Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates T(H)2 differentiation and allergic airway disease

Abstract

Background: IL-4 and signal transducer and activator of transcription 6 (STAT6) play an important role in the progression of allergic airway disease (AAD) or asthma. IL-4 and STAT6 mediate T(H)2 responses in T cells and immunoglobulin class-switching to IgE in B cells. Both T(H)2 responses and IgE promote the asthmatic condition. We have previously demonstrated that poly (ADP-ribose) polymerase (PARP) 14, a member of the PARP family of proteins, regulates the transcription function of STAT6. However, the role of PARP-14 in AAD is not known.

Objective: Here we investigate the role of PARP-14 and the enzyme activity associated with it in a model of AAD dependent on airway hyperresponsiveness and lung inflammation. We also elucidate the mechanism by which PARP-14 regulates AAD.

Methods: The role of PARP-14 and its enzyme activity in AAD and T(H)2 differentiation were examined by using a murine model of AAD and in vitro T(H) cell differentiation.

Results: PARP-14-deficient animals show reduced lung pathology and IgE levels when compared with control animals. Treating mice with a pharmacologic inhibitor for PARP activity reduced the severity of airway hyperresponsiveness and lung inflammation. Mechanistically, our data indicate that PARP-14 and its enzyme activity aid in the differentiation of T cells toward a T(H)2 phenotype by regulating the binding of STAT6 to the Gata3 promoter.

Conclusion: PARP-14 and the catalytic activity associated with it promote T(H)2 differentiation and AAD in a murine model, and targeting PARP-14 might be a potential new therapy for allergic asthma.

Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Figures

Figure 1. PARP-14 and its activity are required for optimal Th2 differentiation

(A–B) Naïve T cell from Parp14−/− and WT mice were cultured under Th2 (A) and Th1 (B) conditions. differentiated cells were re-stimulated and the indicated cytokines were measured by ELISA. (C–D) CD4+CD62Lhigh T cells were cultured under Th2 (C) and Th1 (D) skewing conditions in the presence or absence of PJ34 for 7 days. The indicated cytokines were measured. (E–F) Naïve T cells were nucleofected with the indicated siRNA and then cultured under Th2 conditions for 5 days. Cytokines and PARP-14 expression were measured as indicated. (G–H) Short Hairpins specific for PARP-14 or LacZ were retrovirally transduced into activated T cells and cultured under Th2 conditions. Cytokines and PARP-14 expression were measured as indicated. The results are mean (± SEM) of three different experiments. An asterisk on the graphs indicates a p value of ≤ 0.05 when compared to controls.

Figure 2. PARP-14 plays a role in the development of allergic airway disease

(A–E) Parp14+/+ and Parp14−/− mice were induced for AAD. (A) Lung Resistance (RL) in the indicated mice sensitized and challenged (SC) with OVA or PBS (NSNC) was measured by invasive Resistance and Compliance (RC) system, B – baseline and S – saline. (B) Cells recovered from the BAL fluid from the experimental animals were counted (left graph) and stained with lineage specific markers (middle and right graphs). mRNA levels of Th2 cytokines (C) and chemokines (D) were measured in the lung homogenates of above mice. (E) IgE ELISA was performed on the BAL fluid. (F) Total and OVA specific IgE levels were measured in the serum collected from the experimental animals. (G) Splenocytes isolated from the mice were re-stimulated and the indicated cytokines were measured. The results plotted are mean values (± SEM) from three independent experiments and * indicates a p value of ≤ 0.05 when compared to WT controls.

Figure 3. Inhibition of PARP enzymatic activity during establishment of AAD reduces OVA induced lung disease

(A–G) BALB/c mice were immunized and challenged with OVA and administered with PJ34 as described in online repository Figure E4. BAL fluid was collected from the experimental mice and cells were counted (A), and stained for specific cell populations (B). (C) Relative expression of the indicated cytokines and chemokines was determined. (D) Amount of IgE was measured in BAL fluid. (E) Airway reactivity in each group of mice was determined as lung resistance (RL) to increasing concentrations of methacholine dissolved in saline (S). (F) Splenocytes were re-stimulated and levels of the indicated cytokines was measured. (G) Total and OVA specific IgE were measured in serum collected. Each of the above graphs is means (± SEM) of three independent experiments with each experiment having 5 mice per group. An asterisk on the graphs indicates a p value of ≤ 0.05 when compared to controls.

Figure 4. Administration of PJ34 reduces lung disease in an established AAD model

(A–E)AAD was induced in mice and treated with PJ34 as described in online repository Figure E7. (A) Inflammatory cells in the BAL fluid were counted (left graph) and stained for lineage specific markers (middle and right graph). (B) Lung resistance (RL) in response to increasing doses of bronchoconstrictor was measured. (C) Splenocytes were re-stimulated and cytokines were measured. (D) IgE levels were measured in the BAL fluid. (E) Amount of total and OVA specific IgE in the serum of these mice was also determined. The above results are a mean (± SEM) of two independent experiments with 4–5 mice in each group for each experiment. A p value of ≤ 0.05 is indicated by * symbol.

Figure 5. PARP-14 and activity associated with it is required for ambient GATA-3 expression

Naïve CD4+ T cells from Parp14+/+ and Parp14−/− mice were differentiated under Th2 conditions and then re-stimulated in the presence of IL-4. ChIP-Seq was performed on these cells using an antibody directed towards the active form of RNA Polymerase II antibody. Screen capture views of ChIP-seq signal profile maps for the indicated cytokines (A) and transcriptional factors (B) from the genome browser are shown. (C) CD4+ cells isolated from Parp14+/+ and Parp14−/− mice were cultured for 5 days under Th2 conditions and Gata3 mRNA levels was measured on each day. (D) CD4+ cells were activated in the presence of IL-4 for 1 hr. ChIP was performed using indicated antibodies. The immunoprecipitated DNA was evaluated for Gata3 promoter fragments, S4 (left graph) and S7 (right graph). (E) CD4+ cells were cultured under Th2 conditions with or without 10μM PJ34. Transcripts of Gata3 were quantified using qPCR. (F) CD4+ T cells isolated from WT mice were stimulated with IL-4 in the presence or absence of 10μM PJ34 for 1 hr. ChIP experiments were performed as in (D). The results are mean (± SEM) of three independent experiments. The asterisk indicates a p value of ≤ 0.05 when compared to controls.