Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates TH2 differentiation and allergic airway disease

Poly (ADP-ribose) polymerase 14 and its enzyme activity regulates TH2 differentiation and allergic airway disease Purvi Mehrotra, PhD, Andrew Hollenbeck, BS, Jonathan P. Riley, BS, Fang Li, BS, Ravi J. Patel, MS, Nahid Akhtar, MS, and Shreevrat Goenka, PhD  Indianapolis, Ind 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 TH2 responses in T cells and immunoglobulin class-switching to IgE in B cells. Both TH2 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 TH2 differentiation were examined by using a murine model of AAD and in vitro TH 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 TH2 phenotype by regulating the binding of STAT6 to the Gata3 promoter. Conclusion: PARP-14 and the catalytic activity associated with it promote TH2 differentiation and AAD in a murine model, and targeting PARP-14 might be a potential new therapy for allergic asthma. (J Allergy Clin Immunol 2013;131:521-31.) Key words: IL-4, signal transducer and activator of transcription 6, poly (ADP-ribose) polymerase 14, TH2 cells, Gata3, lung inflammation, airway hyperresponsiveness, poly (ADP-ribose) polymerase inhibitor, allergic airway disease

From the HB Wells Center for Pediatric Research and the Department of Pediatrics, Microbiology and Immunology, Indiana University School of Medicine.  Deceased. Supported by National Institutes of Health grant HL093105 and a Showalter Foundation grant. Disclosure of potential conflict of interest: S. Goenka has received research support, travel support, and payment for writing/reviewing from the National Institutes of Health/National Heart, Lung, and Blood Institute, and has received provision of writing assistance from the Indiana University School of Medicine. The rest of the authors declare that they have no relevant conflicts of interest. Received for publication February 2, 2012; revised June 7, 2012; accepted for publication June 7, 2012. Available online August 1, 2012. Corresponding author: Shreevrat Goenka, PhD, passed away while this article was in press. For correspondence please contact Dr Mark H. Kaplan. E-mail: mkaplan2@ iupui.edu. 0091-6749/$36.00 Ó 2012 American Academy of Allergy, Asthma & Immunology http://dx.doi.org/10.1016/j.jaci.2012.06.015

Abbreviations used AAD: Allergic airway disease AHR: Airway hyperresponsiveness BAL: Bronchoalveolar lavage ChIP: Chromatin immunoprecipitation H&E: Hematoxylin and eosin NSNC: Nonsensitized nonchallenged OVA: Ovalbumin PARP: Poly (ADP-ribose) polymerase PAS: Periodic acid–Schiff qPCR: Quantitative PCR SC: Sensitized challenged siRNA: Small interfering RNA STAT6: Signal transducer and activator of transcription 6 WT: Wild-type

Asthma is a chronic inflammatory airway disease that affects more than 300 million persons worldwide.1 The cardinal features of asthma are airway hyperresponsiveness (AHR), severe tissue inflammation, and goblet cell metaplasia. Traditionally, drugs used to treat asthma promote relaxation of airway smooth muscle (bronchodilators) or suppress airway inflammation (corticosteroids).1 These drugs are directed toward relieving the symptoms of the asthmatic response and not the underlying cause. Dysregulated TH2 immune responses play a key role in the pathogenesis of asthma.2 Therefore a superior approach for asthma therapy might be to target TH2 immunity. IL-4–activated signal transducer and activator of transcription 6 (STAT6) is involved in TH2 responses3 and participates in the progression of asthma.4-7 Thus STAT6 is an attractive therapeutic target for asthma. Previously, we have shown that poly (ADP-ribose) polymerase (PARP) 14, a member of the PARP superfamily, acts as a transcriptional switch for IL-4–dependent STAT6-mediated transcription.8 PARP activity contained in PARP-14 is a reversible posttranslational modification and is characterized by the addition of ADP-ribose moieties onto acceptor proteins.9 PARP-1 is well studied in DNA damage-repair mechanisms and has been observed to play a role in tumor progression. A number of PARP inhibitors are available and are being tested as cancer drugs.10 We have shown that ablation of PARP-14 expression and pharmacologic inhibition of PARP activity both result in reduced expression of STAT6-dependent gene expression.8,11 On the basis of these observations, we hypothesize that PARP-14 promotes TH2 cell differentiation and that targeting the catalytic activity of PARP-14 might attenuate a TH2 response, resulting in the alleviation of allergic airway disease (AAD). In this report we evaluate the role of PARP-14 on AAD and T-cell differentiation. In mice lacking PARP-14 expression, allergen-induced airway disease is attenuated and correlates with reduced TH2 cytokine levels and IgE secretion when compared with control values. Importantly, administration of PJ34, 521

522 MEHROTRA ET AL

a PARP inhibitor, alleviates AAD. We find that PARP-14 modulates the binding of STAT6 to the Gata3 promoter. Taken together, our results provide strong evidence for the involvement of PARP14 in AAD, and our data indicate that inhibiting its activity might be a potential therapy for allergic asthma.

METHODS Mice

Six- to 8-week-old Parp142/2 mice on a C57BL/6 background and wildtype (WT) littermates were used for AAD studies, or BALB/c mice were used in the PARP inhibitor AAD experiments. Mice were maintained in pathogen-free conditions, and all studies were approved by the Indiana University School of Medicine and the Institutional Animal Care and Use Committee and adhere to the ARRIVE (Animal Research: Reporting on In Vivo Experiments) guidelines.

In vitro T cultures

Naive T cells (CD41CD62Lhigh) were isolated from spleens by using the magnetic cell-sorting system, and 1 3 106 cells were cultured in Iscove modified Dulbecco medium–10% FBS under TH1 and TH2 conditions, as described previously.12 Differentiated cells were restimulated overnight with anti-CD3/ CD28, and cytokines in cell-free supernatants were analyzed by means of ELISA.

RNA interference for PARP-14 Two approaches for acutely inhibiting PARP-14 expression were used: small interfering RNA (siRNA) and short hairpin transduction. For detailed methods, see the Methods section in this article’s Online Repository at www. jacionline.org.

Induction of AAD and drug administration Mice were sensitized by using intraperitoneal injections on days 0 and 7 of ovalbumin (OVA)/alum (20 mg of OVA in BALB/c mice and 50 mg of OVA in C56BL/6 mice per 2 mg of alum). On day 14, mice were challenged with OVA (100 mg) intranasally. The progression of AAD was determined by evaluating AHR and analysis of bronchoalveolar lavage (BAL) fluid. Mice were administered PJ34, a PARP inhibitor. For detailed methods, see the Methods section in this article’s Online Repository.

