Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections

Abstract

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Keywords: IFNγ; IL-12; T cell differentiation; TCR sequencing; Th1; cDC1; commensal protist; cryptosporidiosis; cryptosporidium; dendritic cell; intestine; microbiome.

Figure 1.. cDC1-deficient mice develop fulminant cryptosporidiosis

(A) Complete blood cell counts, total protein, and glucose from peripheral blood of a spontaneously sick 7-week-old cDC1-deficient mouse (hLang^DTA Irf8 +32 5’^−/−) compared with a non-sick littermate (Irf8 +32 5’^−/−). (B) Spleen size of a spontaneously sick 6-week-old cDC1-deficient mouse (hLang^DTA Irf8 +32 5’^−/−) compared with a non-sick littermate. (C) Bone marrow and SI histology from mice in (A). Arrowheads indicate Cryptosporidium parasites in the SI epithelium. (D) Alignment and clustering of Ct-STL Gapdh sequence with Gapdh sequences from other Cryptosporidium species (left). The number of nucleotide differences in Gapdh between Cp-AUCP-1, Ct-UGA55, and Ct-STL are shown (right).

Figure 2.. Ct-STL behaves like a commensal in WT mice

(A) Quantification of Ct-STL oocysts in terminal pellets by flow cytometry (FC) using Crypto-a-Glo of a Cryptosporidium-free Charles River (CR) vs a WT mouse from our colony (STL Wild-Type). 10⁵ total events are displayed on the left; oocysts in the gate represent total oocysts in 10⁵ stool events. (B) Ct-STL parasites within the jejunum brush border (arrows) in STL WT mice by histology. (C) Ct-STL quantification by FC and qPCR of stool DNA in whole luminal colon contents from STL WT mice before and after weaning (14–21 days and 3–6 weeks old, respectively). FC and qPCR samples were from different mice. Dotted line in FC represents the limit of specific detection determined using Cryptosporidium-free mice (expt.=2). (D) Ct-STL quantification by FC and qPCR in the terminal pellets of 9–13 month old mice with JAX or CR microbiota versus similarly-aged STL WT mice. FC and qPCR samples were from identical mice (expt.=2). (E) FC quantification of Ct-STL in terminal pellets of WT CR mice colonized at 3.5 weeks of age with 250, 4×10³, or 2.5×10⁴ Ct-STL oocysts (expt.=3). (F) Normalized (left) and absolute (right) weights of CR mice colonized with 4×10³ Ct-STL oocysts at 3.5 weeks of age (expt.=2). (G) Fecundity of Ct-STL colonized mice. Littermate CR mice were gavaged with 4×10³ Ct-STL oocysts or PBS (control) at 3.5 weeks of age and set up as breeding pairs at 6 weeks of age. Number of litters per breeding pair (far left), pups born per litter (mid left), number of pups dead or alive at 21 days of life (mid right), and pup weights at 21 days (far right) were quantified over 6 months (expt.=5). (H) Fecal water weight of terminal pellets (left), transit velocity of gavaged dye (mid), and total gastrointestinal (GI) transit time (right) in Ct-STL colonized vs. control CR mice. WT CR mice were gavaged with 4×10³ Ct-STL oocysts or PBS (control) at 3.5 weeks old and mice were assessed after 4 weeks (expt.=2). Each dot represents data from an individual host. Geometric mean ± geometric S.D. (C–E) or mean ± S.E.M. (F right, G, H) are shown. Student’s t-test (C, D, F right, G, H), repeated measures ANOVA (E, F left), or Fisher’s exact test (G mid right) p-values are shown.

Figure 3.. Both innate and adaptive immunity are required to limit Ct-STL

(A-C) Ct-STL oocyst shedding in terminal pellets by FC (A), weight loss (B), and jejunum histology at 13 weeks (C, arrows indicate Ct-STL parasites) in littermate Rag1^+/− and Rag1^−/− mice after colonization with 4×10³ Ct-STL oocysts at 3 weeks of age (expt.=2). Weights were normalized to starting weights. (D) Quantification of Ct-STL oocysts by FC after depletion of innate immune cells with anti-NK1.1 and anti-Thy1.2 or isotype control treatment in Rag1^−/− mice colonized with Ct-STL at weaning (expt=2). (E) IL-12p40^YFP expression in SILP cDC (MHCII⁺ CD11c⁺ F4/80⁻) subsets 1 week after colonization with Ct-STL. DP = double positive (CD103⁺ CD11b⁺), SP = single positive (expt.=2). (F) Expression of IFNγ^YFP IL17A^GFP Foxp3^IRES-Thy1.1 in SILP CD4⁺ T cells 3 weeks after colonization with Ct-STL (expt.=3). Each dot represents data from an individual host. Geometric mean ± geometric S.D. (A, D) or mean ± S.E.M. (B, C, E, F) are shown. P-values from Student’s t-test (C, F), ANOVA with Sidak’s correction (E), repeated measures ANOVA (A, D), or repeated measures mixed effects analysis (B), are shown.

