Fungal symbiont transmitted by free-living mice promotes type 2 immunity

Abstract

The gut mycobiota is crucial for intestinal homeostasis and immune function¹. Yet its variability and inconsistent fungal colonization of laboratory mice hinders the study of the evolutionary and immune processes that underpin commensalism^2,3. Here, we show that Kazachstania pintolopesii is a fungal commensal in wild urban and rural mice, with an exceptional ability to colonize the mouse gastrointestinal tract and dominate the gut mycobiome. Kazachstania pintolopesii colonization occurs in a bacteria-independent manner, results in enhanced colonization resistance to other fungi and is shielded from host immune surveillance, allowing commensal presence. Following changes in the mucosal environment, K. pintolopesii colonization triggers a type 2 immune response in mice and induces gastrointestinal eosinophilia. Mechanistically, we determined that K. pintolopesii activates type 2 immunity via the induction of epithelial IL-33 and downstream IL-33-ST2 signalling during mucus fluctuations. Kazachstania pintolopesii-induced type 2 immunity enhanced resistance to helminth infections or aggravated gastrointestinal allergy in a context-dependent manner. Our findings indicate that K. pintolopesii is a mouse commensal and serves as a valuable model organism for studying gut fungal commensalism and immunity in its native host. Its unnoticed presence in mouse facilities highlights the need to evaluate its influence on experimental outcomes and phenotypes.

Extended Data Fig. 1.. K. pintolopesii is absent from Jackson Laboratory mice and has commensal characteristics.

a, Related to Fig. 1a. Pie charts showing K. pintolopesii representation (%) in the whole mycobiome for mice fecal samples from the institutions as indicated (ITS sequencing at 0.5 cutoff). Each pie chart represents 5 pooled fecal samples per institution. No other Kazachstania species except K. pintolopesii were present in any of the mice. b, Upper panel: Fecal fungal burden assessed by counting colony-forming unit (CFU) for mice that naturally carried K. pintolopesii in NY Inst B. Fecal samples were collected from seven different cages in two animal rooms; Lower panel: The corresponding ITS seq results for the seven K. pintolopesii positive samples. c, ITS sequencing showing the relative abundance of fungal genera in mice purchased from the Jackson Laboratory before and 3 weeks after K. pintolopesii colonization (n = 6). d, Related to Fig. 1b. Percentage of Kazachstania species K. pintolopesii in the mycobiome of lab mice, wildling mice, wild mice in New York City and Los Angeles. e, f, Principal coordinate analysis (PCoA) plot (e, n=10) and K. pintolopesii CFU (f, n=5) for fecal samples collected from mice purchased from the Jackson lab at the indicated time points after K. pintolopesii W26G colonization. Wk0, pre-colonization; Wk, week(s) after colonization. g, Fungal quantification in lab mice fecal samples was performed by normalizing 18S qPCR amplification against total fecal DNA. Fecal samples were collected at the indicated time point after K. pintolopesii colonization (n=10). h, Related to Fig. 1f. K. pintolopesii CFU count for each time point under each temperature condition as indicated. i, In vitro growth curves for C. albicans at different temperatures as indicated. Each condition included technical triplicates. Left: C. albicans growth measured at OD 600 nm. Right: C. albicans growth measured in CFU/g. j, Hyphae formation or lack thereof for K. pintolopesii and C. albicans after overnight incubation under the indicated conditions. Representative bright field images are shown. SDB: Sabouraud Dextrose Broth. pH7: phosphate-buffered neutral SDB; low O2: microaerophilic hypoxia; GlcNac: 250 mM N-acetyl-glucosamine; Serum: SDB supplemented with 10% fetal bovine serum. Scale bar, 20 μm. k, Schematic for cyclophosphamide-induced gut-disseminated infection of K. pintolopesii or C. albicans. l, Fecal CFU burden for 529L in the presence (529L+K.p, n=5) or absence (529L, n=4) of K. pintolopesii W26G at time points as indicated. P value was calculated by two-way ANOVA with multiple comparisons test. m, Fecal CFU burden for K. pintolopesii after single colonization and co-colonization with different fungal strains as indicated. R.m, R. mucilaginosa, n=5; C.g, C. glabrata, n=3; P.k, P. kudriavzevii, n=3; IDB311, n=4; 529L, n=4. n, Flow cytometry plot for red fluorescent protein-labeled C. albicans (C.a-RFP) abundance from fecal samples collected on day 28 after colonization. C.a-RFP was gated from SYBR-green and calcofluor white (CFW) double positive events. o, Fungal burden for K. pintolopesii from the single- or the co-colonized group with C.a-RFP (n = 5). Fecal samples were collected at the indicated time points. Unless otherwise specified, all data were generated by using K. pintolopesii W26G strain and P values were calculated by two-sided t test. Data represent at least two independent experiments with similar results and were presented as mean ± SD.

