Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii–derived glutamic acid–maintained Tregs
Xinying Wang, Haiyang Sun, Ying Tan, Shaoting Xu, Zishan Liu, Kaile Ji, Ding Qiu, Jianping Deng, Bingbing Feng, Xueting Wu, Yoichiro Iwakura, Minhu Chen, Rui Feng, Chanyan Huang, Ce Tang
Xinying Wang, Haiyang Sun, Ying Tan, Shaoting Xu, Zishan Liu, Kaile Ji, Ding Qiu, Jianping Deng, Bingbing Feng, Xueting Wu, Yoichiro Iwakura, Minhu Chen, Rui Feng, Chanyan Huang, Ce Tang
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Research Article Gastroenterology Microbiology

Colonic Engyodontium fungus triggers neutrophil antimicrobial activity to suppress Lactobacillus johnsonii–derived glutamic acid–maintained Tregs

Abstract

Isolating commensal fungi from mouse intestines has been challenging, limiting our understanding of their role in intestinal immune homeostasis and diseases. Using an Fc fusion protein of the C-type lectin receptor Dectin-2, we successfully purified the commensal Ascomycota fungus Engyodontium sp. from mouse feces. Engyodontium enhances the antimicrobial activity of colonic neutrophils via the CARD9 pathway and exacerbates colitis by impairing the colonization of intestinal Lactobacillus johnsonii WXY strain. L. johnsonii produces high levels of l-glutamic acid by expressing the glutaminase-encoding gene glsA to facilitate Treg expansion via enhancing IL-2 receptor signaling. Patients with Crohn disease (CD) and ulcerative colitis harbored increased Engyodontium and decreased L. johnsonii abundance. Engyodontium directly induced calprotectin in human colonic neutrophils, and patients with CD had lower levels of l-glutamic acid, which also promoted human Treg expansion. These findings highlight the Engyodontium-calprotectin axis against the Lactobacillus-glutamate-Treg cascade to aggravate colitis, suggesting commensal Engyodontium-triggered signaling as a therapeutic target for mucosal inflammatory diseases.

Authors

Xinying Wang, Haiyang Sun, Ying Tan, Shaoting Xu, Zishan Liu, Kaile Ji, Ding Qiu, Jianping Deng, Bingbing Feng, Xueting Wu, Yoichiro Iwakura, Minhu Chen, Rui Feng, Chanyan Huang, Ce Tang

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Figure 7

The Engyodontium–DECTIN-2 axis in the pathogenesis of human IBD.

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The Engyodontium–DECTIN-2 axis in the pathogenesis of human IBD. (A) Phy...
(A) Phylum- and genus-level composition of human fecal commensal fungi isolated using Dectin-2–Fc and identified by qPCR with species-specific primers. (B) Relative contents of Engyodontium sp. in fecal fungi and L.j. in fecal bacteria from patients with CD and healthy individuals (HC), determined by qPCR (Engyodontium: HC, n = 59; CD, n = 62. Lacutobacillus: HC, n = 62; CD, n = 63). (C) Relative contents of Engyodontium sp. in fecal fungi and L.j. in fecal bacteria from patients with UC and healthy individuals, determined by qPCR (HC, n = 36; UC, n = 25). (D) Correlation between Ct value of L.j. and Engyodontium sp. from human individuals as described in B (n = 97 [including HC, n = 48 and CD, n = 49]). (E) Correlation between Ct value of L.j. and Engyodontium sp. from human individuals as described in C (n=61 [including HC, n=36 and UC, n=25]). (F) C57BL/6J SPF mice were orally colonized with Engyodontium sp. (Engyod.) every other day for 3 administrations. Thirteen days later, fecal samples were collected, and the relative abundance of fecal L.j. was quantified by qPCR (n = 10 /group). (G) Relative expression of CLEC6A, S100A8, and S100A9 in colon tissues from patients with CD and healthy control (HC) individuals, determined by bulk RNA-Seq analysis (HC, n = 47; CD, n = 57). (H) Correlation between CLEC6A and S100A8 or S100A9 expression based on bulk RNA-Seq data described in G. (I) Correlation between CLEC6A and S100A8 or S100A9 relative expression in colon tissues of patients with UC, determined by qPCR (n = 25). (J) IHC staining for DECTIN-2 and S100A8/S100A9 proteins in serial inflammatory colon sections obtained from a patient with CD after surgical resection. (K) IHC staining for human DECTIN-2 in paired normal and inflamed colon regions from the patient with CD shown in J. (L) Transcriptional levels of CLEC6A in colon tissues from non-IBD control individuals and patients with colonic CD (cCD), ileal CD (iCD), or UC, analyzed by reprocessing a public bulk RNA-Seq dataset (GSE117993) (not IBD, n = 55; cCD, n = 31; iCD, n = 60; UC, n = 43). (M) Colon tissues from 3 patients with IBD were obtained after resection. cLP CD11b+ cells were isolated and stimulated with α-mannan in vitro for 6 hours. S100A8 and S100A9 relative expression was quantified by qPCR (n = 7). (N) Neutrophils isolated from peripheral blood of 2 patients with IBD were stimulated with α-mannan in vitro for 6 hours, and S100A8 and S100A9 relative expression was measured by qPCR (n = 7 technical replicates from 2 biological replicates/group). (O) Correlation between fecal Engyodontium sp. abundance and colonic expression of CLEC6A, S100A8, or S100A9 in patients with UC (n = 25). (P) Correlation between fecal L.j. abundance and colonic expression of CLEC6A, S100A8, or S100A9 in patients with UC (n = 25). (Q) CD11b+ cells isolated from cLP of resected colon tissues from 2 patients with IBD were stimulated with Engyodontium sp. for 24 hours in vitro, followed by qPCR analysis of S100A8 and S100A9 expression (n = 6 technical replicates from 2 biological replicates/group). non-stimu., nonstimulated. (R) Engyodontium sp. was cultured at room temperature with recombinant S100A8 + S100A9 (1:1 mixture, 5 μg/mL each) for 12, 24, and 48 hours. Fungus growth was quantified by CFU enumeration on PDA plates (n = 3 replicates/group). (S) Relative amount of glutamic acid in fecal samples from patients with CD and healthy control individuals, assessed by liquid chromatography for untargeted metabolomics (HC, n = 80; CD, n = 65). (T) Correlation between 1/Ct value of L.j. and fecal glutamic acid levels in patients with CD (n = 8). (U) Correlation between 1/Ct value of L.j. and glutamic acid concentration in colon tissues from patients with UC (n = 25). (V) Peripheral blood leukocytes from 2 healthy donors were isolated and induced toward Treg differentiation in the presence of l-glutamic acid. After 5 days, the proportion of Foxp3+CD4+ among CD45+ leukocytes was analyzed by flow cytometry (n = 6 technical replicates from 2 biological replicates/group). Data in F, M, N, Q, and V are pooled from 2 independent experiments. Data in B, C, G, and L are presented as mean ± SEM, and in F, N, Q, R, S, and V as mean ± SD. Statistical analysis: 2-tailed Mann-Whitney test (B, C, and G), 2-tailed unpaired Student’s t test (F, N, Q–S, and V), 1-way ANOVA with Kruskal-Wallis and Dunn’s test (L), Spearman’s correlation test (D, E, H, I, O, P, T, and U), or paired Student’s t test (M).