Low-dose ganciclovir ameliorates dextran sulfate sodium-induced ulcerative colitis through inhibiting macrophage STING activation in mice

Abstract

Ganciclovir (GCV) is a prodrug nucleoside analogue and is clinically used as antiviral drug for the treatment of cytomegalovirus (CMV) and other infections. Based on the potential anti-inflammatory activity of GCV, this study aimed to investigate the therapeutic effects of ganciclovir on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC), which may involve cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathways. Our results demonstrated that incubation of GCV (50 μM) inhibited cGAS-STING pathway in macrophage RAW264.7 cells. Then, it was found that intestinal cGAS-STING pathways were upregulated in UC patients, Crohn's disease colitis (CD) patients, and DSS-induced colitis mice. Intraperitoneal injection of low-dose GCV (10 mg/kg/day) attenuated DSS-induced colitis and abdominal pain in mice. GCV treatment significantly inhibited the upregulation of cGAS-STING pathway in DSS-induced colitis mice. Moreover, DSS-induced colitis and gut dysbiosis was markedly attenuated in STING deficient mice compared with that of wild-type (WT) mice. Finally, there was lacking therapeutic effect of GCV on DSS-induced colitis in STING deficient mice. Together, our results indicated that low-dose GCV ameliorated DSS-induced UC in mice, possibly through inhibiting STING signaling in colonic macrophages, indicating that GCV may be useful for the treatment of UC.

Keywords: STING; colitis; ganciclovir; macrophage; microbiome.

FIGURE 1

Effects of GCV on expression of inflammatory factors in RAW264.7 cells. (A–E) Q-PCR analysis showed dose-dependent effects of GCV treatment for 24 h on the mRNA expression of Cgas (A), Sting1 (B), Il10 (C), Ifnb1 (D), and Cxcl10 (E) in RAW264.7 cells. (F) Dose-dependent effects of GCV treatment on the mortality associated with LPS-induced sepsis in mice. (n = 7-8 per group; ^* p < 0.05; Log-rank test). (G) Western blotting analysis showing the effect of GCV on LPS-induced expression of IL-1β and TNF-α in RAW264.7 cells. (H) Statistical analysis results of (G). (I) Q-PCR analysis the effect of GCV on LPS-induced mRNA expression of Il6, Il1b, and Tnf in RAW264.7 cells. (n = 4 each group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. saline; ^&& p < 0.01, ^&&& p < 0.001 vs. LPS group, unpaired Student’s t-test). All data was expressed as Mean ± SEM. n.s., no significance.

FIGURE 2

Low-dose GCV inhibited cGAS-STING pathways in RAW264.7 cells. (A–C) Q-PCR analysis showed the effect of pretreatment of GCV on up-regulation of mRNA expression of Sting1, Il10, Ifnb1 induced by CMA- (A), DMXAA- (B), and cGAMP (C) in RAW264.7 cells. (D) Western blotting analysis showed the effect of GCV on CMA-induced expression changes of cGAS, STING, IFN-β, and p-TBK1 in RAW264.7 cells. (E) Statistical analysis results for (D). (F) Western blotting analysis showed the effect of GCV on DMXAA-induced expression changes of cGAS, STING, IFN-β, and p-TBK1 in RAW264.7 cells. (G) Statistical analysis results for (F). (H) Western blotting analysis showed the effect of GCV on cGAMP-induced expression changes of cGAS, STING, IFN-β, and p-TBK1 in RAW264.7 cells. (I) Statistical analysis results for (H). (n = 4 each group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 vs. saline. ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001 vs. STING agonists group, unpaired Student’s t-test). (n = 4 each group, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, ^&& p < 0.01, ^&&& p < 0.001; unpaired Student’s t-test). All data was expressed as Mean ± SEM. VEH, vehicle.