Gene expression analysis Total RNA was isolated with the TRIzol reagent (Invitrogen, Carlsbad, Calif), and cDNA was synthesized with the reverse transcriptase. PCR was performed by using commercially available primers to evaluate gene expression of various cytokines and chemokines.

Chromatin immunoprecipitation Chromatin immunoprecipitation (ChIP) assays were performed on purified CD4 cells essentially as previously described.8 The immunoprecipitated DNA was analyzed for S4 and S7 gene fragments from the Gata3 promoter13 by using SYBR green quantitative PCR (qPCR) and expressed as a percentage of the total chromatin in the sample.

ChIP-Seq analysis

Naive CD41 T cells were cultured and restimulated essentially as described by Wei et al.14 ChIP-Seq was performed by Active Motif (Carlsbad, Calif) using the TranscriptionPath service on the chromatin isolated from these cells.

RESULTS PARP-14 and the catalytic activity associated with it participate in TH2 differentiation It is well established that STAT6 activated by IL-4 plays an important role in TH2 differentiation.15-17 Because we have

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

shown that PARP-14 enhances STAT6-dependent transcription,18 we hypothesized that PARP-14 also participates in TH2 differentiation. Thus naive T cells (CD41CD62L1) were isolated from the spleens of Parp142/2 mice and WT control animals and cultured under either TH1 or TH2 conditions. The phenotype of the differentiated cells was determined by evaluating the cytokines secreted after secondary stimulation. Although TH2-primed cells with WT expression of PARP-14 secreted high levels of IL-4, IL-5, and IL-13, PARP-14–deficient TH2 cells secreted significantly reduced amounts of TH2 cytokines (Fig 1, A). However, PARP14–deficient TH1 cells produced a similar amount of IFN-g compared with WT TH1 cells (Fig 1, B). To determine whether inhibiting PARP enzyme activity would abrogate TH2 differentiation specifically, WT-naive CD41 T cells were cultured under TH1 or TH2 conditions in the presence or absence of PJ34, a PARP inhibitor. A dose-dependent decrease in IL-4, IL-5, and IL-13 levels was observed in cells treated with PJ34 compared with that seen in untreated control samples (Fig 1, C). However, similar amounts of IFN-g were secreted from TH1-skewed cells treated with or without PJ34 (Fig 1, D). These results indicate that PARP-14 and the enzyme activity associated with it participate in the differentiation of TH cells toward a TH2 but not TH1 phenotype. Naive T cells were nucleofected with PARP-14–specific or scrambled siRNA and cultured under TH2 conditions and the phenotype was evaluated after secondary stimulation to determine the effect of acute disruption of PARP-14 expression on TH2 differentiation. Lower levels of TH2 cytokines were observed in the presence of siRNA specific for PARP-14 (Fig 1, E), and correspondingly, there was lower expression of PARP-14 (Fig 1, F, and see Fig E1 in this article’s Online Repository at www. jacionline.org). A second approach of retrovirally transducing a short hairpin against PARP-14 into activated T cells was used to specifically reduce PARP-14 expression and determine its effect on TH2 differentiation. Consistent with our previous data, significantly lower TH2 cytokine and PARP-14 levels were observed with a short hairpin against PARP-14 compared with LacZ controls (Fig 1, G and H). Previously, it has been demonstrated that the short hairpin used to target PARP-14 significantly reduces protein levels of PARP-14 as well.18 Taken together, these data indicate that acute disruption of PARP-14 expression specifically affects TH2 differentiation.

Parp142/2 mice are protected against AAD Because the expression of PARP-14 increases in the lungs of allergen-sensitized and allergen-challenged mice (see Fig E2 in this article’s Online Repository at www.jacionline.org), we next determined the exact role of PARP-14 in mice with AAD. WT and PARP-14–deficient mice were sensitized and challenged with OVA. Lung function in mice was assessed by measuring their lung resistance dependent on increasing doses of inhaled methacholine. An increase in lung resistance was observed with increasing concentrations of methacholine in sensitized challenged (SC) WT mice (Fig 2, A). Importantly, the magnitude of lung resistance in SC Parp142/2 mice was significantly decreased compared with that seen in WT mice and similar to that seen in control nonsensitized nonchallenged (NSNC) mice (Fig 2, A). We next investigated lung inflammation. The BAL fluid of SC Parp142/2 mice showed significantly lower numbers of total inflammatory cells compared with those seen in Parp141/1 mice (Fig 2, B). Numbers

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

MEHROTRA ET AL 523

FIG 1. PARP-14 and its activity are required for optimal TH2 differentiation. A and B, Naive T cells from Parp142/2 and WT mice were cultured under TH2 (Fig 1, A) and TH1 (Fig 1, B) conditions. Differentiated cells were restimulated, and the indicated cytokines were measured by using ELISA. C and D, CD41CD62Lhigh T cells were cultured under TH2-skewing (Fig 1, C) and TH1-skewing (Fig 1, D) conditions in the presence or absence of PJ34 for 7 days. The indicated cytokines were measured. E and F, Naive 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 and H, Short hairpins specific for PARP-14 or LacZ were retrovirally transduced into activated T cells and cultured under TH2 conditions. Cytokine levels and PARP-14 expression were measured as indicated. The results are presented as mean 6 SEM values of 3 different experi_ .05 compared with control values. ments. *P <

of eosinophils, T cells, neutrophils, and macrophages were significantly reduced in PARP-14–deficient animals compared with those seen in WT mice (Fig 2, B). No significant differences in B-cell numbers in BAL fluid were found between WT and Parp142/2 mice (Fig 2, B). PARP-14–deficient mice displayed reduced peribronchial inflammation when compared with mice with intact PARP-14 expression, as observed with hematoxylin and eosin (H&E) staining of lung sections (see Fig E3 in this article’s Online Repository at www.jacionline.org). Taken together, these data indicate that PARP-14 plays an important role in allergen-induced AHR and inflammation. Consistent with AHR and inflammation data, the expression of IL-4, IL-5, and IL-13 was diminished in the lungs of

