Response to Fungal Dysbiosis by Gut-Resident CX3CR1+ Mononuclear Phagocytes Aggravates Allergic Airway Disease - PubMed (original) (raw)

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. 2018 Dec 12;24(6):847-856.e4.

doi: 10.1016/j.chom.2018.11.003. Epub 2018 Nov 29.

Irina Leonardi 1, Alexa Semon 1, Itai Doron 1, Iris H Gao 2, Gregory Garbès Putzel 3, Youngjun Kim 4, Hiroki Kabata 3, David Artis 5, William D Fiers 1, Amanda E Ramer-Tait 6, Iliyan D Iliev 7

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Response to Fungal Dysbiosis by Gut-Resident CX3CR1+ Mononuclear Phagocytes Aggravates Allergic Airway Disease

Xin Li et al. Cell Host Microbe. 2018.

Abstract

Sensing of the gut microbiota, including fungi, regulates mucosal immunity. Whether fungal sensing in the gut can influence immunity at other body sites is unknown. Here we show that fluconazole-induced gut fungal dysbiosis has persistent effects on allergic airway disease in a house dust mite challenge model. Mice with a defined community of bacteria, but lacking intestinal fungi were not susceptible to fluconazole-induced dysbiosis, while colonization with a fungal mixture recapitulated the detrimental effects. Gut-resident mononuclear phagocytes (MNPs) expressing the fractalkine receptor CX3CR1 were essential for the effect of gut fungal dysbiosis on peripheral immunity. Depletion of CX3CR1+ MNPs or selective inhibition of Syk signaling downstream of fungal sensing in these cells ameliorated lung allergy. These results indicate that disruption of intestinal fungal communities can have persistent effects on peripheral immunity and aggravate disease severity through fungal sensing by gut-resident CX3CR1+ MNPs.

Keywords: CX3CR1(+) mononuclear phagocytes; fungi; gut-lung axis; mycobiome; mycobiota dysbiosis.

Copyright © 2018 Elsevier Inc. All rights reserved.

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Figures

Figure 1.

Figure 1.. Fluconazole-induced gut fungal dysbiosis has persistent immune effects on lung allergic airway disease.

(A) Layout of the experiment setup, all mice from normal drinking water (Ctrl), fluconazole (Fluc) and post-fluconazole treated groups (Post-Fluc) were intranasally immunized with HDM. (B) Blood samples were collected for serum fluconazole assessment by liquid chromatography-mass spectrometry (LC-MS/MS). Ctrl (n=8), Fluc (n=8). (C-E) All mice (n=5 per group) were intranasally immunized with HDM. Representative eosinophil staining (pre-gated on CD45+CD11c− cells; left) and frequencies of Siglec-F+SSChi eosinophils in BAL (right). (D) Left to right: total cells, eosinophil, macrophage and neutrophil numbers in BAL. (E) Frequencies of CD4+ IL-4+ Th2 cells (left) and IL-17+ Th17 cells (right) in the medLN. (F-G) ELISA detection of total serum IgE and anti-HDM IgG1. OD value relative to control group. Ctrl (n=10), Fluc (n=5), Post-Fluc (n=10). (H-I) Feces were collected from mice in A) before (Ctrl, n=10), during (Fluc, n=10), and 3 weeks after removal (Post-Fluc, n=10) of fluconazole. (H) Quantitative real-time PCR (q-PCR) for fungal 18S rDNA in feces. (n=5 per group). (I) Alpha diversity (Observed OTU index) and (J) NMDS plot (Bray-Curtis) derived from ITS amplicon sequencing data. Dots represent individual mice, data are represented as mean ± SEM. Data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA.

Figure 2.

Figure 2.. Antifungal treatment does not exacerbate allergic airway disease in mycobiome-free (MyF) mice while oral supplementation with fungi is detrimental.