FIGURE 3

STING was upregulated following DSS-induced chronic colitis in mice and in UC patients. (A) Body weight loss was induced by DSS-colitis in mice. (B) The quantification of area under the curve (AUC) for (A). (C) DSS-colitis induced changes of the Disease activity index (DAI) score. (D) The quantification of AUC for (C). (E) DSS-colitis induced mechanical pain hypersensitivity in the abdomen. (F) The quantification of AUC for (E). (n = 7-8 per group; *p < 0.05, **p < 0.05, ***p < 0.001, DSS vs. vehicle group; two-way ANOVA with post-hoc Bonferroni test). (G) Representative pictures showed colon shortening induced by DSS. (H) The quantification of colon shortening for (G). (n = 7-8 per group; ***p < 0.001, DSS vs. vehicle group; unpaired Student’s t-test). (I) Representative H&E staining of colon sections from DSS group and vehicle group. (J) Statistical analysis for (I). (n = 4 each group, ***p < 0.001, DSS vs. vehicle group; unpaired Student’s t-test). (K) Double immunostaining of STING and F4/80 in the colon tissue from DSS group and vehicle group. (L) Statistical analysis for (K), scale bar: 50 μm. (n = 4 each group, *p < 0.05, DSS vs. vehicle group; unpaired Student’s t-test). (M) Western blotting analysis of STING expression in colon of mice. (N) Statistical analysis of Western blotting. (n = 3 each group, *p < 0.05, DSS vs. vehicle group; unpaired Student’s t-test). (O) The expression of STING in the colon tissue was increased in patients with ulcerative colitis compared with adjacent tissue of colon cancer. (P) Statistical analysis results for (O). (n = 4; **p < 0.01 vs Normal; unpaired Student’s t-test). The scale bar: 20 μm. All data was expressed as Mean ± SEM. DAI, disease activity index; DSS, dextran sulfate sodium.

FIGURE 4

The up-regulation of gene expression of cGAS-STING pathways in IBD patients, including UC and CD. (A) The relative expression changes of macrophage cGAS-STING signaling pathway in IBD patients. (B) The mRNA levels of cGAS-STING signaling pathway in UC and CD responder/non-responder patients, before and after treatment of anti-TNF (infliximab), compared with non-IBD controls. R., anti-TNF responders; nR., anti-TNF non-responders; Bef αTNF, before anti-TNF treatment; Aft αTNF, after anti-TNF treatment. All data are shown as mean ± SD and are analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. UC, ulcerative colitis; CD, Crohn’s disease.

FIGURE 5

Low-dose GCV attenuated DSS-colitis in mice. (A) DSS-colitis induced body weight loss from different groups. (B) The quantification of AUC for (A). (C) DSS-colitis induced increase of DAI scores in different groups. (D) The quantification of AUC for (C). (E) DSS-colitis induced mechanical pain hypersensitivity in the abdomen in mice from different groups. (F) The quantification of AUC for (E). (n = 6-7 per group; ^* p < 0.05, ^** p < 0.05, ^*** p < 0.001, DSS vs. vehicle group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, GCV + DSS vs. DSS group; ^& p < 0.05, ^&& p < 0.01, ^&&& p < 0.001, STING^gt/gt + DSS vs DSS group; two-way ANOVA with post-hoc Bonferroni test). (G) Representative pictures of colonic length from different groups. (H) Quantification of the colonic length for (G). (n = 5-7 each group; ^* p < 0.05, ^## p < 0.01, ^& p < 0.05; one-way ANOVA with post-hoc Bonferroni test) (I) Representative photographies of H&E staining of colon sections from different groups. (J) Statistical analysis for (I) (n = 4 each group; ^*** p < 0.001, ^# p < 0.05, ^& p < 0.05; one-way ANOVA with post-hoc Bonferroni test). (K,L) The Q-PCR analysis of mRNA expression of colonic Cgas, Il10, Ifnb1, Cxcl10, Il1b, Il6, and Tnf in mice of different group (n = 6 per group, ^** p < 0.01, ^*** p < 0.001, ^## p < 0.01, ^### p < 0.001; unpaired Student’s t-test). All data was expressed as Mean ± SEM. DSS, dextran sulfate sodium; VEH, vehicle.

FIGURE 6

Low-dose GCV attenuated cGAS-STING pathways in the colon of DSS-colitis in mice. (A) Western blotting analysis showed that protein expression of colonic STING, cGAS, p-TBK1, IFN-β, IL-1β, and TNF-α in mice of different treatment group. (B) Statistical analysis for (A) (n = 4 per group, ^* p < 0.05, ^** p < 0.01, DSS vs. vehicle group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, GCV + DSS vs. DSS group; unpaired Student’s t-test). (C) Double immunostaining of STING and F4/80 in the colon tissue. (D) Statistical results for (C). Scale bar = 50 μm. (n = 4 each group, ^*** p < 0.001, ^## p < 0.01; unpaired Student’s t-test). All data was expressed as Mean ± SEM.

