The protozoan commensal Tritrichomonas musculis is a natural adjuvant for mucosal IgA

Abstract

Immunoglobulin (Ig) A supports mucosal immune homeostasis and host-microbiota interactions. While commensal bacteria are known for their ability to promote IgA, the role of non-bacterial commensal microbes in the induction of IgA remains elusive. Here, we demonstrate that permanent colonization with the protozoan commensal Tritrichomonas musculis (T.mu) promotes T cell-dependent, IgA class-switch recombination, and intestinal accumulation of IgA-secreting plasma cells (PC). T.mu colonization specifically drives the expansion of T follicular helper cells and a unique ICOS+ non-Tfh cell population, accompanied by an increase in germinal center B cells. Blockade of ICOS:ICOSL co-stimulation or MHCII-expression on B cells is central for the induction of IgA following colonization by T.mu, implicating a previously underappreciated mode of IgA induction following protozoan commensal colonization. Finally, T.mu further improves the induction of IgA-secreting PC specific to orally ingested antigens and their peripheral dissemination, identifying T.mu as a "natural adjuvant" for IgA. Collectively, these findings propose a protozoa-driven mode of IgA induction to support intestinal immune homeostasis.

Graphical abstract

Figure 1.

T.mu induces mucosal IgA. (A) Serum IgA levels in control and T.mu colonized mice. (B) Representative immunofluorescence images of colonic tissue sections from control (left) and T.mu colonized mice (right) samples. Sections were stained with anti-IgA antibodies. Adjacent bar graph shows the quantification of IgA⁺ cells. (C) IgA levels in fecal samples. (D) Gene expression of Pigr in whole colonic tissue. (E) Representative contour plots of colonic lamina propria leukocytes stained for IgA and CD138. Gates identify IgA⁺CD138⁺ PC. (F) Contour plots show FAS⁺GL-7⁺ GC B cells in the MLN of control or T.mu colonized mice. (G) Representative flow cytometry plots identifying PD-1⁺CXCR5⁺ Tfh cells in the MLN of control or T.mu colonized mice. Numbers adjacent to gates show percentages ± SEM in E–G. Bar graphs adjacent to E–G show absolute numbers of the indicated cell type ± SEM. (H) Representative flow cytometry plots identifying PD-1⁺CXCR5⁺ Tfh cells in the PP of control or T.mu colonized mice. Numbers adjacent to gates show percentages ± SEM and the adjacent bar graph indicates quantification across two independent experiments. (I) Bar graphs show percentages of small and large intestinal IgA⁺CD138⁺ PC in control and T.mu colonized mice. (J) Representative flow cytometry plots identifying PD-1⁺CXCR5⁺ Tfh cells in the MLN on control mice or animals colonized with T.mu for 1 year. Numbers adjacent to gates show percentages ± SEM and adjacent bar graph indicates quantification across two independent experiments. (K) Bar graphs indicate concentrations of IgG isotypes in the serum of control or T.mu colonized mice. Data from at least two to three independent experiments with two to four mice/group are shown. Mann–Whitney U test was performed. * = P ≤ 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, NS = not significant. If not stated otherwise, displayed values include ± SEM.

Figure S1.

Unaltered Tfh cell phenotype in the presence of T.mu. (A) Quantification of FOXP3-expressing CD4⁺ T cells in the colonic lamina propria and MLN of control and T.mu colonized mice. (B) Quantification of bioactive TGF-β in colonic tissue explants from control and T.mu colonized mice. (C) Gene expression analysis of Tnfsf13, Tnfsf13b, and Nos2 in total colonic tissue. (D) Nos2^+/− and Nos2^−/− mice were either left untreated or were colonized with T.mu. IgA⁺CD138⁺ PC were quantified in the colonic lamina propria. (E) Tnfa^+/− and Tnfa^−/− mice were either left untreated or were colonized with T.mu. IgA⁺CD138⁺ PC were quantified in the colonic lamina propria. (F) Representative staining for CD4 on lymphocytes in the blood of mice treated with anti-CD4-Ig. (G) Absolute numbers of T.mu from the caecum of C57BL/6 and Rag2^−/− mice 3 wk after oral gavage with 2 × 10⁶ FACS-sorted T.mu. (H) CD40L expression on MLN PD-1⁺CXCR5⁺ Tfh cells from control or T.mu colonized mice. (I and J) PD-1⁺CXCR5⁺ Tfh cells were sorted from the MLN of control or T.mu colonized mice. Gene expression analysis was performed for (I) transcription factors Gata3 and Tbx21 and (J) cytokines Il4, Il6, Il13, Il21, Il10. Data shown are representative of at least two to three independent experiments with three to four mice/group. Mann–Whitney U test was performed. * = P ≤ 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, NS = not significant. If not stated otherwise, displayed values include ± SEM.