PARP-14–deficient mice in comparison with that seen in control mice (Fig 2, C). The transcripts of the chemokines CCL17/thymus and activation-regulated chemokine, CCL11/ eotaxin-1, and CCL24/eotaxin-2, which are required for the recruitment of T cells and eosinophils, respectively, were lower in Parp142/2 mice compared with those seen in their WT littermates (Fig 2, D). The levels of IgE in BAL fluid were reduced by 70% in the lungs of Parp142/2 mice compared with those seen in WT mice (Fig 2, E). Airways within lung sections of SC Parp141/1 mice showed a significant increase in goblet cell hyperplasia, as indicated by periodic acid–Schiff (PAS) positivity, whereas very few cells positive for PAS were detected in PARP-14–deficient mice (see Fig E3). Taken

524 MEHROTRA ET AL

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

FIG 2. PARP-14 plays a role in the development of AAD. A-E, Parp141/1 and Parp142/2 mice were induced for AAD. Lung resistance (RL) in the indicated mice sensitized and challenged (SC) with OVA or PBS (NSNC) was measured by using the invasive resistance and compliance system (Fig 2, A). Cells recovered from the BAL fluid from the experimental animals were counted (left graph) and stained with lineage-specific markers (middle and right graphs; Fig 2, B). mRNA levels of TH2 cytokines (Fig 2, C) and chemokines

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

together, all of the above data demonstrate that PARP-14 plays a role in AAD by modulating inflammatory mediators and AHR. The amounts of total and OVA-specific IgE in sera were measured and found to be greater in Parp141/1 than in Parp142/2 samples to investigate whether PARP-14 is required for systemic TH2 immune responses (Fig 2, F). Also, restimulated splenocytes isolated from SC mice showed significant reductions in IL-4, IL-5, and IL-13 but not IFN-g secretion with PARP-14 deficiency (Fig 2, G). Thus ablation of PARP-14 led to reduced systemic responses, and these data indicate a proallergic role of PARP-14.

Inhibition of PARP activity during antigen sensitization and challenge reduces AAD symptoms Because PARP-14 contains the PARP catalytic domain and because we have shown previously that the PARP activity of PARP-14 regulates the transcription function of STAT6,8,11 we next tested whether inhibiting the PARP catalytic activity would alleviate AAD. Mice were sensitized and challenged with OVA as before, and a PARP inhibitor (PJ34), which has previously been shown to inhibit PARP-14,8 was administered through 3 different routes during induction of AAD. PJ34 was delivered during allergen challenge (PJ34 C) and during both sensitization and challenge (PJ34 SC) and was injected multiple times during the sensitization phase (mPJ34 S, see Fig E4 in this article’s Online Repository at www.jacionline.org). These groups of mice were analyzed in a similar manner to the WT and Parp142/2 experiment. All of the PJ34-treated experimental groups were compared with the NSNC and SC control animals. For some of the graphs, we have not shown the NSNC control animals because the values for this group were negligible. SC mice showed a robust increase in inflammatory cell numbers in the BAL fluid, whereas all three PJ34 treatment protocols showed a decrease in cellular infiltration (Fig 3, A). Numbers of eosinophils, T cells, B cells, macrophages, and neutrophils mimicked the total BAL fluid cell count data (Fig 3, B). These data were confirmed by means of histologic analysis of lung sections of mice stained with H&E (see Fig E5 in this article’s Online Repository at www.jacionline.org). Consistent with the BAL data, mice in which PJ34 was administered intranasally during challenge showed very modest reductions in inflammation (see Fig E5). The expression of IL-4, IL-5, and IL-13 in lung tissue was reduced with PJ34 treatment, and the most profound effect was observed in mice treated with PJ34 during both sensitization and challenge (Fig 3, C). The expression levels of CCL11, CCL17, and CCL24 followed a similar pattern as the numbers of eosinophils and T cells recruited to the lung (Fig 3, C). IgE levels in BAL fluid in mice treated with PJ34 showed a significant reduction from that seen in the untreated control animals (Fig 3, D). A decrease in AHR to methacholine was also observed in mice treated with PJ34 compared with that seen in untreated control animals (Fig 3, E). Histologic analysis revealed the attenuation of mucus hypersecretion by goblet cells

=

MEHROTRA ET AL 525

in PJ34-treated mice compared with the control mice (see Fig E5). The above data indicate the requirement of PARP activity in the pathogenesis of the lung inflammatory response and lung reactivity against OVA antigen. Splenocytes isolated from the same group of mice were stimulated with 100 mg of OVA for 72 hours, and levels of the secreted cytokines were measured. IL-4, IL-5, and IL-13 levels were all reduced in PJ34-treated mice, with the maximum decrease seen when PJ34 was administered during sensitization and challenge (Fig 3, F). Conversely, levels of IFN-g, a classical TH1 cytokine, were increased in 2 of the PJ34 treatment protocols compared with those seen in control samples (Fig 3, F). Lastly, we evaluated IgE levels in the serum, and administration of PJ34 attenuated OVA-induced IgE levels (Fig 3, G). Taken together, our data indicate that inhibition of PARP enzymatic activity during induction of AAD alleviates allergeninduced airway disease in mice. All 3 routes of PJ34 administration were effective, but the maximum effect was observed when PJ34 was administered during sensitization and challenge. Because PJ34 is a general PARP inhibitor, from the above data, it cannot be inferred that PJ34 is inhibiting the PARP activity of PARP-14 to alleviate AAD. To address this, we treated WT and PARP-14–deficient animals with PJ34 and observed no further decrease in the AAD phenotype when Parp142/2 mice were treated with PJ34 compared with WT mice treated with inhibitor (see Fig E6 in this article’s Online Repository at www.jacionline.org). These data indicate that PJ34 treatment is primarily targeting PARP-14 in this model.

Administration of a PARP inhibitor to mice with established AAD alleviates disease pathology Our data indicate that inhibiting PARP activity during the induction of AAD reduces the disease phenotype. We next tested whether administration of a PARP inhibitor to mice with established AAD would also alleviate the pathology. Mice were sensitized with OVA as before, and then mice were challenged with intranasal OVA for 10 days to establish AAD. On the fifth day of OVA challenge, a group of mice were killed, and the AAD phenotype was evaluated as before. We confirmed that the mice at this time point had full-blown AAD by evaluating recruitment of inflammatory cells, airways resistance, and cytokine and IgE production (see Figs E7 and E8 in this article’s Online Repository at www.jacionline.org). The OVA challenges were continued for an additional 5 days to test whether PJ34 would lessen the disease phenotype, and during this time, mice were either left untreated (SC[2]) or treated with PJ34 (SC PJ34, see Fig E7). The inflammation in the lung was reduced significantly in mice that were treated with PJ34, as evaluated based on BAL cell count and H&E staining of lung sections (Fig 4, A, and see Fig E9 in this article’s Online Repository at www.jacionline.org). Lung function was also significantly improved in mice treated with PJ34 (Fig 4, B). Levels of IL-4, IL-5, IL-13, and IgE were all reduced in mice

(Fig 2, D) were measured in the lung homogenates of the above mice. IgE ELISA was performed on the BAL fluid (Fig 2, E). Total and OVA-specific IgE levels were measured in sera collected from the experimental animals (F). Splenocytes isolated from the mice were restimulated, and levels of the indicated cytokines were _ .05 measured (G). The results are plotted as mean 6 SEM values from 3 independent experiments. *P < when compared with WT control animals.

