TAp63, a methotrexate target in CD4+ T cells, suppresses Foxp3 expression and exacerbates autoimmune arthritis
Kensuke Suga, … , Osamu Ohara, Hiroshi Nakajima
Kensuke Suga, … , Osamu Ohara, Hiroshi Nakajima
Published May 22, 2023
Citation Information: JCI Insight. 2023;8(10):e164778. https://doi.org/10.1172/jci.insight.164778.
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Research Article Immunology

TAp63, a methotrexate target in CD4+ T cells, suppresses Foxp3 expression and exacerbates autoimmune arthritis

Abstract

Methotrexate (MTX) is a standard, first-line therapy for rheumatoid arthritis (RA); however, its precise mechanisms of action other than antifolate activity are largely unknown. We performed DNA microarray analyses of CD4+ T cells in patients with RA before and after MTX treatment and found that TP63 was the most significantly downregulated gene after MTX treatment. TAp63, an isoform of TP63, was highly expressed in human IL-17–producing Th (Th17) cells and was suppressed by MTX in vitro. Murine TAp63 was expressed at high levels in Th cells and at lower levels in thymus-derived Treg cells. Importantly, TAp63 knockdown in murine Th17 cells ameliorated the adoptive transfer arthritis model. RNA-Seq analyses of human Th17 cells overexpressing TAp63 and those with TAp63 knockdown identified FOXP3 as a possible TAp63 target gene. TAp63 knockdown in CD4+ T cells cultured under Th17 conditions with low-dose IL-6 increased Foxp3 expression, suggesting that TAp63 balances Th17 cells and Treg cells. Mechanistically, TAp63 knockdown in murine induced Treg (iTreg) cells promoted hypomethylation of conserved noncoding sequence 2 (CNS2) of the Foxp3 gene and enhanced the suppressive function of iTreg cells. Reporter analyses revealed that TAp63 suppressed the activation of the Foxp3 CNS2 enhancer. Collectively, TAp63 suppresses Foxp3 expression and exacerbates autoimmune arthritis.

Authors

Kensuke Suga, Akira Suto, Shigeru Tanaka, Yutaka Sugawara, Takahiro Kageyama, Junichi Ishikawa, Yoshie Sanayama, Kei Ikeda, Shunsuke Furuta, Shin-Ichiro Kagami, Arifumi Iwata, Koichi Hirose, Kotaro Suzuki, Osamu Ohara, Hiroshi Nakajima

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Figure 4

TAp63 downregulates Foxp3.

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TAp63 downregulates Foxp3. (A) Validation of hTAp63-knockdown vectors. C...
(A) Validation of hTAp63-knockdown vectors. CD4+ T cells from female C57BL/6 mice (8 weeks old) were infected with retroviruses of pMXs-hTAp63-IRES-NGFR or pMXs-IRES-NGFR empty vector along with retroviruses of LMP-hTAp63KD1-IRES-Thy1.1, LMP-hTAp63KD2-IRES-Thy1.1, or LMP-nonsilencing-IRES-Thy1.1. Representative flow cytometry profiles of the expression of NGFR versus Thy1.1 on CD4+ T cells (upper panels) and pan-human p63 versus NGFR on Thy1.1+CD4+ infected cells (lower panels) are shown. (B) Human Th17 cells were infected with retrovirus of either LMP-nonsilencing-IRES-Thy1.1 or LMP-hTAp63KD1-IRES-Thy1.1 and of either pMXs-IRES-NGFR or pMXs-hTAp63-IRES-NGFR. Infected cells were sorted and subjected to RNA-Seq analysis. By comparing between LMP-hTAp63KD1-IRES-Thy1.1 and LMP-nonsilencing-IRES-Thy1.1 and between pMXs-IRES-NGFR and pMXs-hTAp63-IRES-NGFR, the top 500 DEGs were extracted. Among genes shared with 2 comparisons, 3 genes were upregulated and 1 gene was downregulated by TAp63. (C) Normalized count data of the genes upregulated or downregulated by TAp63. (D and E) In the left panels, human Th17 cells were infected with retroviruses of LMP-nonsilencing-IRES-Thy1.1 or LMP-hTAp63KD1-IRES-Thy1.1. In the right panels, human Treg cells were infected with retroviruses of pMXs-IRES-NGFR or pMXs-hTAp63-IRES-NGFR. Representative flow cytometry profiles and the frequency of FOXP3- or RORγt-expressing cells in infected cells are shown. Data are reported as mean ± SEM. n = 3 experiments. *P < 0.05 by unpaired t test. P+I, PMA and ionomycin.