Extended Data Fig. 2.. K. pintolopesii did not change microbial community structure.

a, Relative abundance of bacterial composition (based on 16s seq) in laboratory SPF mouse feces collected before (Wk0) and after K. pintolopesii colonization at each time point indicated (n = 10). Wk, week post colonization. b-d, Mouse bacterial communities remain stable after colonization with K. pintolopesii (n=10). (b) Bray-Curtis dissimilarity (Distance) within individual mice between pre- and post- colonization timepoints (red) and between post-colonization timepoints (blue). Week-to-week variation between pre- and post-colonization timepoints is no larger than the variation between post-colonization timepoints and (c) Bray-Curtis dissimilarity (Distance) between all pre-colonization samples (Neg. vs. Neg.), between all pre- and post-colonization samples (Neg. vs. Pos. and Pos. vs. Neg.), and between all post-colonization samples (Pos. vs. Pos.) for all samples. All box plots in panel b and c are in the style of Tukey. The lower and upper hinges correspond to the first and third quartiles. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR (inter-quartile range) from the hinge. The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are plotted individually. The horizontal line bisecting each box indicates the group median; (d) Three indices of beta diversity were used to measure dissimilarity between samples of bacterial microbiota: Bray-Curtis, Jaccard, and Unweighted Unifrac. Dissimilarity (Distance) was first assessed between Week 0 and each subsequent timepoint within each individual, and a mixed-effect linear regression model was fit to those values to determine if bacterial microbiota became more or less dissimilar from the pre-colonization timepoint as time passed. The shaded area represents a 95% confidence interval. One-sided Wilcoxon rank sum test was used for b, c and two-sided t test for d.

Extended Data Fig. 3.. K. pintolopesii tolerates a broad range of pH fluctuations, which does not require urease activity.

a, K. pintolopesii growth curves under different pH conditions as indicated. Culture mediums collected at 0 hour, 6 hours and 24 hours were tested for its turbidity to indicate the growth (n=6 independent samples). b, c, Mouse stomach pH change (b) and fecal K. pintolopesii burden (CFU counts) (c) before and after antacid cimetidine treatment. P value was calculated by two-sided t test. Control, n = 4; Cimetidine, n=5. d, Genome content of K. pintolopesii. Assembled contig sizes are depicted in the circular plot, with the frequency of heterozygous bases (in 1kb windows) depicted in the inner circle. e, Phylogeny inferred using FastTree of urease amidolyase (DUR12) orthologs in yeast species based on protein alignments. f, In vitro urease activity test for K. pintolopesii. Christensen’s Urea Agar plates and urea-free agar control plates were used to test urease activity. R. mucilaginosa served as a positive control. Pink color shows positive urease activity. g, Fecal K. pintolopesii burden at day 7 in mice treated or not with 50 mg/kg urease inhibitor fluorofamide (n=5). Fluorofamide was gavaged once a day for a whole week. P value was calculated with two-sided t test. Data were presented as mean ± SD. Experiments were repeated at least twice with similar results.

