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 8

Aiolos and STAT1 engage in reciprocal regulation.

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Aiolos and STAT1 engage in reciprocal regulation. (A) Publicly available...
(A) Publicly available ATAC-Seq data (GSE203064) from WT and Ikzf3–/– Th1 cells were assessed for alterations in chromatin accessibility at the Stat1 promoter. Publicly available ChIP-Seq data for Aiolos (GSM5106065) were examined at Stat1. Sequencing tracks were viewed using IGV. The Stat1 promoter region of significant differential accessibility is indicated by a blue box (Padj = 0.0302). A ~500 bp region encompassing the indicated Aiolos DNA binding motifs within the Stat1 promoter was subcloned into a reporter plasmid. (B) Schematic depicting the zinc finger (ZF) domains of WT Aiolos and a DNA-binding mutant (AiolosDBM). (C) EL4 T cells were transfected with a Stat1 promoter-reporter and WT Aiolos, AiolosDBM, or empty vector control. Cells were also transfected with SV40-Renilla as a control for transduction efficiency. Luciferase promoter-reporter values were normalized to Renilla control and presented relative to the empty vector control. Aiolos was assessed via immunoblot with an antibody for the V5 epitope tag. β-Actin serves as a loading control. Data are representative of 3 independent experiments (n = 3, mean ± SEM; *P < 0.05, 1-way ANOVA with Tukey’s multiple comparisons test). (D) Publicly available ATAC-Seq data (GSE203064) from Th1 cells and ChIP-Seq data for STAT1 (GSM994528) were viewed using IGV to identify regions of STAT1 enrichment (blue box) at Ikzf3. Approximate ChIP primer locations are indicated with a gray arrow. (E) The Ikzf3 promoter (prom.) and a negative control region (neg. ctrl.) were analyzed for STAT1 enrichment via ChIP. Data were normalized to total input. Percentage enrichment relative to input was divided by IgG, and data are presented as fold-change relative to IgG. (n = 4 biological replicates from 4 independent experiments, mean ± SEM; *P < 0.05, **P < 0.01, 1-way ANOVA with Tukey’s multiple comparisons test.)