Transcriptional Reprogramming and Resistance to Colonic Mucosal Injury in Poly(ADP-ribose) Polymerase 1 (PARP1)-deficient Mice

Abstract

Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection.

Keywords: Poly (ADP-ribose) polymerase; colitis; dextran sulfate; gene expression; gene knockout; gut microbiota; innate immunity; microarray; regulatory T cells.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Reduced mortality and mucosal inflammation in DSS-treated Parp1−/− mice.A, no mortality was observed in Parp1−/− mice treated with 4% DSS for 7 days. B, representative H&E staining of the proximal and distal colon of WT and Parp1−/− mice treated with 3% DSS for 7 days. C, colonic mucosal cytokine mRNA expression in WT and Parp1−/− mice treated with 3% DSS for 7 days evaluated by real-time RT-PCR (n = 3–7; asterisks indicate statistical significance at p < 0.05 between DSS-treated WT and WT and Parp1−/− mice; ANOVA followed by Fisher protected least significant difference post hoc test). D, secretion of IFNγ and TNFα in colonic explant culture from 3% DSS-treated WT and Parp1−/− mice.

FIGURE 2.

Gene expression patterns in control and 3% DSS-treated WT and Parp1−/− mice; hierarchical condition tree.A, data were normalized to the median of all samples, filtered on expression levels (raw data >20.0 in at least one of 12 analyzed samples) and without statistical analysis or pre-selection selection, were subjected to hierarchical clustering. B, analogous clustering analysis but with a preselected gene list from 2-way ANOVA analysis with the corrected p value for genotype-treatment of <0.05, with Benjamini-Hochberg test used as the multiple testing correction.

FIGURE 3.

The magnitude of transcriptional reprogramming in the colon of Parp1−/− mice at baseline. Data were normalized and filtered as in Fig. 2 and statistically analyzed for transcript IDs that statistically differed between WT and Parp1−/− mice at baseline (without DSS) (moderated t test with Benjamini-Hochberg test used as the multiple testing correction). The transcript IDs identified as significantly different (p < 0.05) were plotted as the number of up-regulated (blue line) or down-regulated (red line) transcripts with -fold change increasing from 2- to 10-fold over WT controls.

FIGURE 4.

Gene ontology (GO) analysis of genes differentially expressed in WT and Parp1−/− mice at baseline. 4,785 transcript cluster IDs (WT versus Parp1−/−; -fold change ≥2, p < 0.05) were converted into 3,587 uniquely annotated proteins and analyzed with the DAVID functional annotation tool with the following options: minimum 10 per category, Ease score <0.01. To reduce the results for presentation, biological process categories with ≥100 genes/proteins in each were selected. Black bars represent the number of genes in a given category (upper horizontal axis), and orange dots represent EASE score for each category (lower horizontal axis).

FIGURE 5.

oPOSSUM analysis of transcription factors binding sites over-represented or under-represented in genes regulated in the colon of Parp1−/− mice at baseline. To limit the query to the allowed list to 2,000 genes, we used a sliding scale of moderated t test p value (untreated WT versus Parp1−/− mice) and arrived at p ≤ 0.00365. From this set, oPOSSUM algorithm selected 1600 known genes for analysis. 53 TFs were found as overrepresented (Z-score >1.0; range 33.54 to 1.12), and 53 were underrepresented (Z score < −1.0;l range −35.92 to −1.21). The relative contribution of the under- and over-represented cis elements grouped into respective transcription factor classes is depicted.

FIGURE 6.

Differential effect of genotype (WT versus Parp1−/−) and treatment (H2O) versus 3% DSS on colonic gene expression.A, data were normalized and filtered as in Fig. 2 and statistically analyzed for transcript IDs that statistically differed between H2O- and DSS-treated WT mice (moderated t test p ≤ 0.05, -fold change cutoff of ≥2.0). 755 transcript IDs identified in WT mice are plotted. B, the same 755 transcript IDs were selected and plotted with respective normalized values derived from H2O- and DSS-treated Parp1−/− mice.

FIGURE 7.

Colonic microbial community in Parp1−/− mice.A, next-generation sequencing analysis of the contributions of the major bacterial orders in the colonic contents of WT and Parp1−/− mice. Increase in the order Clostridiales in PARP1-deficient mice was further analyzed at the family level. Statistically significant differences are indicated with an asterisk (p = 0.028). B, real-time PCR analysis of the relative abundance of Clostridia clusters IX and XIVa in WT and Parp1−/− mice at baseline. Data were normalized to all bacteria/archaea detected with universal 16S primers depicted in Table 1. C, flow cytometry analysis of CD4+CD25+FoxP3+ Tregs in the colonic lamina propria of WT and Parp1−/− mice. Representative dot plots (left panels) and summary graph (right panel) are shown; p value from unpaired two-tail t test is indicated.

FIGURE 8.

Loss of protective effects of Parp1 deficiency in Parp1−/−x Rag2−/− DKO mice.A, body weight loss and mortality in response to treatment with 4% DSS for 7 days. B, representative H&E histology staining of the proximal and distal colon of Rag2−/− and DKO mice treated with H2O or 3% DSS for 7 days. C, mucosal TNFα and IFNγ expression in the proximal and distal colon of Rag2−/− and DKO mice treated with 3% DSS for 7 days (N.S., not statistically significant).