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
View: Text | PDF
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

×

Figure 1

Engyodontium sp. colonization modifies intestinal immune profiles and commensal microbiota in steady-state mice.

Options: View larger image (or click on image) Download as PowerPoint
Engyodontium sp. colonization modifies intestinal immune profiles and c...
(A) Flow cytometric analysis of Dectin-2-Fc binding to mouse fecal commensal microorganisms. (B) Phylum- and genus-level composition of mouse fecal commensal fungi isolated using Dectin-2-Fc and identified by ITS1–2 sequencing. (C) Representative images of purified Engyodontium sp. colonies cultured on antibiotic-containing medium (kanamycin, gentamicin, colistin, metronidazole, vancomycin, and erythromycin) at room temperature for 4 days. Left, top view of culture plate; right, bottom view of culture plate. (D) Light microscopy images of Engyodontium sp. cultured on PDA plates for 48 hours and stained with lactophenol cotton blue. (E) C57BL/6J SPF mice were treated with fluconazole for 1 week and orally administered Engyodontium sp. every other day for 3 doses. Mice were euthanized 7 days after the final administration, and fungal colonization was assessed by FISH using probes specific for fungal 28S rDNA on colon tissue sections. (F) Scanning electron microscopy (SEM) images of hyphae and conidia of Engyodontium sp. cultured on PDA plates for 2 days. (G and H) SPF mice were pretreated with combined antibiotics and/or fluconazole for 1 week, followed by Engyodontium colonization every other day for 3 doses. Colons were harvested 13 days (G) or 8 days (H) after the initial colonization and analyzed by SEM to visualize intestinal microorganisms. (I–P) SPF mice were colonized with Engyodontium sp. every other day for 3 doses and euthanized 13 days later. (I) Representative images of the cecum and colon. (J) Representative flow cytometry plots showing frequencies of Ly6G+CD11b+ neutrophils and Foxp3+CD25+ Treg cells in cLP. (K) Absolute numbers of cLP neutrophils and Treg cells quantified by flow cytometry (I–K: PBS n = 13, Engyodontium n = 12). (L) Heatmap depicting mRNA expression of immune-related genes in colon tissues by qPCR. (M–P) Fecal bacterial microbiota analyzed by 16S rDNA-Seq, shown as stacked bar plots at the phylum level (M), relative abundance of indicated bacterial phyla (N), Bacteroidetes/Firmicutes ratio (O), and relative abundance of indicated bacterial orders (P) (L–P: n = 5/group). Data in I–K are pooled from 3 independent experiments. Data in L and M are from 1 of 2 independent experiments. Data in I–K and N–P are presented as mean ± SD. Statistical analysis: 2-tailed unpaired Student’s t test (I–P). Engyod., Engyodontium; ABX, antibiotics; FCZ, Fluconazole.