Melatonin Mitigates Oxazolone-Induced Colitis in Microbiota-Dependent Manner

Abstract

Levels of type 2 cytokines are elevated in the blood and intestinal tissues of ulcerative colitis (UC) patients in the active phase; this phenomenon indicates the participation of type 2 immune response in UC progression. The beneficial effects of melatonin in dextran sodium sulfate (DSS) and 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis models have been illustrated, but its role in the oxazolone (Oxa)-induced colitis model (driven by type 2 immune response) remains relatively unknown. We investigated the relationship between melatonin concentration and the severity of UC, revealing a significantly negative correlation. Subsequently, we investigated the effects of melatonin in Oxa-induced colitis mice and the potential underlying mechanisms. Administration of melatonin significantly counteracted body weight loss, colon shortening, and neutrophil infiltration in Oxa-induced colitis mice. Melatonin treatment mitigated Oxa-induced colitis by suppressing type 2 immune response. In addition, melatonin attenuated intestinal permeability by enhancing the expression of ZO-1 and occludin in colitis mice. Interestingly, the protective effect of melatonin was abolished when the mice were co-housed, indicating that the regulation of gut microbiota by melatonin was critical in alleviating Oxa-induced colitis. Subsequently, 16S rRNA sequencing was performed to explore the microbiota composition. Decreased richness and diversity of intestinal microbiota at the operational taxonomic unit (OTU) level resulted from melatonin treatment. Melatonin also elevated the abundance of Bifidobacterium, a well-known probiotic, and reduced proportions of several harmful bacterial genera, such as Desulfovibrio, Peptococcaceae, and Lachnospiraceae. Fecal microbiota transplantation (FMT) was used to explore the role of microbiota in the function of melatonin in Oxa-induced colitis. Microbiota transplantation from melatonin-treated mice alleviated Oxa-induced colitis, suggesting that the microbiome participates in the relief of Oxa-induced colitis by melatonin. Our findings demonstrate that melatonin ameliorates Oxa-induced colitis in a microbiota-dependent manner, suggesting the therapeutic potential of melatonin in treating type 2 immunity-associated UC.

Keywords: ILC2; inflammatory bowel disease; melatonin; microbiota; ulcerative colitis.

Figure 1

Melatonin in the colon decreases and negatively associates with disease severity in ulcerative colitis (UC) patients. (A) Melatonin concentrations in healthy control (HC) and UC patients were detected by ELISA. (B) Representative H&E staining of colonic sections (×20). (C) Correlation of melatonin concentration and histological severity scores of all 11 UC patients and 4 random HCs. (D, E) AANAT and HIOMT mRNA expression levels in HCs and UC patients (all values were normalized to the HC group). ***p < 0.001; ****p < 0.0001.