526 MEHROTRA ET AL

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

FIG 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 Fig E4. BAL fluid was collected from the experimental mice, and cells were counted (Fig 3, A) and stained for specific cell populations (Fig 3, B). Relative expression of the indicated cytokines and chemokines was determined (Fig 3, C). Amount of IgE was measured in BAL fluid (Fig 3, D). Airway reactivity in each group of mice was determined as lung resistance (RL) to increasing concentrations of methacholine

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

MEHROTRA ET AL 527

FIG 4. Administration of PJ34 reduces lung disease in an established AAD model. A-E, AAD was induced in mice treated with PJ34, as described in Fig E7. Inflammatory cells in the BAL fluid were counted (left graph) and stained for lineage-specific markers (middle and right graphs; Fig 4, A). Lung resistance (RL) in response to increasing doses of bronchoconstrictor was measured (Fig 4, B). Splenocytes were restimulated, and cytokine levels were measured (Fig 4, C). IgE levels were measured in BAL fluid (Fig 4, D). Amount of total and OVA-specific IgE in the sera of these mice was also determined (Fig 4, E). The above results are mean 6 SEM _ .05. values of 2 independent experiments, with 4 to 5 mice in each group for each experiment. *P <

administered a PARP inhibitor, with the exception of IFN-g (Fig 4, C-E). Inflammation around the airways and goblet cell hyperplasia were consistent with these data (see Fig E9). Taken

=

together, our data indicate that inhibiting PARP activity during the induction of AAD or during an ongoing allergen challenge can be effective in alleviating the disease.

dissolved in saline (S; Fig 3, E). Splenocytes were restimulated, and levels of the indicated cytokines were measured (Fig 3, F). Total and OVA-specific IgE levels were measured in collected serum (Fig 3, G). Each of the above graphs show mean 6 SEM values of 3 independent experiments, with each experiment having 5 _ .05 compared with control animals. mice per group. *P <

528 MEHROTRA ET AL

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

FIG 5. PARP-14 and activity associated with it are required for ambient GATA3 expression. Naive CD41 T cells from Parp141/1 and Parp142/2 mice were differentiated under TH2 conditions and then restimulated in the presence of IL-4. ChIP-Seq analysis was performed on these cells by using an antibody directed

MEHROTRA ET AL 529

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

PARP-14 and its enzyme activity regulate expression of Gata3 by modulating the binding of STAT6 to its promoter To define the mechanism by which PARP-14 participates in TH2 cell differentiation and hence affects AAD, we compared the global expression profile of genes between WT and Parp142/2 TH2 cells. Naive T cells were isolated from WT and Parp142/2 mice and differentiated under TH2 conditions. These cells were then used for ChIP-Seq analysis with an antibody directed against the active form of RNA polymerase II. Using this approach, we were able to capture the active transcription profile of genes on a genomic scale in TH2 cells. We evaluated the peak profiles and average peak values of the TH2 cytokine genes Il4, Il5, and Il13 in WT and Parp142/2 TH2 cells (Fig 5, A, and Table I). Consistent with our ELISA data, the peak intensity of all 3 cytokines was markedly reduced with PARP-14 deficiency (Figs 1, A, and 5, A, and Table I). We next screened for the RNA polymerase II binding profile of the Batf, Gfi1, Irf4, Gata3, and Maf genes, which are implicated in TH2 differentiation.19-23 The peak profile and average peak value of only Gata3 was reduced because of PARP-14 deficiency, and the other transcription factor genes had comparable values in WT and Parp142/2 samples (Fig 5, B, and Table I). These data suggest that PARP-14 might affect TH2 differentiation by regulating the expression of GATA3. To confirm whether Gata3 expression is regulated by PARP-14, we cultured CD41 T cells from WT and Parp142/2 mice under TH2 conditions and determined the level of Gata3 expression by using qPCR during the differentiation process. Gata3 expression was significantly reduced at the later time points during TH2 differentiation in the absence of PARP-14 expression (Fig 5, C). Consistent with these data, the silencing of PARP-14 by means of short hairpin RNA targeting PARP-14 resulted in reduced Gata3 expression (see Fig E10 in this article’s Online Repository at www.jacionline.org). To determine whether PARP-14 affected the binding of STAT6 to the Gata3 promoter so as to regulate its expression, we determined the binding ability of STAT6 to 2 binding sites within the Gata3 regulatory region13 with and without PARP-14 expression. STAT6 ChIP experiments were performed on T cells from WT and Parp142/2, and the level of the Gata3 promoter fragments was quantified by using qPCR. Consistent with the Gata3 expression data, the binding of STAT6 to both promoter regions was significantly reduced in PARP-14–deficient T cells (Fig 5, D). Next we determined whether PARP enzyme would also regulate Gata3 expression. Inhibiting PARP activity had a profound effect on Gata3 expression throughout the differentiation process (Fig 5, E). Consistent with these data, inhibition of PARP activity resulted in decreased STAT6 binding to the Gata3 promoter (Fig 5, F). Taken together, these data indicate that PARP-14 and the enzyme activity associated with it

=

TABLE I. Indicated values are average peak intensities of the ChIP-Seq signal for each gene Gene

Parp141/1

Parp142/2

Il4 Il5 Il13 Ifng Batf Gfi1 Irf4 Gata3 Maf

19.065 5.224 10.730 2.972 5.904 6.088 19.016 18.697 5.413

7.469 0.732 3.569 2.933 6.560 5.662 18.963 14.529 4.745

Values in boldface indicate significant reduction in signal between WT and PARP-14deficient mice.

participate in TH2 differentiation by regulating the binding ability of STAT6 to the Gata3 promoter.