Extended Data Fig. 4.. K. pintolopesii did not trigger immune response at steady state.

a, Upper panel: Gating strategy for eosinophils (Siglec-F⁺CD11b⁺), dendritic cells (CD11c⁺MHCII⁺), macrophages (F4/80⁺MHCII⁺), and neutrophils (Ly6G⁺CD11b⁺) from live CD45⁺ Lin⁻ cells presented in Fig. 2g–j, 2m, 2n, 2p, 3j–m and 4j. Lin: CD3ε, CD5, CD19, NK1.1; Lower panel: gating strategy for CD4⁺GATA3⁺ and CD4⁺RORγt⁺ T cell presented in Fig. 2f, 2k, 2o, 3n and 4g. b-g, Immune cell populations in small intestine-draining lymph nodes (ssLN) of regular chow (RC)-fed mice colonized with and without K. pintolopesii colonization for two weeks (n = 5). b, The total number of CD45⁺ hematopoietic cells. c, Frequency of CD4⁺, CD8⁺ T cell and CD19⁺B220⁺ B cell in CD45⁺ cells. d, Frequency of the Foxp3⁺ population among CD4⁺ T cells. e, Frequency of T helper cell sub-populations indicated by corresponding transcription factor. f, Frequency of myeloid cell populations among CD45⁺ hematopoietic cells. g, Frequency of neutrophil (Ly6G⁺CD11b⁺) in CD45⁺ cells. h, Representative flow cytometry plots for eosinophils (Siglec-F⁺CD11b⁺) in CD45⁺Lin⁻ cells isolated from duodenum lamina propria of RC-fed mice with and without K. pintolopesii colonization. i, Quantification of eosinophil frequency among CD45⁺ cells in duodenal lamina propria. n=5. j, Circulating IgA level from mice colonized (RC/K.p) or not (RC) with K. pintolopesii. n=6. k, K. pintolopesii burden (CFU counts) in feces collected from WT mice (n = 8), Rag1^−/− (n = 3) and Card9^−/− (n = 3) mice on the indicated days after K. pintolopesii colonization. l, K. pintolopesii burden (CFU counts) in feces collected from WT mice (n=4), Rag2 gamma chain^−/− mice (n=5), TSLP^−/− mice (n=4) and IgA^−/− mice (n=5) on the indicated days after K. pintolopesii colonization. Unless otherwise specified, all data were generated by using K. pintolopesii W26G and presented as mean ± SD and two-sided t test was used for the statistical analysis. All experiments were repeated at least twice with similar results.

Extended Data Fig. 5.. K. pintolopesii induces immune response under FFD condition without changing microbiome structure before and after colonization.

a, Relative abundance of bacterial genera (16s seq) in feces of mice under RC or FFD condition. Fecal samples were collected before (week 0) and after K. pintolopesii colonization at the indicated time points. Compositional weekly sampling (week 0,1,2,3) for each individual mouse under RC (RC1, RC2, RC3, RC4, RC5) or FFD (FFD1, FFD2, FFD3, FFD4, FFD5) is depicted. n=5. b, PCoA plot showing distinct bacterial composition clustering based on dietary conditions (RC or FFD). c, Colonization with K. pintolopesii does not induce greater changes in bacterial microbiota than the observed in normal week-to-week variation. Bray-Curtis dissimilarities (“Distance”) of bacterial microbiota between pre- and post-colonization timepoints (Negative vs. Positive) and among post-colonization timepoints (Positive vs. Positive) within each individual mouse. The microbiota changes within individuals from pre- to post-colonization timepoints are no larger than the changes among different post-colonization timepoints, according to the one-sided Wilcoxon rank sum test (RC: p = 1.0, FFD: p = 0.54). d, Fungal CFU counts in mucosal scraping samples collected from stomach (St mucus), duodenum (Du mucus) and colon (Co mucus) of mice under the indicated conditions (n = 5). e, K. pintolopesii CFU counts in feces collected from WT mice (n = 5), Rag1^−/− (n = 3) and Card9^−/− (n = 5) mice under FFD on the indicated days after K. pintolopesii colonization. f-j, Frequency of dendritic cells and macrophages (f); neutrophils (g); CD4⁺, CD8⁺ T cell and CD19⁺B220⁺ B cell (h) within CD45⁺ cells or Foxp3⁺ population within CD4⁺ T cells (i) and eosinophil cell number (j) in ssLN of FFD-fed mice colonized with (FFD/ K.p, n=5) or without (FFD, n=5) K. pintolopesii, assessed by flow cytometry. Unless otherwise specified, all data were generated by using K. pintolopesii W26G strain and P value was calculated by two-sided t test. Data were presented as mean ± SD. All experiments were repeated at least twice with similar results.

