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Mycobiota-induced IgA antibodies regulate fungal commensalism in the gut and are dysregulated in Crohn’s disease

Abstract

Secretory immunoglobulin A (sIgA) plays an important role in gut barrier protection by shaping the resident microbiota community, restricting the growth of bacterial pathogens and enhancing host protective immunity via immunological exclusion. Here, we found that a portion of the microbiota-driven sIgA response is induced by and directed towards intestinal fungi. Analysis of the human gut mycobiota bound by sIgA revealed a preference for hyphae, a fungal morphotype associated with virulence. Candida albicans was a potent inducer of IgA class-switch recombination among plasma cells, via an interaction dependent on intestinal phagocytes and hyphal programming. Characterization of sIgA affinity and polyreactivity showed that hyphae-associated virulence factors were bound by these antibodies and that sIgA influenced C. albicans morphotypes in the murine gut. Furthermore, an increase in granular hyphal morphologies in patients with Crohn’s disease compared with healthy controls correlated with a decrease in antifungal sIgA antibody titre with affinity to two hyphae-associated virulence factors. Thus, in addition to its importance in gut bacterial regulation, sIgA targets the uniquely fungal phenomenon of hyphal formation. Our findings indicate that antifungal sIgA produced in the gut can play a role in regulating intestinal fungal commensalism by coating fungal morphotypes linked to virulence, thereby providing a protective mechanism that might be dysregulated in patients with Crohn’s disease.

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Fig. 1: Under homoeostatic conditions, the majority of the mouse and human gut mycobiota is coated by sIgA primarily induced by C. albicans.
Fig. 2: sIgA antibodies preferentially bind fungal hyphae and influence C. albicans morphotypes in the gut.
Fig. 3: C. albicans hyphal morphotypes induce potent sIgA responses.
Fig. 4: C. albicans sIgA responses are mediated through interaction with DC2 and CX3CR1+ MNPs.
Fig. 5: C. albicans-induced sIgA that targets hyphae-associated virulence factors is decreased in patients with CD.

Data availability

Source data are provided with this paper. The data that support the findings of this study are available from the corresponding author on request.

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Acknowledgements

We thank members of the Iliev laboratory for their critical reviews of the manuscript. We thank Ramnik Xavier for discussion and analysis that helped us with shaping the hypothesis. We thank all contributing members of the JRI IBD Live Cell Bank Consortium, and the Microbiome Core Laboratory of Weill Cornell Medicine. Support for human sample acquisition through the JRI IBD Live Cell Bank is provided by the Jill Roberts Institute, Jill Roberts Center for IBD, Cure for IBD, the Rosanne H. Silbermann Foundation and Weill Cornell Medicine Division of Pediatric Gastroenterology and Nutrition. J.P. and E.R. were funded by PGC2018-095047-B-I00 from MINECO and InGEMICS (B2017/BMD-3691) from CAM. Research in the Iliev laboratory is supported by US National Institutes of Health (R01AI163007, R01DK113136 and R01DK121977), the Leona M. and Harry B. Helmsley Charitable Trust, the Irma T. Hirschl Career Scientist Award, Crohn’s and Colitis Foundation, Pilot Project Funding from the Center for Advanced Digestive Care (CADC) and the Burrough Welcome Trust PATH Award.

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Contributions

I.D. and I.D.I. conceived and designed the experiments. I.D., M.M., D.G.S., X.V.L., I.L., T.K., W.D.F., W.-Y.L., E.R. and M.B.-D. performed the experiments. J.P., R.S.L. and P.C.W., generated key research materials and contributed to interpretation of the experiments. I.D. and I.D.I. generated figures and legends from analysed data. I.D.I acquired funding for the project. I.D. and I.D.I. wrote the manuscript.

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Correspondence to Iliyan D. Iliev.