Figure 2.

T.mu induces IgA in an ICOS and T cell-dependent fashion. (A) Serum IgA levels and colonic lamina propria IgA⁺CD138⁺ plasma cell numbers in control or T.mu-colonized Tcrb^+/− and Tcrb^−/− mice. (B) Serum IgA levels and colonic lamina propria IgA⁺CD138⁺ plasma cell numbers in control or T.mu colonized with and without anti-CD4-Ig treatment. (C) Percentages and absolute number of colonic lamina propria IgA⁺CD138⁺ PC in control or T.mu colonized Tcrb^+/− and Tcrb^−/− after injection of CD4⁺ T cells. (D) Contour plots show ICOS expression on non-Tfh cells in MLN from control mice or mice colonized with T.mu. (E) Adjacent bar graph shows quantification of absolute ICOS⁺ CD4⁺ non-Tfh cells. F) ICOS expression on PD-1⁺CXCR5⁺ Tfh cells (blue), CD4⁺ non-Tfh cells (red), and isotype control staining on T cells (black) from control or T.mu colonized mice. Adjacent bar graphs show quantification of ICOS mean fluorescent intensity on PD-1⁺CXCR5⁺ Tfh cells and CD4⁺ non-Tfh cells. (G) Adoptive transfer of purified ICOS⁺ PD-1⁻ CXCR5⁻ CD4⁺ non-Tfh cells into T.mu colonized Tcrb^−/− mice. Contour plots show representative staining for CD3 and CD4 in Tcrb^−/− mice injected with PBS (black) or purified ICOS⁺ PD-1⁻ CXCR5⁻ CD4⁺ non-Tfh cells (red). CD4⁺ T cells in the MLN of mice injected with ICOS⁺ PD-1⁻ CXCR5⁻ CD4⁺ non-Tfh cells were analyzed for the expression of PD-1 and CXCR5. Numbers within plots show percentages ± SEM. (H) ICOS expression on ICOS⁺ PD-1⁻ CXCR5⁻ CD4⁺ non-Tfh cells after adoptive transfer into T.mu colonized Tcrb^−/− mice. (I) Percentages of FAS⁺GL-7⁺ GC B cells in the MLN of T.mu colonized Tcrb^+/−, Tcrb^−/−, or Tcrb^−/− mice injected with ICOS⁻ PD-1⁻ CXCR5⁻ CD4⁺ T cells or ICOS⁺ PD-1⁻ CXCR5⁻ CD4⁺ T cells. (J) Quantification of serum IgA levels and colonic IgA⁺CD138⁺ PC in control mice, mice colonized with T.mu, and T.mu colonized mice injected with blocking anti-ICOSL-Ig. Data shown are from two to three independent experiments with three to four mice/group. Mann–Whitney U test was performed. * = P ≤ 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, NS = not significant. If not stated otherwise, displayed values include ± SEM.

Figure S2.

T.mu promotes ICOS⁺ CD4⁺ non-Tfh cells independent of the microbiota. (A) Concentrations of FITC-Dextran in the serum of control or T.mu colonized mice. (B) Total T.mu numbers in the caecum of T.mu colonized C57BL/6 mice, 2, 7, 14, 21, and 28 days after colonization. Staining of T.mu with anti-IgA was performed at the indicated time points. Red numbers adjacent to the curve indicate percentages of IgA-coated T.mu ± SEM. (C and D) Groups of germ-free mice were colonized with B. ovatus strains (A, H, N, I, C, and L). Three weeks after colonization ICOS⁺ CD4⁺ non-Tfh cells and PD-1⁺ CXCR5⁺ Tfh cells were quantified in the MLN. (E) Groups of germ-free mice were either left untreated, or were colonized with purified T.mu, purified T.mu, and a complete SPF microbiota, or a complete SPF microbiota only. ICOS⁺ CD4⁺ non-Tfh cells were quantified in the MLN and serum IgA levels were assessed 3 wk after colonization. (F) Frequencies of IL-17 producing Th cells and ICOS⁺ CD4⁺ non-Tfh cells were assessed in the MLN of control of T.mu-colonized mice. Data shown are representative of at least two to three independent experiments with three to four mice/group. If not stated otherwise, displayed values include ± SEM.

Figure 3.

