Fungal β-glucan instructed miR-32-5p modulates Dectin-1 signaling mediated inflammation, reactive oxygen species and apoptosis through polarization of "M2a-like" macrophage in Candida colitis

Abstract

Ulcerative colitis (UC) is a chronic and easy-to-relapse intestinal disease characterized by colon inflammation and microbial dysbiosis. Candida albicans is the most common fungal resident in the human gut. Overgrowth of C. albicans has been linked to the aggravation of UC. Previously, we demonstrated that miR-32-5p was the most differentially expressed microRNA in DSS-induced colitis model supplemented with C. albicans and might be a potential target in the treatment of Candida colitis. However, the underlying pathogenic and therapeutic mechanisms remain unclear. Here, we firstly used the ITS technique to analyse intestinal mycobiota. The miR-32-5p adenovirus in company with extracted Candida cell wall β-glucan were then employed to monitor the impacts of miR-32-5p and fungal β-glucan on the severity of Candida colitis. Subsequently, gene silencing together with inhibitors of reactive oxygen species (ROS) and apoptosis was used to survey the modulation of miR-32-5p on macrophage polarization. According to these results, C. albicans became the dominant intestinal fungal species in Candida colitis model. Candida β-glucan could instruct miR-32-5p expression to affect the severity of Candida colitis in a concentration-dependent manner. Interestingly, high fungal β-glucan with pro-inflammatory effects hindered further increases in gut inflammation. Further analysis showed that overexpression of miR-32-5p could effectively inhibit inflammation and apoptosis and enhance phagocytosis and ROS production through Dectin-1 signalling in macrophages. A panel of representative gene expressions verified the polarization of the M2-like phenotype induced by miR-32-5p. Mechanistically, our results reveal the therapeutic potential of miR-32-5p in the amelioration of Candida colitis.

Keywords: Candida albicans; Dectin-1; Ulcerative colitis; miR-32-5p; oxidative stress; β-glucan.

Figure 1.

Candida albicans causes intestinal mycobiota dysbiosis. (a) Workflow of DSS induced colitis establishment (n = 5). (b) ACE index. (c) Shannon index. (d) Simpsoneven index. (e) Venn diagram based on identified OTUs. (f) PCoA analysis based on weighted UniFrac distance. (g) Relative abundance of dominant fungi phyla. (h) Distribution of microbial communities at the phylum level for each sample. Data is visualized by Circos. The bar width of each phylum represents the relative abundance of that phylum in the sample. (i) Community heatmap analysis at the genus level. b was analysed by ANOVA using the Tukey’s test. c and d were analysed by ANOVA using Dunn’s post-hoc test. Data are shown as the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2.

Increased miR-32-5p is protective against DSS induced colitis with C. albicans supplementation. (a) Workflow of colitis establishment in mice. The mice are intraperitoneally injected with miR-32-5p adenovirus followed by being normally given water and food at liberty for 2 weeks before DSS gavage. b. Weight loss (n = 5). c. Disease activity index (n = 5). d. Colon length (n = 5). e. Histopathologic changes of colon tissue (n = 3). Scale bar: 50 μm. f-h. Relative gene expressions of miR-32-5p, IL-1β and TNF-α in colon tissues (n = 3). i-l. Correlations of colon inflammation and intestinal barrier integrity with miR-32-5p in the presence (i, j) or absence (k, l) of C. albicans (n = 5). i, k. TNF-α versus miR-32-5p (p < 0.01,rs = -0.806; p = 0.263 > 0.01, rs = -0.394), j, l. FITC-dextran versus miR-32-5p (p < 0.01,rs=-0.956; p = 0.464 > 0.05, rs = 0.232). (m). Fungal burdens of colon, kidney, liver, lung, spleen, stomach and brain in the presence of C. albicans (n = 3). n, o. Immunoblots of tight-junction protein. (n). Occludin, (o). Claudin-1. Each experiment was repeated three times. Data are shown as the mean ± SD. B,C,D,F-H,N,O were analysed by one-way analysis of variance using Least Significant Difference (LSD) post hoc test. I-L were analysed by Spearman analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.

Figure 3.