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Peer review information Nature Microbiology thanks Kathy McCoy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identification of gut fungi from fecal material by flow cytometry and anti-C. albicans sIgA dynamics.

a, Microbes in fecal material from SPF WT WCM-CE mice were distinguished as a Sybrhi population that is absent in GF mouse feces. b, Fungi (SybrhiCFW+) were enriched from bacteria (SybrhiCFW) through size separation by 900g centrifugation and calcofluor white (CFW) staining of the resulting pellet. c, C. albicans cultured for 18 hours in hyphae-inductive media was stained with fecal supernatant from C. albicans-colonized GF mice (N = 6) collected at 0, 2-, 4, 8- and 14-days post colonization, followed by sIgA staining. Analysis of IgA binding representative of two independent experiments, one-way ANOVA, followed by Sidak’s test. d, Representative flow cytometry plots of frequency of B220+IgA+ among Live CD45+CD4 cells in the PP of germ-free (GF) mice orally gavaged with PBS (GF) or colonized for two weeks with C. albicans (+Ca). Data in (c) represents mean ± SEM.

Source data

Extended Data Fig. 2 CFW+Sybrhi FSChiSSChi C. albicans population in feces represents hyphal/ pseudohyphal fungal morphologies that are preferentially bound by sIgA.

a, CFW+Sybrhi fungal population from feces of SPF mice colonized with CAF2-RFP C. albicans was sorted into FSChiSSChi and FSCl°SSClo fractions. Constitutive expression of RFP in this strain allows for high visibility and resistance to signal quenching upon prolonged light exposure during flow cytometry and microscopy on the same material. b, Immunofluorescence microscopy of sorted material from (a). Composite images at 20X magnification of FSChiSSChi and FSCloSSClo shown in left and right panels, respectively. Scale bar represents 25μm. Data representative of two independent experiments. c, CFW+Sybrhi fungal population from feces of SPF mice colonized with CAF2-RFP was sorted into IgA+ and IgA populations. Gray histograms represent IgAisotype control staining used to distinguish sorted populations. d-e, Area (d) and perimeter length (e) of CAF2-RFP were compared between IgA+ and IgA sorted populations. Data represents two independent experiments, mean ± SEM. Two-sided Mann-Whitney test. N = 5.

Source data

Extended Data Fig. 3 Assessment of Ca-dREP C. albicans double reporter strain upon IgA staining and hyphae forming deficiency of efg1Δ/Δ cph1Δ/Δ C. albicans strain.

a-b, Immunofluorescence microscopy of Ca-dREP incubated with human fecal supernatant as a source of sIgA and stained with DAPI and anti-human IgAAPC (a) or an APC isotype control (b). Single channel staining of 2 samples shown. Left to right: DAPI, constitutive ENO1-GFP expression, hyphae-specific HWP1-RFP expression, and anti-human IgAAPC (a) or APC isotype control (b). Top rows in a and b correspond to composite images in Fig. 2d,e, representing three independent experiments. Scale bar represents 50μm. c, Hyphae-competent (WT), but not hyphae-deficient (yeast-locked; efg1Δ/Δ cph1Δ/Δ) strains of C. albicans forms hyphae upon hyphae-inducing stimuli in vitro. Scale bar represents 25μm.

Extended Data Fig. 4 Flow cytometry gating strategy in PPs, LP and in feces.

a-b, Cell gating startegy for assessment of IgA+ GC B cell in PPs (a) and IgA+ plasmablasts in lamina propria (b). c, gating strategy of C.albicans cells in feces pre- and post- C.albicans (C.a) colonization.

Extended Data Fig. 5 Graphical abstract for the model of antifungal IgA induction by and regulation of intestinal fungal commensalism.

(Credit: Created with BioRender.com).

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2. Supplementary Table 1. Reagent sources. Supplementary Table 2. Mucosal washings and serum metadata.

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Source data

Source Data Fig. 1

Flow cytometry and ELISA data.

Source Data Fig. 2

Flow cytometry, microscopy and microbial counts data.

Source Data Fig. 3

Flow cytometry, microbial counts and ELISA data.

Source Data Fig. 4

Flow cytometry data.

Source Data Fig. 5

Flow cytometry and ELISA data.

Source Data Extended Data Fig. 1

Flow cytometry data.

Source Data Extended Data Fig. 2

Flow cytometry and microscopy data.

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Doron, I., Mesko, M., Li, X.V. et al. Mycobiota-induced IgA antibodies regulate fungal commensalism in the gut and are dysregulated in Crohn’s disease. Nat Microbiol 6, 1493–1504 (2021). https://doi.org/10.1038/s41564-021-00983-z

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