FIGURE 7

STING deficiency attenuated DSS-colitis in mice. (A) Representative Western blotting detected cGAS and STING expression in STING^gt/gt and WT mice. (B) Statistical analysis for (A). (C) Representative immunofluorescence detected STING and F4/80 expression in STING^gt/gt and WT mice. (D) Statistical analysis for (C). (n = 3 each group, ^** p < 0.01, STING^gt/gt vs. vehicle group; unpaired Student’s t-test). Wild-type (WT) and STING^gt/gt mice were given 3% dextran sulfate sodium (DSS) in drinking water for 8 days. (E) Body weight changed following DSS administration in mice. (F) The quantification of AUC for (E). (G) DSS-induced changes of DAI score in WT and STING^gt/gt mice. (H) The quantification of AUC for (G). (I) DSS-induced changes of mechanical pain sensitivity in the abdomen in STING^gt/gt mice and WT mice. (J) The quantification of AUC for (I). (n = 5-7 per group; ^** p < 0.01, ^*** p < 0.001, DSS vs. vehicle group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, STING^gt/gt + DSS vs DSS group; two-way ANOVA with post-hoc Bonferroni test). (K) Representative pictures of colons from WT and STING^gt/gt mice on day 8. (L) Quantification of the colon length in (K). (n = 5-7 per group; ^*** p < 0.001, ^### p < 0.001; unpaired Student’s t-test). (M) Representative photographies of H&E staining of colon sections from 4 different groups. (N) Statistical analysis for (M) (n = 4 each group; ^*** p < 0.001, ^# p < 0.05; unpaired Student’s t-test). (O) The Q-PCR analysis of mRNA expression of colonic Cgas, Il10, Ifnb1, Cxcl10, Il1b, and Tnf in mice from different group (n = 6 each group, ^** p < 0.01, ^*** p < 0.001, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001; unpaired Student’s t-test). All data was expressed as Mean ± SEM. DSS, dextran sulfate sodium; n.s., no significance. WT, wild type.

FIGURE 8

STING deficiency prevented dysbiosis induced by DSS-colitis in mice. (A) Heatmap analyses of differentially abundant phylum between the vehicle group, WT + DSS group, vehicle + STING^gt/gt group, STING^gt/gt + DSS groups. (B) Heatmap analyses of relative abundant bacterial family in each four group. (C) Relative abundance of the main bacterial phylum in different group. (D) LEfSe score plot of the discriminative microbial taxa that are more enriched in the vehicle group (red), DSS (green), STING^gt/gt (blue), and STING^gt/gt + DSS(violet) groups. (E) Identification of differentially abundant microbial taxa using linear discriminant analysis effect size (LEfSe) analysis. LEfSe cladogram of the discriminative microbial taxa; the size of the circle showed the relative abundance of the taxa, and the colour shows the different group (red, vehicle group; green, DSS group; blue, STING^gt/gt group; violet, STING^gt/gt + DSS group). (F,G) Quantification of the relative abundance of Gram-negative communities (F) and Gram-positive communities (G) from (A). (H–K) Quantification of the relative abundance of Lactobacillales (H), Bifidobacteriales (I), Bacteroidaceae (J), Ruminococcaceae (K) from (B). (n = 3–10 each group, n.s., no significance, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, unpaired Student’s t-test). All data was expressed as Mean ± SEM. WT, wild type. DSS, dextran sulfate sodium; WT, wild type.

FIGURE 9

Low-dose GCV had no therapeutic effects on DSS-colitis in mice. (A) DSS-colitis induced body weight changes in different groups. (B) The quantification of AUC for (A). (C) DSS-colitis induced DAI score changes of mice in different groups. (D) The quantification of AUC for (C). (E) DSS-colitis induced mechanical pain hypersensitivity in the abdomen in mice from different groups. (F) The quantification of AUC for (E). (n = 5-7 per group; n.s., no significance, ^# p < 0.05, ^### p < 0.001, GCV + DSS vs. DSS group; ^& p < 0.05, ^&&& p < 0.001, STING^gt/gt + DSS vs DSS group; two-way ANOVA with post-hoc Bonferroni test). (G) Representative pictures of colons from different groups. (H) Quantification of the colonic length for (G). (n = 5-7 each group; ^## p < 0.01; ^### p < 0.001; unpaired Student’s t-test). (I) Representative photographies of H&E staining of colon sections from different groups. (J) Statistical results for (I) (n = 4 each group; ^# p < 0.05; unpaired Student’s t-test). (K) Q-PCR analysis showed the mRNA expression changes of Cgas, Il10, Cxcl10, Ifnb1, Tnf, Il6, and Il1b from different group (n = 6 per group, unpaired Student’s t-test). All data was expressed as Mean ± SEM. n.s., no significance.

FIGURE 10

The working hypothesis of this study.