Systemic anti-commensal response to fungi analyzed by flow cytometry is related to gut mycobiome ecology

Abstract

Background: Interest for the study of gut mycobiota in relation with human health and immune homeostasis has increased in the last years. From this perspective, new tools to study the immune/fungal interface are warranted. Systemic humoral immune responses could reflect the dynamic relationships between gut mycobiota and immunity. Using a novel flow cytometry technology (Fungi-Flow) to determine immunoglobulin (Ig) responses to fungi, we studied the relationships between gut mycobiota and systemic humoral anti-commensal immunity.

Results: The Fungi-Flow method allows a sensitive and specific measurement of systemic IgG responses against 17 commensal and environmental fungi from the two main divisions; Ascomycota and Basidiomycota. IgG responses exhibited a high inter-individual variability. Anti-commensal IgG responses were contrasted with the relative abundance, alpha-diversity, and intra-genus richness of fungal species in gut mycobiota of twenty healthy donors. Categorization of gut mycobiota composition revealed two differentiated fungal ecosystems. Significant difference of anti-Saccharomyces systemic IgG responses were observed in healthy donors stratified according to the fungal ecosystem colonizing their gut. A positive and significant correlation was observed between the variety of IgG responses against fungal commensals and intestinal alpha-diversity. At the level of intra-genus species richness, intense IgG responses were associated with a low intra-genus richness for known pathobionts, but not commensals.

Conclusions: Fungi-Flow allows an easy and reliable measure of personalized humoral responses against commensal fungi. Combining sequencing technology with our novel Fungi-Flow immunological method, we propose that there are at least two defined ecosystems in the human gut mycobiome associated with systemic humoral responses. Fungi-Flow opens new opportunities to improve our knowledge about the impact of mycobiota in humoral anti-commensal immunity and homeostasis. Video Abstract.

Keywords: Flow cytometry; Humoral immunity; ITS rRNA gene sequencing; Immunoglobulin G; Mycobiota; Systemic anti-commensal responses.

Fig. 1

Fungi-Flow discriminates between sporulated and budding fungal forms. a Overnight culture of sporulated forms in RPMI medium. b Acquisition by flow cytometry permits discrimination of two populations according to size (FSC) and granularity (SSC). c Calcofluor White staining of both fungal populations. d Fluorescence-Activated Cell Sorting of two C. albicans and A. fumigatus populations shows a yeast or spores enriched population, which is well differentiated from the budding form population (Light microscopy imaging × 400).

Fig. 2

Fungi-Flow optimal conditions and specificity. a, b Dose response relationship was evaluated by plotting median fluorescence intensity (MFI) obtained for IgG responses at different combinations of C. albicans and A. fumigatus and serum antibody concentrations. Serum was obtained from patients with a confirmed hepatosplenic candidosis (HSC) or aspergilloma (AG). c, d Specificity of antibodies in sera from HSC, AG patients and healthy donor (HD). Results correspond to the mean and standard deviation (n = 3). Statistical analysis was performed using Student’s t test (**p < 0.05, ***p < 0.005)

Fig. 3

Overview of the Fungi-Flow protocol. Step 1: Fungal sporulated forms are cultured overnight at 30 °C to obtain budding forms. They were stored at − 80 °C until use. Step 2: Antibody samples are diluted to normalized concentrations. Step 3: Fungi are incubated with serum. Step 4: After washing, fungi are incubated with secondary antibodies. Step 5: Data acquisition. Acquisition by flow cytometry permits discrimination of fungi according to size (FSC-A) and granularity (SSC-A). Step 6: Data analysis. Histograms show the median fluorescence intensity (MFI) for yeast forms of C. albicans after incubation with different dilutions of serum from a healthy donor followed by immunostaining with fluorescently-labeled anti-human IgG antibodies. This protocol can be applied to any culturable fungi. Relevant negative controls must be used to detect unspecific fluorescence. None = unstained fungal forms. NRA non-relevant antibody, IC isotype control

Fig. 4

Fungi-Flow method allows an easy and reliable measure of human anti-commensal IgG responses. a Systemic IgG responses to 15 fungal genera, measured using the sporulated forms, are displayed as median fluorescence intensity (MFI) values and categorized according to phylogenetic relatedness. b Systemic IgG responses to 4 fungal general, measured using the budding forms. c Ratio between MFI IgG responses measured against budding forms versus sporulated forms of Candida, Aspergillus, Acremonium, and Fusarium. Results represent the mean of two independent assays. y yeast, s spore, bf budding form

Fig. 5

Gut fungal ecosystems in healthy donors have an impact in systemic anti-commensal response. a Hieratical clustering of the 50 most abundant species in gut mycobiome of healthy donors. This analysis revealed that our healthy population could be shared in two clusters showing two well-differentiated ecosystems (Eco). b Comparison of relative abundances between both ecosystems. Differences were mainly driven by the more abundant fungi, Saccharomyces but also by less abundant fungi such as Cyberlindnera. c Comparison of anti-commensal IgG responses between both ecosystems. Statistical analysis was performed using Mann-Whitney test (**p < 0.05)

Fig. 6

Diversity of the systemic anti-commensal IgG response is associated with alpha diversity of gut mycobiome in healthy donors. a Analysis of gut mycobiome diversity and its association with the IgG response index observed healthy donors. IgG response index was calculated using the Shannon index formula applied to the MFI values obtained for Saccharomyces, Debaryomyces, Candida, Cyberlindnera, Malassezia, Mycosphaerella, Penicillium, Botrytis, Yarrowia, Cladosporium, Kluyveromyces, Cryptococcus, and Aspergillus. b, c Analysis of alpha-diversity from gut mycobiome and IgG response index in healthy donors harboring different ecosystems. Statistical analysis was performed using Mann-Whitney test (**p < 0.05)

Fig. 7

Relationship between systemic anti-commensal responses and intestinal species or strains richness. IgG response in donors harboring gut a pathobionts or b commensal fungi with less or more than one different amplicon sequence variant (ASV). Statistical analysis was performed using the Mann-Whitney Wilcoxon test (**p < 0.05). Ns non-significant difference