DISCUSSION In this study we have provided evidence that a PARP family member, PARP-14, regulates the pathogenesis of AAD in a murine model. Our data demonstrate that with PARP-14 deficiency and inhibition of the catalytic activity associated with PARP-14, the severity of AHR and airway inflammation induced by an allergen are markedly reduced. We have found that PARP14 modulates TH2 differentiation through its catalytic activity. Mechanistically, we demonstrate that PARP-14 regulates binding of STAT6 to the Gata3 promoter. Our data indicate that there is residual responsiveness to the allergen in the absence of PARP14, indicating that PARP-14 might provide part of the transcription activation potential to STAT6. The other cofactors that aid STAT6 are still available in the absence of PARP-14 to mediate the allergic response. On the basis of these studies, we propose that PARP-14 is an important mediator of TH2-mediated AAD and that targeting its catalytic activity might be a potential therapy for allergen-induced asthma. Current asthma therapies target the symptoms of asthma rather than the underlying cause. TH2 cells activated by innocuous antigens are initiators of the allergic asthmatic condition.2 Therefore targeting TH2 cells through PARP-14 might be a more effective therapy for this form of asthma. Here we show that administration of the PARP inhibitor during ongoing allergen exposure alleviates AAD symptoms in mice, suggesting that administering PARP inhibitors to a patient undergoing allergen challenge might be effective in alleviating the disease. Moreover, when the PARP inhibitor is administered during the sensitization phase of our AAD protocol, the allergic disease symptoms in the mice are also reduced. Our data indicate that there is a reduction in TH2 responses and that this is responsible in turn for the alleviation of the allergic

toward the active form of the RNA polymerase II antibody. A and B, Screen-capture views of ChIP-Seq signal profile maps for the indicated cytokines (Fig 5, A) and transcriptional factors (Fig 5, B) from the genome browser are shown. C, CD41 cells isolated from Parp141/1 and Parp142/2 mice were cultured for 5 days under TH2 conditions, and Gata3 mRNA levels were measured on each day. D, CD41 cells were activated in the presence of IL-4 for 1 hour. ChIP was performed with the indicated antibodies. The immunoprecipitated DNA was evaluated for the Gata3 promoter fragments S4 (left graph) and S7 (right graph). E, CD41 cells were cultured under TH2 conditions with or without 10 mmol/L PJ34. Transcripts of Gata3 were quantified by using qPCR. F, CD41 T cells isolated from WT mice were stimulated with IL-4 in the presence or absence of 10 mmol/L PJ34 for 1 hour. ChIP experiments were performed as in Fig 5, D. The results are mean 6 SEM _ .05 when compared with control animals. values of 3 independent experiments. *P <

530 MEHROTRA ET AL

disease symptoms. Taken together, our data provide a proof of principle that inhibiting PARP catalytic activity can affect both local and systemic TH2 responses and that this might be an effective therapy for allergic asthma. The PARP inhibitor PJ34 used in these studies is a general PARP inhibitor and does not discriminate among different PARP enzymes. Therefore our data with PJ34 itself do not prove that it is targeting PARP-14 specifically to reduce the allergic response. However, we provide evidence that when PJ34 is used in Parp142/2 mice, there is no further reduction in AAD symptoms, suggesting that PJ34 targets predominantly PARP-14 (see Fig E6). Interestingly, the expression of IL-5 in in vitro differentiated Parp142/2 TH2 cells is significantly reduced in the presence of PJ34 compared with that seen without the inhibitor. These data suggest that PJ34 might be inhibiting another PARP enzyme other than PARP-14 to decrease the expression of IL-5. Indeed, PARP-1 has been shown to regulate IL-5 expression by regulating the calpain-dependent degradation of STAT6.24 This study showed that when PARP-1 was absent, there were reduced levels of STAT6 protein that affected the expression of IL-5. The same group had shown that PARP-1 deficiency or inhibition of PARP activity with another PARP inhibitor (TIQ-A) resulted in reduced IL-5 but not IL-4 production in their OVA/alum AAD model.25 There are several differences in this study and ours. First, our study uses 6 to 10 intranasal challenges with OVA, which mimics a more severe model of allergen challenge compared with 1 aerosolized administration of the antigen. Second, our data show that PARP-14 and inhibition of PARP activity play a role in TH2 differentiation, which affects the expression of all TH2 cytokines and not only IL-5. Third, the route of administration of PARP inhibitor in the other study was limited because it was administered as a single intraperitoneal injection 2 hours before challenge with antigen. This would not attenuate the TH2 differentiation process that takes place during the sensitization phase. In the present study we have used several protocols for administration of the PARP inhibitor and show that PARP inhibition targets TH2 differentiation systemically and not only the inflammatory response that ensues right after local challenge with an antigen. Nevertheless, both the previous work and the present study indicate that inhibition of PARP activity is an attractive asthma therapy. Moreover, both these studies indicate that there is a need to identify specific inhibitors for the different PARP enzymes because each enzyme might play a distinct biological role. Our ex vivo data demonstrate that PARP-14 deficiency and inhibition of PARP activity results in the attenuation of all signature TH2 cytokines. We have shown that among all of the transcription factors involved in TH2 differentiation, only the expression of Gata3 is dependent on PARP-14 and its catalytic activity (Fig 5). Because the expression of GATA3 is dependent on IL-4 and STAT6,26 our data imply that PARP-14 might regulate the expression of Gata3 through STAT6. Indeed, we observe that the binding of STAT6 to 2 regions within the Gata3 promoter is diminished in the absence of PARP-14 or its catalytic activity (Fig 5). These data are consistent with our previous finding that PARP-14 and its enzymatic activity affect the expression of Ie and Fcer2a by modulating the binding of STAT6 to the promoter elements of these genes.8 Hence we can speculate that the reduced expression of IgE in our in vivo model might be due to the B cell–specific intrinsic role of PARP-14 in immunoglobulin class-switching to IgE. From this current study, we cannot rule out the possibility that the reduction in IgE levels is due to

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

reduced help from the T cell. Further studies will be needed to address the exact role of PARP-14 in immunoglobulin class-switching. Here we have provided strong evidence that PARP-14 and the catalytic activity associated with it play a role in AAD dependent on IL-4 and STAT6. There are several other allergic conditions, such as atopic dermatitis and eosinophilic esophagitis, the pathologies of which are associated with IL-4 and STAT6. It will be important to study whether PARP-14 plays a role in the pathogenesis of these other diseases and whether targeting PARP activity could be a potential therapy for these conditions as well. We thank Drs Mark Kaplan, Rebecca Shilling, and Robert Tepper for their critical review of this article.

