1. 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, Shreevrat Goenka, PhD 
Journal of Allergy and Clinical Immunology 
Volume 131, Issue 2, Pages e12 (February 2013)
DOI: /j.jaci
Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
PARP-14 and its activity are required for optimal TH2 differentiation. A and B, Naive T cells from Parp14−/− 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, CD4+CD62Lhigh 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 ± SEM values of 3 different experiments. *P ≤ .05 compared with control values.
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3. Fig 2
PARP-14 plays a role in the development of AAD. A-E, Parp14+/+ and Parp14−/− 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 (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 measured (G). The results are plotted as mean ± SEM values from 3 independent experiments. *P ≤ .05 when compared with WT control animals.
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4. 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 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 ± SEM values of 3 independent experiments, with each experiment having 5 mice per group. *P ≤ .05 compared with control animals.
Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci )
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5. 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 ± SEM values of 2 independent experiments, with 4 to 5 mice in each group for each experiment. *P ≤ .05.
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6. Fig 5
PARP-14 and activity associated with it are required for ambient GATA3 expression. Naive CD4+ T cells from Parp14+/+ and Parp14−/− 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 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, CD4+ cells isolated from Parp14+/+ and Parp14−/− mice were cultured for 5 days under TH2 conditions, and Gata3 mRNA levels were measured on each day. D, CD4+ 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, CD4+ cells were cultured under TH2 conditions with or without 10 mmol/L PJ34. Transcripts of Gata3 were quantified by using qPCR. F, CD4+ 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 ± SEM values of 3 independent experiments. *P ≤ .05 when compared with control animals.
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7. 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 β-actin as an endogenous control (lower panel). The blot shows a representative of 2 independent experiments.
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8. 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–β-actin. The blot shows results of 2 mice per group. B, Parp14+/+ and Parp14−/− 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.
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9. 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) Parp14+/+ and Parp14−/− 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 ×100, and the bar is 50 μm.
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10. 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.
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11. 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 μm (upper panels). Lung sections of mice treated as indicated were stained with PAS. The photomicrographs are representative of 15 mice per group. Scale bar = 50 μm (lower panels).
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12. Fig E6
Inhibition of PARP activity in PARP-14–deficient animals does not further alleviate the pathogenesis of allergic inflammation. A, CD4+CD62L+ cells isolated from the WT and Parp14−/− mice were cultured for 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, Parp14+/+ and Parp14−/− 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 β-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 ± SEM values of 2 independent experiments with 5 mice per group for each experiment. *P ≤ .05.
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13. 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.
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14. 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 = 50 μm. Each of the graphs are mean ± SEM values of 7 to 9 mice analyzed in 2 independent experiments.
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15. 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 [−]) 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 ×100.
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16. 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 β-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 ± SEM values of 2 independent experiments. *P ≤ .05.
Journal of Allergy and Clinical Immunology  , e12DOI: ( /j.jaci )
Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions