Proc Natl Acad Sci U S A 116(3): 982-987

PMC: PMC6338861

PMID: 30593560

Phosphatase PP2A is essential for T<sub>H</sub>17 differentiation

Richard Sloan+4 authors

Supplementary Material

^{Institute of Immunology, and Department of Dermatology and Rheumatology in Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310058, P. R. China;}

^{Program in Molecular and Cellular Biology, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China;}

^{Zhejiang University–University of Edinburgh Institute, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China;}

^{School of Medicine, Lishui University, Lishui 323000, Zhejiang Province, P. R. China;}

^{Division of Infection and Pathway Medicine, The University of Edinburgh, EH16 4SB Edinburgh, Scotland, United Kingdom;}

^{Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China;}

^{Key Laboratory of Model Animals for Disease Study of the Ministry of Education, Model Animal Research Center, Nanjing University, 210061 Nanjing, P. R. China;}

^{Innovation Center for Cell Signaling Technology Network, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China;}

^{Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, 310058 Hangzhou, P. R. China}

^{To whom correspondence may be addressed. Email:}nc.ude.ujn@gnaixoag or nc.ude.ujz@gnornil_ul.

Edited by Masato Kubo, Tokyo University of Science, Research Institute for Biological Science, Noda, Japan, and accepted by Editorial Board Member Tadatsugu Taniguchi November 26, 2018 (received for review May 6, 2018)

Author contributions: Q.X., M.Z., S.W., H.H., X.G., and L.L. designed research; Q.X., X.J., M.Z., D.R., G.F., Z.W., and J.L. performed research; Q.X., X.J., and L.L. analyzed data; and Q.X., X.J., R.S., L.W., H.H., X.G., and L.L. wrote the paper.

^{Q.X. and X.J. contributed equally to this work.}

Published under the PNAS license.

Significance

By using a gene knockout that leads to T cell-specific deletion, we reveal the essential role of Ser/Thr phosphatase PP2A in T_H17 differentiation. We also show that this works through the regulation of SMAD2/3 phosphorylation status, which elucidates molecular pathways by which PP2A modulates the expression of T_H17 phenotypes. This finding extends our understanding of the close relationship between PP2A overexpression and inflammatory disease. PP2A is a Ser/Thr phosphatase shown to be capable of controlling T_H17 differentiation via modulating R-SMADs activity. We also demonstrate the translational potential of these findings by showing a therapeutic effect of PP2A inhibitors in controlling autoimmune disease in the encephalomyelitis model.

Keywords: TH17, TGFβ, PP2A, SMAD2/SMAD3, EAE

Significance

Abstract

Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling T_H17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for T_H17 differentiation. These PP2A cKO mice had reduced T_H17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on T_H17 cells as were observed in PP2A cKO mice, i.e., decreased T_H17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for T_H17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling T_H17-driven autoimmune diseases.

Abstract

T helper type 17 (T_H17) cells, a subset of CD4^{T cells defined by IL17, IL22, and IL21 production, are essential for control and clearance of extracellular bacterial and fungi (}1, 2). However, excessive T_H17 responses are involved in chronic inflammation and development of many human autoimmune diseases (3). Upon encountering antigen in the context of a local cytokine milieu including transforming growth factor β (TGFβ) and IL6, naïve CD4^{T cells undergo differentiation into effective T}_H17 cells. TGFβ is the principal, essential factor promoting the differentiation of T_H17 cells (4, 5).

Through two related transmembrane Ser/Thr kinase receptors, TGFβ induces Ser/Thr signal cascades in activated T cells. Recent work including work from our laboratory has revealed the regulatory roles of some other Ser/Thr kinases in this process. For example, both MEKK2/3 and MINK1 suppress T_H17 differentiation through direct phosphorylation of the TGFβ signaling components SMAD2 and SMAD3 (6, 7). Precise regulation of SMAD2/3 Ser/Thr phosphorylation status is thus important in driving T_H17 differentiation (6 –8). Dephosphorylation of SMAD2/3 is equally critical in this process, but the specific phosphatases that catalyze SMAD2/3 dephosphorylation remains unknown.

As one of the major Ser/Thr phosphatases in eukaryotes, phosphatase PP2A is critical for many cellular functions including cell survival, proliferation, activation, and differentiation (9). It has been reported that elevated PP2A expression levels are linked to the up-regulation of IL17A production by CD4^{T cells in human systemic lupus erythematosus patients (}10). Studies in the PP2Ac transgenic mouse model also demonstrated the relationship and mechanism linking of PP2A and Il17-dependent immunopathology (11, 12). PP2A is composed of three polypeptide chains, the structural A, the regulatory B, and the catalytic C subunits (13). The heterodimer of the A subunit and the C subunit (PP2A_A-PP2A_C) forms the PP2A core enzyme that associates with one regulatory B subunit, thus determining the substrate specificity of the holoenzyme complex (13).

In TGFβ signaling, two related regulatory B subunits, Bα (Ppp2r2a) and Bδ (Ppp2r2d), opposingly modulate TGFβ/Activin/Nodal signaling (14), while carboxy terminal phosphorylation of MAD (the SMAD homolog protein in Drosophila) is negatively regulated by the PP2A inhibitor okadaic acid (OA) (15). By analogy, these observations suggest that PP2A might be a Ser/Thr phosphorylation modulator involved in controlling T_H17 differentiation.

Here, we present data showing that T_H17 cell polarization was largely impaired when Ppp2ca was ablated in mature T cells and rendered resistance toward MOG-induced experimental autoimmune encephalomyelitis (EAE). We also show that PP2A knockout led to altered activation of R-SMADs (specifically decreasing SMAD2 activation and increasing SMAD3 activation). This synergistically inhibited RORγt mediated Il17a transcription. This work thus reveals a specific role of PP2A in regulating the canonical TGFβ−R-SMADs–RORγt signaling process during T_H17 differentiation and indicates a possible therapeutic approach for controlling T_H17-driven autoimmune diseases via inhibition of PP2A.

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Acknowledgments

We thank Dr. Dawang Zhou for providing plasmids in luciferase assay; Dr. Xinhua Feng for providing ALK5(202D)-HA plasmid; Youfa Zhu, Yanwei Li, Jiajia Wang, and Yingying Huang from the core facilities (Zhejiang University School of Medicine) for technical assistance in histology and FACS analysis. This work was supported by National Natural Science Foundation of China Grants 31770954 and 31530019 (to L.L.), 81570013 (to S.W.), and 31500708 (to M.Z.); National Key R&D Program of China Grant 2018YFC1105102 (to L.L.); and Fundamental Research Funds for the Central Universities Grant 2018XZZX001-12 (to L.L.).

Acknowledgments

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. M.K. is a guest editor invited by the Editorial Board.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. {"type":"entrez-geo","attrs":{"text":"GSE119836","term_id":"119836","extlink":"1"}}GSE119836).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1807484116/-/DCSupplemental.

Footnotes

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