Key messages d

PARP-14, a cofactor for STAT6, participates in AAD by affecting allergen-induced AHR and inflammation in the lung.

d

Inhibiting the catalytic activity associated with PARP-14 alleviates the pathogenesis of AAD in mice, implicating this as a potential new therapy for asthma.

d

PARP-14 and the enzyme activity associated with it participate in AAD by promoting TH2 differentiation and regulating Gata3 expression by modulating the DNA binding of STAT6 to the Gata3 promoter.

REFERENCES 1. Fanta CH. Asthma. N Engl J Med 2009;360:1002-14. 2. Broide DH, Finkelman F, Bochner BS, Rothenberg ME. Advances in mechanisms of asthma, allergy, and immunology in 2010. J Allergy Clin Immunol 2011;127: 689-95. 3. Goenka S, Kaplan MH. Transcriptional regulation by STAT6. Immunol Res 2011; 50:87-96. 4. Kuperman DA, Schleimer RP. Interleukin-4, interleukin-13, signal transducer and activator of transcription factor 6, and allergic asthma. Curr Mol Med 2008;8: 384-92. 5. Sehra S, Bruns HA, Ahyi AN, Nguyen ET, Schmidt NW, Michels EG, et al. IL-4 is a critical determinant in the generation of allergic inflammation initiated by a constitutively active Stat6. J Immunol 2008;180:3551-9. 6. Kuperman D, Schofield B, Wills-Karp M, Grusby MJ. Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigeninduced airway hyperresponsiveness and mucus production. J Exp Med 1998; 187:939-48. 7. Akimoto T, Numata F, Tamura M, Takata Y, Higashida N, Takashi T, et al. Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice. J Exp Med 1998;187:1537-42. 8. Mehrotra P, Riley JP, Patel R, Li F, Voss L, Goenka S. PARP-14 functions as a transcriptional switch for Stat6-dependent gene activation. J Biol Chem 2011;286: 1767-76. 9. Schreiber V, Dantzer F, Ame JC, de Murcia G. Poly(ADP-ribose): novel functions for an old molecule. Nat Rev Mol Cell Biol 2006;7:517-28. 10. Virag L, Szabo C. The therapeutic potential of poly(ADP-ribose) polymerase inhibitors. Pharmacol Rev 2002;54:375-429. 11. Goenka S, Cho SH, Boothby M. Collaborator of Stat6 (CoaSt6)-associated poly(ADP-ribose) polymerase activity modulates Stat6-dependent gene transcription. J Biol Chem 2007;282:18732-9. 12. Stritesky GL, Muthukrishnan R, Sehra S, Goswami R, Pham D, Travers J, et al. The transcription factor STAT3 is required for T helper 2 cell development. Immunity 2011;34:39-49. 13. Onodera A, Yamashita M, Endo Y, Kuwahara M, Tofukuji S, Hosokawa H, et al. STAT6-mediated displacement of polycomb by trithorax complex establishes longterm maintenance of GATA3 expression in T helper type 2 cells. J Exp Med 2010; 207:2493-506.

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

14. Wei L, Vahedi G, Sun HW, Watford WT, Takatori H, Ramos HL, et al. Discrete roles of STAT4 and STAT6 transcription factors in tuning epigenetic modifications and transcription during T helper cell differentiation. Immunity 2010;32: 840-51. 15. Kaplan MH, Schindler U, Smiley ST, Grusby MJ. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 1996;4: 313-9. 16. Shimoda K, van Deursen J, Sangster MY, Sarawar SR, Carson RT, Tripp RA, et al. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 1996;380:630-3. 17. Takeda K, Tanaka T, Shi W, Matsumoto M, Minami M, Kashiwamura S, et al. Essential role of Stat6 in IL-4 signalling. Nature 1996;380:627-30. 18. Goenka S, Boothby M. Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor. Proc Natl Acad Sci U S A 2006;103:4210-5. 19. Betz BC, Jordan-Williams KL, Wang C, Kang SG, Liao J, Logan MR, et al. Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J Exp Med 2010;207:933-42.

MEHROTRA ET AL 531

20. Zhu J, Guo L, Min B, Watson CJ, Hu-Li J, Young HA, et al. Growth factor independent-1 induced by IL-4 regulates Th2 cell proliferation. Immunity 2002; 16:733-44. 21. Ahyi AN, Chang HC, Dent AL, Nutt SL, Kaplan MH. IFN regulatory factor 4 regulates the expression of a subset of Th2 cytokines. J Immunol 2009;183:1598-606. 22. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 1997;89:587-96. 23. Ho IC, Hodge MR, Rooney JW, Glimcher LH. The proto-oncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 1996;85:973-83. 24. Datta R, Naura AS, Zerfaoui M, Errami Y, Oumouna M, Kim H, et al. PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model. Allergy 2011;66:853-61. 25. Oumouna M, Datta R, Oumouna-Benachour K, Suzuki Y, Hans C, Matthews K, et al. Poly(ADP-ribose) polymerase-1 inhibition prevents eosinophil recruitment by modulating Th2 cytokines in a murine model of allergic airway inflammation: a potential specific effect on IL-5. J Immunol 2006;177:6489-96. 26. Paul WE. What determines Th2 differentiation, in vitro and in vivo? Immunol Cell Biol 2010;88:236-9.

531.e1 MEHROTRA ET AL

METHODS RNA interference for PARP-14 Nucleofection of siRNA. Naive T cells were nucleofected with scrambled siRNA (SC-37007; Santa Cruz Biotechnology, Santa Cruz, Calif) and PARP-14 siRNA (SC-76057, Santa Cruz Biotechnology) with the Amaxa kit, according to the manufacturer’s protocol. Four hours after transfection, cells were cultured under TH2 conditions for 5 days. Transfected cells were harvested and analyzed for PARP-14 expression and restimulated for cytokine ELISA. Retroviral transduction. Transduction experiments were performed with a pSIREN retrovector coexpressing green fluorescent protein and short hairpin targeting N-terminal region of PARP-14 (Parp14; 59-GCA GATGTGTACAAAGTAAAG-39)E1 and LacZ as a control. PlatE cells were transiently transfected with the calcium phosphate method. Naive T cells were cultured under TH0 conditions and transduced on day 2 and further expanded with murine IL-4 (10 ng/mL) and 50 U of human IL-2. On day 5, green fluorescent protein–positive cells were sorted and restimulated with anti-CD3/ CD28. Cytokine levels were measured in supernatants by means of ELISA, and cells were lysed in Trizol for RNA expression.