Figure 4.. Ct-STL induces an antigen-specific Th1 response

(A-C) CD4⁺ T cell TCR repertoire changes in anti-IFNγ-treated Ct-STL colonized mice. Fixed TCRβ Foxp3^GFP Tcra^+/− mice were gavaged with 2.5–5×10⁴ Ct-STL oocysts or PBS and treated weekly with anti-IFNγ to increase Ct-STL burden. TCRα chain sequencing was performed on CD4⁺ Teff (CD62L^lo CD44^hi Foxp3⁻) and Treg (Foxp3⁺) populations after 2–3 weeks (expt.=3). (A) Bray-Curtis NMDS analysis of TCRα sequences from the MLN (top) or SILP (bottom). (B) Heatmaps representing the mean frequencies of the top 10 Teff (top) or Treg (bottom) TCRs in the SILP in Ct-STL colonized mice. TCRs selected for functional studies are noted (RG1–3, LA3). RG1-like TCRs with TRAV differences are indicated by (RG1) (n=5 for each group). (C) TRAV and CDR3 amino acid sequences for Ct-STL specific TCRs are shown. (D) RG1 and RG2 TCR-transduced T cell hybridoma cells were cultured together with the indicated antigen presented by splenic Flt3L-induced CD11c⁺ cells. Anti-CD3 was used as a positive control. NFAT-GFP expression was analyzed after 40 hours (expt.=3). (E-H) In vivo analysis of RG1 TCR. 2×10⁴ (E-G) or 10⁵ (H) retroviral RG1-expressing TCli-αβ Foxp3^IRES-GFP T cells were transferred into Ct-STL colonized mice and analyzed 1 week later (expt.=2). (E) CTV-labeled RG1 T cell expansion in mice colonized with Ct-STL for 3 days. (F) RG1 T cell expression of CXCR3, CD25, and Foxp3 in Ct-STL colonized mice for 3 days. CXCR3 and CD25 on host polyclonal MLN CD4⁺ T cells are also shown. (G) CTV-labeled RG1 T cell expansion and expression of CXCR3 and Foxp3 in mice colonized with Ct-STL for 4–6 weeks. (H) RG1 T cell expression of Tbet and FoxP3 transcription factor staining in mice colonized with Ct-STL for 3 days. Each dot represents data from an individual host (A, E-H) or mean of 2 in vitro co-cultures (D). Mean ± S.E.M. is shown. Student’s t-test (E-H) or ANOVA with Dunnett’s correction (D) p-values are shown.

Figure 5.. Ct-STL colonization alters the small intestine TCR repertoire at homeostasis

(A-D) CD4⁺ T cell TCR repertoire analysis of the SILP and MLN. Fixed TCRβ IFNγ^YFP IL17^GFP Foxp3^IRES-Thy1.1 Tcra^+/− mice were colonized with Ct-STL at weaning for 3 weeks before sorting CD4⁺ CD62L^lo CD44^hi Th1 (Foxp3⁻ IFNγ^YFP+ IL17^GFP−), Teff-DN (Foxp3⁻ IFNγ^YFP− IL17^GFP−) and Treg (Foxp3⁺) populations (n=5–6, expt.=3 for SILP and MLN DN and Treg; n=4, expt.=2 for MLN Th1). (A) Bray-Curtis NMDS analysis of Teff TCRα sequences. Each dot indicates a T cell subset from an individual mouse. (B) Comparison of non-Cryptosporidium dependent TCR frequencies in control and Ct-STL colonized mice. Mean frequency of TCRs found in both control and Ct-STL data sets are plotted (see Methods). Each dot represents the mean frequency of an individual TCR that was found in at least 1 control mouse and at least one Ct-STL colonized mouse. (C) Identification of TCRs that distinguish between Ct-STL and control by machine learning. GBM was used to classify control vs Ct-STL using the data from Th1 and Teff-DN cells from the MLN, SILP, and colon (see Methods). Data shown are the average prediction values for each sample (left) and the average relative influence for each TCR. (D) Analysis of SILP top 10 Th1 and Treg TCRs by heatmap (left, average frequency) and frequency bar plot (right). RG3-like TCR with CDR3 amino acid difference is indicated by superscript. Each dot represents data from an individual host (A, D right) or individual TCR (B-C). Violin plot with median and quartiles (C left) or mean ± S.E.M (D right) are shown. Paired t-test (B) and Mann-Whitney U (C left) p-values are shown. N.D. = not detected.