(A) Q-PCR for bacterial 16S rDNA in feces from mice in Figure 1H. (B) Alpha diversity (Observed OTU index) by 16S rDNA sequencing. (C) NMDS plot (Bray-Curtis) for bacterial OTUs in feces. (D) Relative bacterial abundance at the family level (n=5 per group). (E) Q-PCR for 16S rDNA (left) and 18S rDNA (right) in feces from germ-free (GF) mice (n=5), MyF-ASF mice (n=8) and WT SPF mice (n=6). (F) Species specific q-PCR for bacterial species of altered Schaedler flora in feces from MyF-ASF mice administrated normal water (n=4) or fluconazole for 3 weeks (n=5). (G) MyF-ASF mice were administrated water (Ctrl, n=4) or fluconazole water (Fluc, n=4) 7 days prior to HDM immunizations. H&E stained lung sections (20X), scale bar= 100 μM. (H-K) Mice were fed either with PBS (n=7) or a mixture of three fungal species (A. amstelodami, E. nigrum and W. sebi; n=7) for a week prior to exposure to HDM. (H) H&E stained lung histology (20X), scale bar= 100 μM. (I) Representative eosinophil staining (left), frequencies (right), (J) number of eosinophils and (K) total number of cells in BAL. Dots represent individual mice, data are represented as mean ± SEM. Data are representative of at least two or three independent experiments. *p < 0.05, **p < 0.01, Mann-Whitney test.

Figure 3.

Figure 3.. The systemic effects of gut fungal dysbiosis on lung allergy are mediated by CX3CR1+ MNPs.

(A) _Cd11c-Cre_−/− Cx3cr1 DTR littermates (Litt) (n=14) and Cd11c-Cre+/− Cx3cr1 DTR (ΔCX3CR1) (n=12) mice were treated with DT as described in Figure S4A. CD11b+CX3CR1+ MNPs were gated within live CD45+MHC+II+CD11c+ cells (left) and CX3CR1+MNP frequencies of in the colonic lamina propria (cLP) and lung are shown (right). (B-F) Litt and ΔCX3CR1 mice were administrated normal water (open bars, Litt (n=6), ΔCX3CR1 (n=6)) or fluconazole water (filled black bars, Litt (n=7), ΔCX3CR1 (n=6)) and immunized with HDM (Figure S4D). (B-C) Representative eosinophil staining (B, left), frequencies (B, right) and (C) total number of cells in BAL. (D) medLN total cell numbers. (E) Representative staining (left) and frequencies (right) of CD4+ GATA3+Th2 cells in the lung. (F) Frequencies of CD4+IL-4+ Th2 cells in medLN. Dots represent individual mice, data are represented as mean ± SEM. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, Mann-Whitney test.

Figure 4.

Figure 4.. Syk signaling in intestinal CX3CR1+ MNPs contributes to allergic airway inflammation during gut fungal dysbiosis.

(A-B) Mice were treated following experimental setup outlined in Figure 1A, Ctrl (n=6), Fluc (n=8). Frequencies of CD4+ T cells (A) and CX3CR1+MNPs (B) in the lung are shown. (C-D) Littermates (Litt, n=8) and Syk fl/fl Cx3cr1-Cre-ERT mice (ΔSyk, n=7) were treated (i.p) with 4-OHT as described in Figure S4F. (C) Representative Syk staining of CX3CR1+ MNPs and (D) mean fluorescence intensity (MFI) in intestinal cLP and lung. (E-I) Litt and ΔSyk mice were administrated normal water (open bars, Litt (n=5), ΔSyk (n=8)) or fluconazole water (filled black bars, Litt (n=8), ΔCX3CR (n=7)) and immunized with HDM. (E) Representative plots and frequencies of eosinophils in BAL, (F) Representative plots (left) and frequencies (right) of CD4+ GATA3+ Th2 cells in the lung. (G) H&E stained lung sections. (20X), scale bar= 100 μM. (H-I) Representative plots (H, left), frequencies (H, right) and total number of CD4+ GATA3+ Th2 cells (I) in the cLP. Dots represent individual mice, data are represented as mean ± SEM. Data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 Mann-Whitney test.

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