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 2

Engyodontium induces epithelial chemokine production to recruit neutrophils and exacerbates colitis.

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Engyodontium induces epithelial chemokine production to recruit neutrop...
(A and B) C57BL/6J mice were orally colonized with Engyodontium sp. every other day for 3 doses and simultaneously treated intraperitoneally with the CXCR1/2 inhibitor reparixin (15 mg/kg) for 3 doses. On day 8 after the initial colonization, colons were harvested. (A) Immunofluorescent staining of Ly6G, Dectin-2, and CXCL2 on serial colon tissue sections. (B) Colonic epithelial cells were isolated after EDTA treatment, and chemokine mRNA expression was quantified by qPCR (n = 7 technical replicates/group). (C) NCM460 epithelial cells were cocultured with Engyodontium sp. for 12 hours in the presence of inhibitors targeting ERK (GDC-0994), NF-κB (BAY11-7082), TLR2 (C29), TRAF6 (C25-140), or SYK (R406). CXCL1 and CXCL2 mRNA expression was measured by qPCR (n = 6 technical replicates/group). (D–F) WT and Clec4n–/– mice were analyzed under physiological conditions. (D and E) Frequencies (D) and absolute numbers (E) of cLP neutrophils (WT n = 10, Clec4n–/– n = 9) and Tregs (WT n = 7, Clec4n–/– n = 6) measured by flow cytometry. (F) Heatmap showing qPCR analysis of cytokine and antimicrobial gene expression in colon tissues (WT n = 4, Clec4n–/– n = 3). (G and H) Body weight change (G) and colitis DAI (H) after Engyodontium colonization. (I–L) Fluconazole-pretreated mice were colonized with Engyodontium sp. and treated with 1.5% DSS for 7 days starting from the third fungal administration. TRAF6 inhibitor C25-140 was administered intraperitoneally every other day for 4 doses. (I) Body weight loss, (J) DAI, (K) gross colon morphology and length, and (L) cLP neutrophil frequencies assessed by flow cytometry (PBS n = 5, Engyodontium n = 4; Engyodontium-C25-140 n = 5). Data in B–E (Treg panel) are pooled from 2 independent experiments. Data in D and E (neutrophil panel) are pooled from 3 independent experiments. Data in F–L are from 1 of 2 independent experiments. Data in B–E and G–L are presented as mean ± SD. Statistical analysis: 2-tailed unpaired Student’s t test (B, and D–H), 1-way ANOVA with Bonferroni’s multiple-comparison test (I and J), and 1-way ANOVA with Tukey’s multiple-comparison test (C, K, and L). Engyod., Engyodontium.