Airway reactivity AHR was measured by using the invasive Resistance and Compliance FinePointe system (Buxco Research Systems, Wilmington, NC) 24 hours after the last challenge. The mice were anesthetized and intubated, and airway reactivity was measured by determining the lung resistance to aerosolized methacholine.

Collection and analysis of BAL fluid Lungs were washed 3 times with 1 mL of PBS, which was collected as BAL fluid 48 hours after the last challenge. Cell-free supernatants were analyzed for IgE by means of ELISA, and the cells collected from BAL fluid were stained with surface markers and analyzed by using flow cytometry to identify the cells.E2

Administration of PJ34 during induction of AAD Mice were sensitized with OVA plus alum on days 0 and 7 and challenged with intranasal OVA for 6 consecutive days. PJ34 was administered either (30 mg) intranasally during all days of the OVA challenges (PJ34 C), as 2 intraperitoneal injections (200 mg) during sensitization and intranasally during challenges (PJ34 SC), or intraperitoneally on alternate days during sensitization (mPJ34 S, see Fig E4).

Administration of PJ34 to mice with established AAD Mice were sensitized with OVA plus alum on days 0 and 7 and challenged with intranasal OVA for 10 consecutive days. Thirty milligrams of intranasal and 200 mg of intraperitoneal PJ34 was administered to mice from the sixth to the tenth days of the OVA challenge (see Fig E7).

Histology Experimental animals were killed, and lungs were harvested, fixed in formalin, and embedded in paraffin. The lungs were sectioned and stained with H&E or PAS by using standard protocols.

ChIP-Seq using TranscriptionPath ChIP was performed with an antibody against the active form of RNA polymerase II, and the immunoprecipitated DNA fragments were sequenced with the Illumina Analyzer II (Illumina, San Diego, Calif). The average peak values were determined as the average fragment densities within the gene interval.

RESULTS Expression of PARP-14 increases in lung tissue after antigen exposure AAD is dependent on TH2 immune responses, and we have determined that PARP-14 plays a role in TH2 differentiation.

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

Therefore the expression profile of PARP-14 was evaluated in a murine model of AAD. Mice were sensitized with 2 intraperitoneal injections of OVA and alum, and 1 week after the second injection, mice were challenged intranasally with OVA for 6 days (SC mice). Simultaneously, control mice were injected and challenged with PBS (NSNC mice). We compared the expression levels of PARP-14 in lung tissue of NSNC and OVA SC animals using 2 approaches. As determined by using immunoblot analysis, the levels of PARP-14 in lung tissue were significantly increased when mice were sensitized and challenged with OVA (see Fig E2, A). Next, we performed immunohistochemical analysis of the lung sections to determine the exact location of PARP-14 expression. PARP-14 was expressed in the airway epithelia of NSNC animals (see Fig E2, B). Importantly, in SC animals an increase in PARP-14 expression was observed that was mainly localized to the inflammatory infiltrate (see Fig E2, B). To control for specificity for antibody reactivity, we used PARP-14–deficient lung tissue that displayed only background staining (see Fig E2, B). Taken together, these data indicate that on antigenic challenge, the expression of PARP-14 is enhanced in the lung because of infiltration of immune cells.

Inhibition of PARP activity in Parp142/2 mice does not result in further decrease in OVA-induced pulmonary pathogenesis Ablation of PARP-14 expression or inhibition of PARP activity by PJ34 showed a marked reduction in OVA-induced AAD symptoms. These data imply that PJ34 might be inhibiting the PARP activity of PARP-14 to mediate its effect. However, because PJ34 is a general PARP inhibitor that potentially inhibits other PARP enzymes, from the data obtained thus far, it is not possible to delineate whether PJ34 is specifically inhibiting PARP-14. Hence to determine whether the PARP activity of PARP-14 was being inhibited by PJ34, we carried out both in vitro and in vivo studies in which WT and Parp142/2 mice were treated with PJ34. Naive T cells from PARP-14–deficient and WT mice were cultured under TH2 conditions in the presence or absence of 10 mmol/L PJ34. Consistent with our previous findings, both PJ34-treated WT cells and PARP-14–deficient cells showed a significant reduction in levels of all 3 TH2 cytokines (IL-4, IL-5, and IL-13; see Fig E6, A). Importantly, PJ34 treatment had no further significant effect on IL-4 and IL-13 secretion from PARP-14–deficient cells. A modest but significant reduction in IL-5 levels was observed in PJ34-treated Parp142/2 cells compared with those seen in untreated cells (see Fig E6, A). These data suggest that the expression of IL-4 and IL-13 is regulated by the PARP activity of PARP-14. However, the expression of IL-5 might be regulated by the PARP activity of another PARP than PARP-14. To confirm the role of PARP-14 enzyme activity during AAD, we administered PJ34 both during sensitization and challenge in Parp142/2 mice and their WT littermates. The mRNA levels of IL-4, IL-5, IL-13, CCL11, CCL17, and CCL24 in the lung homogenates from WT mice treated with PJ34 were significantly reduced when compared with those of untreated WT mice, and this was comparable with the levels measured in the lungs of Parp142/2 mice that were treated or not with PJ34 (see Fig E6, B). BAL fluid cell numbers were reduced in PARP-14–deficient mice and in PJ34-treated WT mice in comparison with those seen in untreated control animals. Importantly, the absence of PARP-14

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

expression and PJ34 treatment did not result in a further reduction in inflammatory cell numbers in the BAL fluid (see Fig E6, C). A similar trend for IgE was observed in the BAL fluid of these experimental animals (see Fig E6, D). No further decrease in serum IgE levels was observed in PJ34-treated Parp142/2 mice (see Fig E6, E), and there was no reduction in TH2 cytokine levels after restimulation of splenocytes from PJ34-treated Parp142/2 mice (see Fig E6, F). Taken together, these data strongly suggest that PARP-14 and its activity are critical players in eliciting allergen-dependent pulmonary disease. Importantly, our data

MEHROTRA ET AL 531.e2

demonstrate that PARP-14 might be an attractive therapeutic target for allergic asthma. REFERENCES E1. Goenka S, Boothby M. Selective potentiation of Stat-dependent gene expression by collaborator of Stat6 (CoaSt6), a transcriptional cofactor. Proc Natl Acad Sci U S A 2006;103:4210-5. E2. van Rijt LS, Kuipers H, Vos N, Hijdra D, Hoogsteden HC, Lambrecht BN. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma. J Immunol Methods 2004;288:111-21.

