Aiolos promotes CXCR3 expression on Th1 cells via positive regulation of IFN-γ/STAT1 signaling
Melissa R. Leonard, Devin M. Jones, Kaitlin A. Read, Srijana Pokhrel, Jasmine A. Tuazon, Robert T. Warren, Jacob S. Yount, Kenneth J. Oestreich
Melissa R. Leonard, Devin M. Jones, Kaitlin A. Read, Srijana Pokhrel, Jasmine A. Tuazon, Robert T. Warren, Jacob S. Yount, Kenneth J. Oestreich
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Research Article Immunology Infectious disease

Aiolos promotes CXCR3 expression on Th1 cells via positive regulation of IFN-γ/STAT1 signaling

Abstract

CD4+ T helper 1 (Th1) cells coordinate adaptive immune responses to intracellular pathogens, including viruses. Key to this function is the ability of Th1 cells to migrate within secondary lymphoid tissues, as well as to sites of inflammation, which relies on signals received through the chemokine receptor CXCR3. CXCR3 expression is driven by the Th1 lineage-defining transcription factor T-bet and the cytokine-responsive STAT family members STAT1 and STAT4. Here, we identify the Ikaros zinc finger (IkZF) transcription factor Aiolos (Ikzf3) as an additional positive regulator of CXCR3 both in vitro and in vivo using a murine model of influenza virus infection. Mechanistically, we found that Aiolos-deficient CD4+ T cells exhibited decreased expression of key components of the IFN-γ/STAT1 signaling pathway, including JAK2 and STAT1. Consequently, Aiolos deficiency resulted in decreased levels of STAT1 tyrosine phosphorylation and reduced STAT1 enrichment at the Cxcr3 promoter. We further found that Aiolos and STAT1 formed a positive feedback loop via reciprocal regulation of each other downstream of IFN-γ signaling. Collectively, our study demonstrates that Aiolos promotes CXCR3 expression on Th1 cells by propagating the IFN-γ/STAT1 cytokine signaling pathway.

Authors

Melissa R. Leonard, Devin M. Jones, Kaitlin A. Read, Srijana Pokhrel, Jasmine A. Tuazon, Robert T. Warren, Jacob S. Yount, Kenneth J. Oestreich

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Figure 7

IFN-γ/STAT1 signaling induces Aiolos expression.

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IFN-γ/STAT1 signaling induces Aiolos expression. (A) Schematic of cultur...
(A) Schematic of culturing system. WT naive CD4+ T cells were stimulated with α-CD3/CD28 under Th1-polarizing conditions (IL-12, α–IL-4). Some cells were also treated with α–IFN-γ to inhibit IFN-γ/STAT1 signaling. (B) At day 3, transcript analysis was performed via qRT-PCR. Transcript was normalized to Rps18 and presented as fold-change compared with WT control (n = 4 biological replicates from 4 independent experiments, mean ± SEM; **P < 0.01, ****P < 0.0001, 2-tailed unpaired Student’s t test). (C) Representative flow cytometric analysis at day 3 for CXCR3 expression on WT Th1 cells treated with or without α–IFN-γ. Data are displayed as percentage positive for CXCR3 (n = 3 biological replicates from 3 independent experiments, mean ± SEM; *P < 0.05, 2-tailed unpaired Student’s t test). (D) An immunoblot was performed to assess the relative abundance of the indicated proteins. β-Actin serves as a loading control (n = 4 independent experiments, mean ± SEM; *P < 0.05, **P < 0.01, ****P < 0.0001, 2-tailed unpaired Student’s t test). (E) At day 3, transcript and flow cytometric analyses were performed for Ikzf3 and Aiolos protein expression, respectively. Flow cytometric data are displayed as MFI fold-change compared with WT controls (n = 3 biological replicates from 3 independent experiments, mean ± SEM; **P < 0.01, 2-tailed unpaired Student’s t test).