Figure 6.. cDC1s are important for Ct-STL control and Th1 differentiation

(A-C) Littermate cDC subset-deficient hLang^DTA Irf8 +32 5’ mice were colonized at weaning with Ct-STL (expt.=4) and tracked for (A) oocyst shedding using flow cytometry, and (B) weight change. “X” symbols in (B, right) indicate mice that died between 7–12 weeks after Ct-STL colonization. (C) Histologic analysis of jejunums 13 weeks after Ct-STL colonization. (D-F) Analysis of polyclonal CD4⁺ T cells and Ct-STL reactive RG1-expressing T cells in the SILP of cDC subset-deficient mice (expt.=2). 10⁵ retrovirally-transduced RG1 T cells were transferred 3 days after Ct-STL colonization, and assessed after 1 week for transcription factor expression by intracellular staining. FACS plots are representative of high FoxP3 and RORγt mice. Each dot represents data from an individual host. Geometric mean ± geometric S.D. (A) or mean ± S.E.M. are shown. Repeated measures mixed effects analysis (A, B left) or ANOVA with Dunnett’s correction (B right, C-F) p-values are shown.

Figure 7.. Homeostatic Ct-STL induces colon Th1 responses and facilitates immunity to pathogens

(A-D) Analysis of mice 3 weeks after colonization with Ct-STL at weaning. (A) Relative quantification of Ct-STL Gapdh to mouse Trac DNA in different segments of intestinal epithelium in mice 3 weeks after Ct-STL colonization. Ct Gapdh fold change is normalized to the average of all samples. (expt.=2). (B-C) IFNγ expression in cLP polyclonal CD4⁺ T cells by (B) flow cytometry of IFNγ^YFP IL17^GFP Foxp3^Thy1.1 mice (expt.= 3) or (C) qPCR of sorted CD4⁺ T cells for Ifng and Tbx21 expression (expt.=2). (D) Cytokine analysis of colons by multiplex Luminex assay. PCA using RANTES, IL12p40, and Figure S7G cytokines is displayed (expt.=2). (E) Comparison of the top 10 colon Th1 TCRs vs SILP TCRs in Ct-STL colonized mice (see also Figure 5, expt.=5). TCRs tested are indicated (n=5 for Ct-STL cLP, n=3 for control cLP, n=5–6 for SILP). (F) In vitro reactivity of Ct-STL-colonized colon Th1 TCRs shown in (E) to Cp oocyst antigens as per Figure 4D (expt.=2). (G) Cumulative frequencies of known Ct-STL-reactive TCRs in the MLN, SILP, and cLP Th1 TCR repertoires at homeostasis. (H) 2×10⁵ retroviral RG1-expressing IFNγ^YFP TCli-β T cells were transferred into 3.5-week-old CR WT mice 3 days after Ct-STL colonization, and analyzed for CTV dilution and IFNγ^YFP expression after 2 weeks in the pLN (axillary, inguinal, and brachial), spleen, MLN, SILP, and cLP. (expt.=2). (I) C. rodentium infection of 2 strains of C3H from 2 different vendors 3 weeks after colonization at 3.5 weeks of age. Survival is based on euthanasia at 20% weight loss (n=10 per group, expt.=2 per C3H strain). C. rodentium CFUs in stool are shown (right, expt.=3). (J) Colon injury in DSS and anti-IL10R-treated, Ct-STL-colonized and control mice. 3.5 week old CR WT mice were colonized with Ct-STL as in (I) and then treated with 1 mg of anti-IL10R one time plus 1% DSS in drinking water for 7 days. Survival, weight loss, and colon length at end of experiment or euthanasia threshold (20% weight loss or morbidity) were assessed (n=16–19 per group, expt.=3). Each dot represents data from an individual host (A-D, G-H, I right, J right) or individual hybrid experiment well (F). Mean ± S.E.M. error bars are shown. Student’s t-test (B-C, H bottom, J right), Kaplan-Meier (I, J), one-way ANOVA with Dunnet’s (F) or Sidak’s (H) correction, Mann-Whitney test (I right), or repeated measures mixed effects analysis (J middle) p-values are shown. Multiple t-tests with FDR-correction (Benjamini, Krieger, and Yekutieli) q-values are shown in (D).