T.mu shapes the anti-bacterial IgA reactome. (A) Fecal bacteria were isolated from control or T.mu colonized mice and stained with anti-IgA antibodies. Histograms show staining of anti-IgA antibodies. Adjacent bar graph shows the quantification of IgA-coated bacteria. (B) Western blot analysis of IgA reactivity on wild type C57BL/6 fecal bacterial protein lysates. Membranes were developed after incubation with serum from control mice or T.mu colonized mice. Numbers adjacent to membranes indicate molecular weight. (C) Serum from control mice or T.mu colonized mice was collected and used to stain fecal bacteria obtained from Rag2^−/− mice. Histogram shows coating of fecal bacteria with serum IgA. Adjacent bar graphs show quantification of IgA-coated bacteria and the serum staining intensity displayed as mean fluorescent intensity normalized to controls. (D) Fecal bacteria obtained from Rag2^−/− mice were stained with serum from Rag2^−/− mice (blue histogram). (E) PCoA of 16S rDNA gene sequencing data obtained from fecal samples collected from control and T.mu colonized littermates and Iga^−/− mice. (F) Pairwise distance comparison of microbial communities in littermate controls and Iga^−/− mice free of or colonized with T.mu. (G) Relative abundance of bacterial OTUs in littermate control and Iga^−/− mice free of or colonized with T.mu. colored by phylum as indicated. (H) PCoA of 16S rDNA gene sequencing data obtained from fecal samples or sorted IgA⁻ and IgA-coated bacteria, collected from control and T.mu colonized mice. (I) Bacterial richness of fecal and flow-sorted microbiota from control or T.mu colonized mice Bacterial evenness of fecal and flow-sorted microbiota from control or T.mu colonized mice. (J) Relative abundance of bacterial OTUs in fecal and flow sorted microbiota from control or T.mu colonized mice. Data shown is representative of at least one to three independent experiments with three to four mice/group. Mann–Whitney U test was performed. * = P ≤ 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, NS = not significant. If not stated otherwise, displayed values include ± SEM. Source data are available for this figure: SourceData F3.

Figure S3.

Purity of sorted IgA-coated bacteria. (A) Post sort analysis of IgA-coated bacteria purified from control or T.mu colonized mice. Values adjacent to gates indicate percentages.

Figure 4.

T.mu-driven IgA requires antigen-presentation by B cells. (A) Quantification of ICOS⁺ CD4⁺ non-Tfh cells in the MLN and serum IgA levels in control and T.mu colonized MHCII^−/− mice. (B) Quantification of MLN ICOS⁺ CD4⁺ non-Tfh cell and Tfh cell numbers in µMT^−/− control mice or µMT^−/− mice colonized with T.mu. (C) Analysis of bone marrow chimeric mice. Groups of mice received a 50:50 mixture of either MHCII^−/− µMT^−/− or MHCII^+/− µMT^+/− bone marrow cells after lethal irradiation and were colonized with T.mu after full bone marrow reconstitution. Absolute numbers of colonic IgA⁺CD138⁺ PC, MLN FAS⁺GL-7⁺ GC B cells, PD-1⁺CXCR5⁺ Tfh cells, and ICOS⁺ CD4⁺ non-Tfh cells were quantified. (D) Serum and fecal IgA levels in control or T.mu colonized MHCII^−/− µMT^−/− mice and T.mu-colonized MHCII^−/− µMT^+/− mice. Data shown are representative of at least three independent experiments with three to four mice/group. Mann–Whitney U test was performed. * = P ≤ 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, NS = not significant. If not stated otherwise, displayed values include ± SEM.

Figure 5.

T.mu promotes peripheral dissemination of IgA-secreting plasma cells primed against gut antigens. (A) Groups of control or T.mu colonized mice were treated with OVA-containing drinking water ad libitum for 3 wk. Total serum and fecal IgA levels were assessed. (B) OVA-specific IgA levels were determined in the serum or feces of OVA-treated mice in the presence or absence of T.mu. (C) MLN, spleens, and bone marrow were harvested from mice used in A, and numbers of OVA-specific anti-IgA–secreting cells (ASC) were determined. (D) Purified CD4⁺ OTII^Tg T cells were adoptively transferred into control or T.mu colonized Tcrb^−/− mice followed by treatment with OVA-containing drinking water. MLNs were analyzed for FAS⁺GL-7⁺ GC B cells, PD-1⁺CXCR5⁺ Tfh cells, and ICOS expression on non-Tfh cells. (E) MLN, spleens, and bone marrow were harvested from animals used in D, and numbers of OVA-specific anti-IgA secreting cells (ASC) determined. Data shown are representative of at least two to three independent experiments with three to four mice/group. Mann–Whitney U test was performed. * = P ≤ 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, NS = not significant. If not stated otherwise, displayed values include ± SEM.