The intestinal commensal fungus Wallemia mellicola enhances asthma in mice through Dectin-2

Abstract

Overgrowth of the fungus Wallemia mellicola in the intestines of mice enhances the severity of asthma. Wallemia mellicola interacts with the immune system through Dectin-2 expressed on the surface of myeloid and intestinal epithelial cells. Using Dectin-2-deficient mice, we show that the interaction of W. mellicola with Dectin-2 is essential for the gut-lung pathways, enhancing the severity of asthma in mice with W. mellicola intestinal dysbiosis. These findings offer better insight into dysbiosis-associated inflammation and highlight the role pattern recognition receptors have in immune recognition of commensal fungi in the gut, leading to alterations in immune function in the lungs.

Figure 1.

(A) Wallemia mellicola spores submerged in modified Dixon's (mDixon) medium interacted with macrophages (RAW 264.7) and intestinal epithelial cells (Caco-2), resulting in the production of TNFα and IL-6, respectively. Controls (Ctl) represent RAW 264.7 cells cultured in Dulbecco's minimal essential medium (MEM), 10% fetal bovine serum (FBS), Antibiotic-Antimycotic (Gibco^TM), and Caco-2 cells cultured in Eagle's MEM and 20% FBS, respectively. (B) An ELISA-based assay using fusion proteins combining common myeloid C-type lectin receptors (CLRs) with human IgG Fc (hFc) was performed to investigate W. mellicola's recognition by macrophages and intestinal cells. The CLR Dectin-2, which recognizes high mannose structures, bound W. mellicola spores. This binding was visualized under fluorescent microscopy using an immunofluorescence assay of Dectin-2-hFc stained with FITC-conjugated anti-hFc antibodies. (C) The production of the inflammatory cytokines TNFα and IL-6 resulting from the interaction of W. mellicola spores with murine macrophages and human intestinal epithelial cells, respectively, was significantly attenuated by hindering Dectin-2 expression using Dectin-2 small interfering RNA (siDec-2). (D) CARD-9 is a downstream signaling protein of Dectin-2 and other CLRs; the stimulation of bone marrow-derived macrophages (BMDM) from CARD9-deficient (CARD9^−/−) mice resulted in significantly less TNFα secretion compared to BMDM from Wild-Type (WT) mice. Abbreviations: Ctl: control, siCtl: control siRNA, MR: mannose receptor, DC-SIGN: Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin, MCL: Macrophage C-type lectin.

Figure 2.

(A) Experiments were conducted using wild-type (WT) and Dectin-2-deficient (Dectin-2 ^−/−) C57BL/6 female mice treated with cefoperazone drinking water for 7 days to deplete intestinal bacteria. Wallemia mellicola (Wal) spores were administered to half the mice through gavage feeding three times weekly, while the other half received distilled water gavage. House dust mite (HDM) sensitization was performed once weekly for a total of three times. Mice were sacrificed at day 23. (B) The enhanced eosinophilic airway inflammation after HDM sensitization of mice with W. mellicola intestinal colonization is not observed in Dectin-2-deficient (Dectin-2 ^−/−) mice, indicating the C-type lectin receptor Dectin-2 may be necessary for the gut-lung cross talks aggravating asthma in mice with W. mellicola-induced intestinal dysbiosis. (C) All four groups of mice were phenotypically normal and displayed similar growth rates. Wallemia mellicola DNA was not detected above control baseline by real-time PCR in the lungs of mice with dysbiosis. (D) These experiments demonstrate that Dectin-2 is essential for Gut-Lung Axis interactions that enhance asthma severity in mice.