Peptidoglycan recognition protein 3 and Nod2 synergistically protect mice from dextran sodium sulfate-induced colitis - PubMed (original) (raw)

Peptidoglycan recognition protein 3 and Nod2 synergistically protect mice from dextran sodium sulfate-induced colitis

Xuefang Jing et al. J Immunol. 2014.

Abstract

Aberrant immune response and changes in the gut microflora are the main causes of inflammatory bowel disease (IBD). Peptidoglycan recognition proteins (Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4) are bactericidal innate immunity proteins that maintain normal gut microbiome, protect against experimental colitis, and are associated with IBD in humans. Nucleotide-binding oligomerization domain 2 (Nod2) is an intracellular bacterial sensor and may be required for maintaining normal gut microbiome. Mutations in Nod2 are strongly associated with Crohn's disease, but the causative mechanism is not understood, and the role of Nod2 in ulcerative colitis is not known. Because IBD is likely caused by variable multiple mutations in different individuals, in this study, we examined the combined role of Pglyrp3 and Nod2 in the development of experimental colitis in mice. We demonstrate that a combined deficiency of Pglyrp3 and Nod2 results in higher sensitivity to dextran sodium sulfate-induced colitis compared with a single deficiency. Pglyrp3(-/-)Nod2(-/-) mice had decreased survival and higher loss of body weight, increased intestinal bleeding, higher apoptosis of colonic mucosa, elevated expression of cytokines and chemokines, altered gut microbiome, and increased levels of ATP in the colon. Increased sensitivity to dextran sodium sulfate-induced colitis in Pglyrp3(-/-)Nod2(-/-) mice depended on increased apoptosis of intestinal epithelium, changed gut microflora, and elevated ATP. Pglyrp3 deficiency contributed colitis-predisposing intestinal microflora and increased intestinal ATP, whereas Nod2 deficiency contributed higher apoptosis and responsiveness to increased level of ATP. In summary, Pglyrp3 and Nod2 are both required for maintaining gut homeostasis and protection against colitis, but their protective mechanisms differ.

Copyright © 2014 by The American Association of Immunologists, Inc.

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Figures

Figure 1

Figure 1. _Pglyrp3_−/−_Nod2_−/− mice are more susceptible to DSS-induced colitis than Pglyrp3 and Nod2 single-deficient mice

WT, _Nod2_−/−, _Pglyrp3_−/−, and _Pglyrp3_−/−_Nod2_−/− mice were treated with 5% DSS in drinking water and development of colitis was evaluated. (A) DSS-treated mice were monitored over time for survival, change in body weight, and stool and rectal bleeding. (B and C) DSS-treated mice were sacrificed on day 7 of treatment and hematoxylin-eosin stained sections of the colon were (B) qualitatively and (C) quantitatively evaluated for severity of colitis. Epithelial cells (E), lamina propria (LP), goblet cells (G), muscularis mucosa (MM), submucosa (SM), muscularis externa (ME), and inflammatory cell infiltrations (IN) are indicated; size bar = 100 μm. The data are (A) mean ± SEM of 19-20 mice/group; and (B) representative images or (C) mean ± SEM from 4 mice/group. Significance of differences between _Pglyrp3_−/−_Nod2_−/− and WT mice is indicated by asterisks (*); and between _Pglyrp3_−/−_Nod2_−/− and _Pglyrp3_−/− is indicated by the number sign (#); *, #, P ≤ 0.05; **, ##, P ≤ 0.005.

Figure 2

Figure 2. _Pglyrp3_−/−_Nod2_−/− mice have higher permeability and increased apoptosis in the colon

(A to D) WT, _Nod2_−/−, _Pglyrp3_−/−, and _Pglyrp3_−/−_Nod2_−/− mice were treated with 5% DSS and (A) gavaged with FITC-dextran on days 0, 3, 6, and 7 and assayed for FITC-dextran in the serum; (B) 4 days later injected with BrdU and colon sections were stained for BrdU; (C and D) 4 days later colon sections were stained for (C) TUNEL; or (D) cleaved caspase-3. (E) _Pglyrp3_−/− _Nod2_−/− mice were treated with 5% DSS and gavaged daily with the caspase inhibitor Q-VD-Oph or with PBS for the control group and development of colitis was monitored by survival, change in body weight, and stool and rectal bleeding scores or 4 days later colon sections were stained for TUNEL. The data are mean ± SEM of (A) 12, (B, C, and D) 4 to 6, and (E) 9 to 10 mice/group for colitis and 6 mice/group for TUNEL staining; representative histological images are shown in (B-D). Significance of differences for (A to D) knockout versus WT mice or (E) inhibitor treated versus control group is indicated by asterisks (*); and between _Pglyrp3_−/−_Nod2_−/− or _Nod2_−/− versus _Pglyrp3_−/− is indicated by the number sign (#); *, #, P ≤ 0.05; **, ##, P ≤ 0.005.

Figure 3

Figure 3. _Pglyrp3_−/−_Nod2_−/− mice express higher levels of chemokine and cytokine mRNA in the colon following DSS treatment

WT, _Nod2_−/−, Pglyrp3−/−, and _Pglyrp3_−/−_Nod2_−/− mice were treated with 5% DSS for indicated times and expression of inflammatory genes in the colon was measured by qRT-PCR. The results are means ± SEM of the ratio of the amount of mRNA in DSS-treated to untreated mice from 9 mice per group, 3 mice pooled per array. Significance of differences between knockouts and WT mice is indicated by asterisks (*) and between _Pglyrp3_−/− _Nod2_−/− and _Pglyrp3_−/− is indicated by the number sign (#); *, #, P ≤ 0.05; **, ##, P ≤ 0.005. The data are selected from the 96 inflammatory gene expression array shown in supplemental Table S1.