Figure 2

Melatonin mitigates oxazolone (Oxa)-induced colitis. (A) Body weight changes after 3% Oxa-treated (day 0). (B) Weight changes at day 1. (C, D) Colon length at day 1. (E–G) Representative H&E staining (×20) and pathological score of the inflamed gut epithelium. (H, I) Flow cytometry analysis of colonic CD45⁺CD11b⁺Ly6G⁺ neutrophil subsets in melatonin-treated and melatonin-untreated mice at day 1 after 3% oxazolone enema. (J, K) qPCR of Tnfa and Il-1b mRNA in colonic tissues in melatonin-treated and melatonin-untreated mice at day 1 after 3% oxazolone enema (all values were normalized to the melatonin-untreated group). (L) Immunoblot for ZO-1 and occludin in colonic tissue and (M, N) the densitometric analysis of ZO-1 and occludin immunoblot of melatonin-treated and melatonin-untreated mice. Data are representative of at least two independent experiments. Data are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Melatonin inhibits type 2 immunity in oxazolone (Oxa)-induced colitis. (A, B) Flow cytometry analysis of IL-5 and IL-13 in colonic CD45⁺Lin⁻GATA3⁺ ILC2s and quantification of IL-13 in colonic ILC2s in melatonin-treated and melatonin-untreated mice at day 1 after 3% oxazolone enema. (C, D) qPCR of Il-5 and Il-13 mRNA of colonic tissue in melatonin-treated and melatonin-untreated mice at day 1 after 3% oxazolone enema (all values were normalized to the melatonin-untreated group). (E, F) Representative flow cytometry plots and quantification of large intestinal CD45⁺Lin⁻GATA3⁺ ILC2 function treated with melatonin for 16 h in LPLs in vitro. (G, H) Representative flow cytometry plots and quantification of purified large intestinal CD45⁺Lin⁻CD127⁺KLRG1⁺ ILC2 function treated with melatonin for 16 h in vitro. Data are representative of at least two independent experiments. Data are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Effect of melatonin on oxazolone (Oxa)-induced colitis depends on gut microbiota. (A) Weight changes and colon length (B, C) in co-housed mice at day 1 after 3% oxazolone enema. (D–F) Representative H&E staining of colonic sections (×20) and pathological score of inflamed colonic tissue. (G, H) Flow cytometry analysis of colonic CD45⁺CD11b⁺Ly6G⁺ neutrophil subsets in co-housed mice at day 1 after 3% oxazolone enema. (I, J) qPCR of Tnfa and Il-1b mRNA in colonic tissues in co-housed mice at day 1 after 3% oxazolone enema (all values were normalized to the melatonin-untreated group). (K–M) Immunoblot and densitometric analysis of ZO-1 and occludin in co-housed mice. Data are representative of at least two independent experiments. Data are shown as mean ± SD. ns, no significance.

Figure 5

Melatonin alters microbiota composition in alpha and beta diversity. (A) Principal coordinate analysis (PCoA) of fecal microbiota in separately housed mice treated or untreated with melatonin for 7 days. (B) PCoA of fecal microbiota in co-housed mice treated or untreated with melatonin for 7 days. (C) PCoA of fecal microbiota among separately housed melatonin-treated mice, separately housed melatonin-untreated mice, and co-housed mice. PCoA is based on binary Jaccard distance. ANOSIM and 999 times permutation tests were utilized. (D–F) Shannon, Chao, and Ace indices of operational taxonomic unit (OTU) levels. Data are expressed as mean ± SD; statistical significance was determined by a two-sided Student’s t-test or one-way ANOVA. *p < 0.05; ***p < 0.001; ****p < 0.0001. ns, no significance.

Figure 6

Melatonin regulates certain microbiota on genus level. (A) Kruskal–Wallis H test for differences between separated and co-housed mice for 7-day melatonin treatment on genus levels. (B–E) Relative abundance of Bifidobacterium, Desulfovibrio, Lachnospiraceae_UCG-006, and Peptococcaceae on genus levels. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ****p < 0.0001. ns, no significance.

Figure 7

Microbiota transplantation from melatonin-treated mice alleviates oxazolone (Oxa)-induced colitis. (A) The process of fecal microbiota transplantation. The mice were treated with antibiotics for 7 days to clear the gut microbiota (day 1 to day 7) and transplanted feces from 7-day melatonin-treated or melatonin-untreated mice (day 8 to day 14) before 3% oxazolone enema (day 15). Mice were sacrificed on day 16. (B) Body weight changes in fecal microbiota transplantation (FMT) mice at day 16. (C, D) Colon length in FMT mice at day 16. (E–G) Representative H&E staining (×20) and pathological score of the inflamed gut epithelium. (H, I) qPCR of Tnfa and Il-1b mRNA in colonic tissues in FMT mice at day 16 after 3% oxazolone enema (all values were normalized to MT0 group). (J–L) Immunoblot and densitometric analyses of ZO-1 and occludin in FMT mice at day 16 after 3% oxazolone enema. (M, N) qPCR of Il-5 and Il-13 mRNA in colonic tissues in FMT mice at day 16 after 3% oxazolone enema (all values were normalized to MT0 group). Data are representative of two independent experiments. Data are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ns, no significance.