Poly-ADP-ribosyl polymerase-14 promotes T helper 17 and follicular T helper development

Abstract

Transcription factors are critical determinants of T helper cell fate and require a variety of co-factors to activate gene expression. We previously identified the ADP ribosyl-transferase poly-ADP-ribosyl polymerase 14 (PARP-14) as a co-factor of signal transducer and activator of transcription (STAT) 6 that is important in B-cell and T-cell responses to interleukin-4, particularly in the differentiation of T helper type 2 (Th2) cells. However, whether PARP-14 functions during the development of other T helper subsets is not known. In this report we demonstrate that PARP-14 is highly expressed in Th17 cells, and that PARP-14 deficiency and pharmacological blockade of PARP activity result in diminished Th17 differentiation in vitro and in a model of allergic airway inflammation. We further show that PARP-14 is expressed in T follicular helper (Tfh) cells and Tfh cell development is impaired in PARP-14-deficient mice following immunization with sheep red blood cells or inactivated influenza virus. Decreases in Th17 and Tfh development are correlated with diminished phospho-STAT3 and decreased expression of the interleukin-6 receptor α-chain in T cells. Together, these studies demonstrate that PARP-14 regulates multiple cytokine responses during inflammatory immunity.

Keywords: T helper cell; differentiation; knockout; transcription factor.

© 2015 John Wiley & Sons Ltd.

Figures

Figure 1

Poly (ADP) ribose polymerase 14 (PARP‐14) regulates T helper type 17 (Th17) differentiation: (a) Naive T cells isolated from C57BL/6 mice were cultured under Th0 or Th17 conditions. Parp14 expression was measured in helper T‐cell subsets using quantitative RT‐PCR. (b, c) Naive wild‐type and Parp14−/− CD4+ T cells were cultured under Th17 polarizing conditions with or without transforming growth factor‐β (TGF‐β) and harvested on day 5. Differentiated cells were stimulated with PMA and ionomycin for 4 hr before intracellular cytokine staining for interleukin‐17A (IL‐17A) and IL‐17F production (b) or with anti‐CD3 stimulation for 24 hr before supernatants were harvested for analysis by ELISA (c). (d–e) TGF‐β‐induced Th17 cells from wild‐type and Parp14−/− mice were stained for intracellular RORγt (d) and gene expression for transcription factors by quantitative PCR (e). (f) Naive CD4+ T cells were cultured under Th17 polarizing conditions in the presence or absence of PJ34 and stained for intracellular cytokines. Data are mean ± SEM of four independent experiments *P < 0·05.

Figure 2

Poly (ADP) ribose polymerase 14 (PARP‐14) regulates T helper type 17 (Th17) cell cytokine production in allergic inflammation. (a–j) Parp14+/+ and Parp14−/− mice were sensitized with ovalbumin and alum and challenged with ovalbumin to induce allergic inflammation. The number of CD4+ T cells and γδ T cells secreting interleukin‐17 (IL‐17) in the lungs (a, b) and bronchoalveolar lavage (BAL) cells (c, d) are depicted. Cytokines were measured in the BAL fluid (e) or in the supernatant of antigen‐stimulated splenocytes (f) by ELISA. Expression of cytokines in the lung tissue of mice was assessed by quantitative PCR (g). (d–f) Allergic inflammation was induced in C57BL/6 mice and treated with or without PJ34. Cytokines were measured in the BAL fluid (h) and in antigen‐stimulated splenocytes (i) by ELISA. (j) Cytokine expression in the lung tissue was measured by quantitative PCR. Data are means ± SEMs of three independent experiments, *P < 0·05.

Figure 3

Poly (ADP) ribose polymerase 14 (PARP‐14) promotes T follicular helper (Tfh) and germinal centre (GC) B‐cell development in response to sheep red blood cell (SRBC) immunization. Wild‐type (WT) and Parp14−/− mice were immunized with SRBC. On day 9, splenocytes were analysed for Tfh cell (a) and GC B‐cell (d) populations with quantitative analysis shown next to the representative gating. Data are gated on CD4+ and B220+ for Tfh and GC B cells, respectively. (b) Absolute mRNA levels of Parp14 from sorted Tfh cells and non‐Tfh cells were measured by quantitative PCR. (c) Expression of transcription factors in Tfh (CD4+ CXCR5+ PD‐1high) cells sorted from SRBC immunized WT and Parp14−/− mice. (e) Serum from WT and Parp14−/− mice was used to measure SRBC‐specific antibody titres by ELISA. Data are mean ± SE of four or five mice per group and representative of three independent experiments. *P < 0·05.

Figure 4

Poly (ADP) ribose polymerase 14 (PARP‐14) promotes T follicular helper (Tfh) and germinal centre (GC) B‐cell development in response to heat‐inactivated influenza immunization. Wild‐type (WT) and Parp14−/− mice were immunized with heat‐inactivated influenza virus. On day 9, splenocytes were analysed for Tfh cell (a) and GC B‐cell (b) populations with quantitative analysis shown next to the representative gating. Data are gated on CD4+ and B220+ for Tfh and GC, respectively. Data are mean ± SE of four or five mice per group and representative of three independent experiments. *P < 0·05.

Figure 5

Decreased signal transducer and activator of transcription (STAT3) activation in the absence of poly (ADP) ribose polymerase 14 (PARP‐14) (a, b) Naive CD4+ T cells were isolated from wild‐type (WT) and Parp14−/− mice and differentiated under T helper type 17 (Th17) polarizing conditions. (a) The levels of phospho‐STAT3 (pSTAT3) were measured by intracellular cytokine staining daily during differentiation. (b–d) Expression of the indicated genes was measured daily by quantitative PCR during Th17 differentiation. (f, g) Naive T cells from C57BL/6 mice were differentiated under Th17 polarizing condition in the presence or absence of PJ34. (f) The pSTAT3 was measured daily by intracellular cytokine staining. (g) Gene expression in the differentiated Th17 cells was measured by quantitative PCR. (h) Parp14−/− and wild‐type mice were immunized with sheep red blood cells (SRBC). On day 9, splenocytes were analysed by flow cytometry with histograms of pSTAT3 staining on gated CD4+ CXCR5+ PD‐1+ cells. Percentages are mean ± SE of four or five mice per group and representative of three independent experiments. *P < 0·05.