative control. The 3 groups of cells were washed and rested in fresh medium for 4 hours, and then IL-23 or PBS were added as secondary stimulations for 30 minutes. Nuclear protein was extracted for use in a co-IP assay performed with Ab for total STAT3 or IgG control. Precipitated protein complexes and inputs were electrophoresed with SDS-PAGE. p-STAT4, p-STAT3, and total STAT3 were analyzed sequentially by Western blot, and images shown are from the same blot. (E) Splenocytes from naive Vα2.3/Vβ8.2 TCR Tg mice were activated with MBP Ac1-11 with and without IL-23 for 64 hours, and then CD4+ T cells were purified. Nonactivated T cells were the negative control. Nuclear protein was extracted for use in a co-IP assay performed with Ab for total STAT3 and an isotype control. Precipitated protein complexes and inputs were electrophoresed with SDS-PAGE. p-STAT4 (top) and p-STAT3 (bottom) were detected by Western blot.