531.e3 MEHROTRA ET AL

FIG E1. PARP-14 levels in siRNA nucleofected T cells. Naive T cells were nucleofected with scrambled or PARP-14 siRNA and cultured under TH2 conditions for 5 days. PARP-14 protein levels were assessed by means of immunoblot analysis (upper panel). The blots were stripped and probed for b-actin as an endogenous control (lower panel). The blot shows a representative of 2 independent experiments.

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

FIG E2. Expression of PARP-14 increases after antigen sensitization and challenge. A, BALB/c mice were sensitized with OVA absorbed on alum and challenged intranasally with OVA (SC) or only PBS (NSNC), as described. Mice were killed, and cell-free extracts of the lungs were analyzed by means of Western blotting with anti–PARP-14. The blots were stripped and reprobed with anti–b-actin. The blot shows results of 2 mice per group. B, Parp141/1 and Parp142/2 mice were treated as mentioned above. Immunohistochemistry was performed on paraffin-embedded lung sections of these mice with anti–PARP-14. The photomicrographs are representative of 5 mice per group.

MEHROTRA ET AL 531.e4

531.e5 MEHROTRA ET AL

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

FIG E3. Inflammation in the lung and goblet cell hyperplasia are reduced with PARP-14 deficiency. Lungs from the OVA-sensitized and OVA-challenged (SC) Parp141/1 and Parp142/2 mice and control nonsensitized and nonchallenged mice (NSNC) were fixed, and paraffin-embedded sections were stained with H&E (upper panel) and PAS (lower panel). The photomicrographs are representative of 15 mice per group. Original magnification is 3100, and the bar is 50 mm.

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

MEHROTRA ET AL 531.e6

FIG E4. Administration of PJ34 to mice during induction of AAD. In all cases mice were sensitized with OVA plus alum on days 0 and 7 of the protocol. Starting on day 14, the mice were challenged with OVA for 6 consecutive days. PJ34 was administered as indicated, and on day 21 of the experiment, the mice were analyzed for AAD. IP, Intraperitoneal.

531.e7 MEHROTRA ET AL

FIG E5. Lung inflammation and goblet cell hyperplasia are diminished with treatment with a PARP inhibitor. Paraffin-embedded lung sections of mice were stained with H&E. The photomicrographs are representative of 15 mice per group. The scale bar represents 50 mm (upper panels). Lung sections of mice treated as indicated were stained with PAS. The photomicrographs are representative of 15 mice per group. Scale bar 5 50 mm (lower panels).

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

MEHROTRA ET AL 531.e8

FIG E6. Inhibition of PARP activity in PARP-14–deficient animals does not further alleviate the pathogenesis of allergic inflammation. A, CD41CD62L1 cells isolated from the WT and Parp142/2 mice were cultured for

531.e9 MEHROTRA ET AL

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

FIG E7. Administration of PJ34 to mice with established AAD. Mice were sensitized with OVA plus alum on days 0 and 7 of the protocol. Starting on day 14, the mice were challenged with OVA for 10 consecutive days. PJ34 was administered as indicated from days 19 to 23 of the experiment, and the mice were analyzed for AAD on day 24. A group of mice were analyzed for AAD on day 19 before PJ34 treatment, as indicated by the asterisk. IP, Intraperitoneal.

=

7 days under TH2 conditions with and without PJ34. Equal numbers of differentiated cells were restimulated with anti-CD3 and anti-CD28 overnight, and cell-free supernatants were analyzed for cytokines. B-F, Parp141/1 and Parp142/2 mice were sensitized and challenged with OVA and alum. Half the number of mice from each group were treated with PJ34 during sensitization and challenges. Cytokine and chemokine message levels normalized to b-actin were measured in the lungs of the mice by using qPCR (Fig E6, B). Inflammatory cells in the BAL fluid from these mice were counted (Fig E6, C). IgE levels were measured in the BAL fluid from each mouse by using ELISA (Fig E6, D). The amount of total and OVA-specific IgE in the serum of these mice was also determined (Fig E6, E). Splenocytes isolated from these mice were restimulated with anti-CD3 and anti-CD28 for 48 hours, and cytokine levels were measured in the cell-free supernatants (Fig E6, F). The above results are mean 6 SEM values of 2 independent experiments with _ .05. 5 mice per group for each experiment. *P <

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

MEHROTRA ET AL 531.e10

FIG E8. BALB/c mice were sensitized with OVA alum (SC) or PBS twice (NSNC) and challenged with OVA or PBS for 5 days, and different parameters of airway disease were analyzed after the last challenge. A, Cells collected from the BAL fluid of these mice were counted (left graph) and stained for lineage-specific markers (middle and right graphs). DCs, Dendritic cells. B, Lung resistance (RL) in response to increasing doses of methacholine was measured by using the invasive resistance and compliance method. C, Isolated splenocytes were restimulated with OVA (100 mg) for 72 hours, and cytokine levels were measured by using ELISA. D, IgE levels in BAL fluid were also measured by using ELISA. N.D., Not detected. E, Total and OVA-specific IgE levels were quantified in the sera collected from the above treated mice. F and G, Lung sections were stained with H&E (Fig E8, F) or PAS (Fig E8, G). The photomicrographs are representative of 7 mice per group. Scale bars 5 50 mm. Each of the graphs are mean 6 SEM values of 7 to 9 mice analyzed in 2 independent experiments.

531.e11 MEHROTRA ET AL

FIG E9. Inhibition of PARP activity reduces inflammation and mucus production in an established AAD model. Lung sections from mice sensitized and challenged for 10 days with OVA and treated with PBS (SC [2]) or PJ34 (SC PJ34) for the last 5 days of the challenge were stained with H&E (upper panel) and PAS (lower panel). The photomicrographs are representative of 7 mice per group. Original magnification is 3100.

J ALLERGY CLIN IMMUNOL FEBRUARY 2013

J ALLERGY CLIN IMMUNOL VOLUME 131, NUMBER 2

MEHROTRA ET AL 531.e12

FIG E10. Gata3 expression is reduced in the presence of PARP-14–specific RNA interference. A, TH0-primed cells were retrovirally transduced with short hairpins specific for LacZ or PARP-14 on day 2 of a 5-day TH2 culture. Sorted cells were harvested and analyzed for Gata3 expression by means of real-time PCR. Gata3 mRNA levels were normalized to b-actin. B, Naive T cells were nucleofected with either scrambled or PARP-14–specific siRNA and then cultured under TH2 conditions. On day 5, cells were lysed in Trizol, and Gata3 levels were measured. The above graphs are mean 6 SEM values of 2 independent experiments. _ .05. *P <