Cell wall β-glucan is the major component to stimulate pro- and anti-inflammatory dual responses through miR-32-5p in Candida colitis. a,b. Relative expression of miR-32-5p in (a) Caco2 and (b) RAW264.7 cells (both at 3 × 10⁵ cells/mL) treated with heat-killed C. albicans(HK-CA, 9 × 10⁵CFU), cell wall of C. albicans(CW-CA, 100 μg/mL), cellular inclusion of C. albicans(CI-CA, 9 × 10⁵CFU), supernatant from C. albicansat 9 × 10⁵CFU/mL (SN-CA, 1 mL), CW-CA (100 μg/mL) hydrolysed by β-glucanase (0.1 mg/mL) for 4 h. c. Relative expression of miR-32-5p in RAW264.7 cells (3 × 10⁵ cells/mL) treated, respectively, with zymosan at 50 (Zym50) and 200 μg/mL (Zym200) for 4 h. d. Workflow of colitis establishment in the presence of C. albicans. The mice were treated with SN-CA (200 μL per mouse from 1 × 10⁸CFU), CW-CA (50 mg/kg), cell wall β-glucan of C. albicansat 20 mg/kg (CWGlu-CA-20), CWGlu-CA-20 hydrolysed by β-glucanase (4 mg/mL), curdlan (25 mg/kg), cell wall β-glucan of C. albicansat 80 mg/kg (CWGlu-CA-80) on the day 1, 3, 5, 7. e. Weight loss (n = 5). f. Disease activity index (n = 5). g. Colon length (n = 5). h. Histopathologic changes of colon tissue (n = 3). Scale bar: 50 μm. i. Relative expression of miR-32-5p in colon tissues (n = 3). j. Fungal burdens of colon, kidney, stomach, spleen, lung, and liver (n = 3). k,l. Immunoblots of tight-junction protein Occludin and Claudin-1. Each experiment was repeated three times. Data are shown as the mean ± SD. A-C, E-H and J-L were analysed by one-way analysis of variance using the Least Significant Difference (LSD) post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.

Figure 4.

Fungal β-glucan restricts ever-increasing inflammation. (a–c). Relative expressions of miR-32-5p, TNF-α and IL-1β in RAW264.7 cells (3 × 10⁵ cells/mL) treated with isometric supernatant from C. albicans at 9 × 10⁵CFU/mL (SN-CA, 1 mL) and/or β-glucanase (0.01 mg/mL) for 4 h. (d). Flow cytometry analysis of exposed cell wall β-glucan of C. albicans (1 × 10⁶ CFU/mL) induced by caspofungin at 0.005, 0.01 and 0.02 μg/mL for 24 h. (e). Fungal counting in RAW264.7 (3 × 10⁵ cells/mL) infected with C. albicans (9 × 10⁵ CFU/mL) after co-culture with caspofungin at 0.005, 0.01 and 0.02 μg/mL for 24 h. (f-h). Relative expressions of miR-32-5p, TNF-α and IL-1β. The experimental conditions are the same as E. Each experiment was repeated three times. Data are shown as the mean ± SD. (a-h) were analyzed by one-way analysis of variance using Least Significant Difference (LSD) post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.

Figure 5.

MiR-32-5p facilitate fungal removal through Dectin-1 mediated ROS.

Figure 6.

miR-32-5p influences macrophage apoptosis in a Dectin-1 receptor independent manner.

Figure 7.

MiR-32-5p induces polarization of M2a phenotype. a. Representative fluorescent photos of CD206 expression stained by DAPI and anti-CD206 in RAW264.7 treated with miR-32-5p mimic. Magnification: ×200.b. Flow cytometry analysis of CD206 in RAW264.7 treated with miR-32-5p mimic.c-k. Relative expressions of a panel of M2a phenotype genes. (c) TGF-β, (d) Arg1, (e) Ym1, (f) IL-4, (g) IL-13, (h) TNF-α, (i) IL-1β, (j) IL-6, (k) IL-10. Each experiment was repeated three times. Data are shown as the mean ± SD. B-K were analysed by one-way analysis of variance using the Least Significant Difference (LSD) post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: no significance.

Figure 8.

Schematic workflow of this study.