Figure 4

Figure 4. _Pglyrp3_−/−_Nod2_−/− mice have changes in intestinal bacterial flora, however only _Pglyrp3_-dependent changes contribute to the increased sensitivity to colitis in _Pglyrp3_−/− _Nod2_−/− mice

(A) Changes in the composition of bacterial flora in the stools of WT and knockout mice in all Eubacteria and the indicated bacterial groups, measured by qPCR; (B) WT germ-free mice were treated with 4% DSS and gavaged daily with stool homogenates from conventionally raised WT, _Nod2_−/−, _Pglyrp3_−/−, or _Pglyrp3_−/−_Nod2_−/− mice or (C) _Nod2_−/− mice were pre-treated with antibiotics for 3 weeks and then gavaged daily with stool homogenates from conventionally raised WT or _Pglyrp3_−/− and treated with 4% DSS. Development of colitis in B and C was evaluated by measuring survival, body weight, and stool score. Gross rectal bleeding in mice gavaged with stools from _Pglyrp3_−/− mice but not from WT mice is shown in (C). The results are means ± SEM of (A) 18 mice/group and (B and C) 6 mice/group from 2 experiments. Significance of differences for (A) *, knockout versus WT; (B) *, _Pglyrp3_−/− versus _Nod2_−/− and WT; #, _Pglyrp3_−/−_Nod2_−/− versus _Nod2_−/− and WT; ^, _Pglyrp3_−/−_Nod2_−/− versus _Pglyrp3_−/−; (C) *, _Pglyrp3_−/− versus WT; *, #, ^, P ≤ 0.05; **, ##, ^^, P ≤ 0.005.

Figure 5

Figure 5. WT (Nod+/+) but not _Nod2_−/− macrophages and colon cells produce more chemokines and cytokines in response to stools from _Pglyrp3_-deficient mice

(A) Colon cells and colon fragments or (B) bone marrow-derived macrophages from WT or _Nod2_−/− mice were stimulated with stools from WT or _Pglyrp3_−/− mice or with PBS (no stimulant). Supernatants were assayed for the indicated chemokines or cytokines. The results are means ± SEM (N = 6 experiments); significance of differences for _Nod2_−/− versus WT cells; *, P ≤ 0.05; **, P ≤ 0.005.

Figure 6

Figure 6. _Pglyrp3_−/−_Nod2_−/− and _Pglyrp3_−/− mice have higher levels of ATP in the colon, and intestinal ATP preferentially increases sensitivity of _Nod2_−/− mice to DSS-colitis

(A) Amounts of ATP in the stools of WT, _Nod2_−/−, _Pglyrp3_−/−, and _Pglyrp3_−/−_Nod2_−/− mice. (B) WT and _Nod2_−/− mice treated with 5% DSS and gavaged with the ATP analog, α,β-ATP, have lower survival and body weight, and higher stool scores than PBS-treated mice, and _Nod2_−/− mice are more sensitive to ATP than WT mice. (C) Increased percentages of the indicated cell types in colon lamina propria and mesenteric lymph nods (MLN) in _Nod2_−/− mice, gated for CD45 or CD4. (D) Representative dot plots for Th17, Th1, and Th2 cells for the data shown in (C). The results are means ± SEM of (A) 6-8, (B) 10-11, and (C) 6-7 mice/group from 2 experiments. Significance of differences for (A) knockouts versus WT; (B) *, **, ATP- versus PBS-treated _Nod2_−/− mice; ^, ^^, ATP- versus PBS-treated WT mice; #, _Nod2_−/− versus WT ATP-treated mice; or (C) ATP versus PBS; *, ^, #, P ≤ 0.05; **, ^^, P ≤ 0.001.

Figure 7

Figure 7. Lack of Nod2 in bone marrow-derived cells combined with presence of Nod2 in structural cells predisposes _Pglyrp3_−/− mice to severe colitis

(A) Chimeric (_Pglyrp3_−/− > _Pglyrp3_−/−_Nod2_−/− and _Pglyrp3_−/−_Nod2_−/− > _Pglyrp3_−/−) and transplanted control (_Pglyrp3_−/− > _Pglyrp3_−/− and _Pglyrp3_−/−_Nod2_−/− > _Pglyrp3_−/− _Nod2_−/−) mice were treated with 4% DSS in drinking water and development of colitis was evaluated. DSS-treated mice were monitored over time for survival, change in body weight, and stool and rectal bleeding. The results are means ± SEM of 5-6 mice/group. Statistically significant differences between the indicated groups are shown as *, P ≤ 0.05; **, P ≤ 0.005. (B) Presence of Nod2 KO (1 kb amplified fragment) or Nod2 WT (300 bp amplified fragment) alleles was assessed by PCR of genomic DNA from blood cells of chimeric (_Pglyrp3_−/−_Nod2_−/− > _Pglyrp3_−/− and _Pglyrp3_−/− > _Pglyrp3_−/−_Nod2_−/−) or non-transplanted _Pglyrp3_−/− and _Nod2_−/− mice.

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