Extended Data Fig. 6.. K. pintolopesii colonization induced epithelial derived IL-33 expression and related immune response.

a, Heatmap for top 50 genes significantly upregulated or downregulated by K. pintolopesii colonization in FFD fed mice (n=5). b, IL-33 gene expression for duodenum collected from FFD fed, K. pintolopesii colonized or non-colonized mice (n=5). c, Related to Fig. 3f. Long exposure showing IL-33 activation in K. pintolopesii W26G colonized mice. d, Western blots and quantification for IL-33 expression in stomach tissue collected from regular chow-fed mice colonized with and without K. pintolopesii (n=5 for each group) as indicated. Full length IL-33 (35kD) and fragments below 25kD were all used for the quantification. e, Western blots and quantification for IL-33 expression in stomach tissue collected from mice (n=3 for each group) treated as indicated. P values were calculated by one way ANOVA with multiple comparisons. f, Related to Fig. 3h. Quantification for mucosa IL-33 relative intensity against actin (n=5 biologically independent samples). g, Related to Fig. 3i. TSNE plot showing IL-33 expression in different cell populations based on the density. h, Total IL-33 expression level in different cell populations as indicated by multiplying density with total cell numbers. i, Eosinophil frequencies in ssLN from mice as indicated. Each dot represents a mouse. IL-33^litt, n=3; IL-33^litt/K.p, n=9; IL-33^ΔIEC, n=3; IL-33^ΔIEC /K.p, n=6. One-way ANOVA with multiple comparisons were used to calculate the p values. ns, not significant. Lin: CD3, CD5, CD19, NK1.1. j, K. pintolopesii abundance in fecal samples collected from mice with IL-33 deficiency in GI epithelium (n=5 mice, IL33^ΔIEC) and their littermate controls (n=5 mice, IL33^litt) under RC and FFD respectively. Unless otherwise specified, all data were generated by using K. pintolopesii W26G strain and P value was calculated by two-sided t test. All experiments were repeated at least twice with similar results. Data were presented as mean ± SD. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 7.. K. pintolopesii induced type 2 immunity through IL-33/ST2 signaling pathway, potentially via its secreted proteases.

a, Schematic for ST2 neutralization experiment in mice colonized with K. pintolopesii. b, Frequency of eosinophils in Lin⁻ CD45⁺ hematopoietic cells (n=5 mice for each group). Statistical analysis was performed by two-sided t test. Lin: CD3, CD5, CD19, NK1.1. c, Frequency of GATA3⁺ T cell in CD4⁺ T cell (n=5 mice). d, Mouse fecal K. pintolopesii abundance in mice treated with anti-ST2 and its IgG isotype control under RC and FFD condition (n=5 mice for each group). e-h, Mice were treated with anti-ST2 antibody (n=4 mice) and its IgG isotype control (n=5 mice) two weeks after K. pintolopesii colonization under regular chow condition. (e) CD11b⁺ Siglec-F⁺ cell frequency in Lin⁻ CD45⁺ cells and (f) its cell counts; (g) Gata3⁺ cell frequency in total CD4⁺ T cells and its cell counts (h) from mouse small intestine draining lymph nodes. i, Secretome analysis for K. pintolopesii secreted proteases in the culture supernatant (n=3 biologically independent samples). Left panel: Pie chart for proteases detected in K. pintolopesii culture supernatant by liquid chromatography–mass spectrometry (LC–MS); Right panel: Relative abundance for representative proteases detected. j, Heatmap showing the inhibition effects (% inhibition) of different protease inhibitors on K. pintolopesii protease activity as indicated. Three representative experiments are shown. k, Western blotting analysis for in vitro cleavage of recombinant IL-33 by K. pintolopesii-secreted proteases in the presence or absence of different protease inhibitors. l, In vitro cleavage of IL-33-GST tagged recombinant protein by K. pintolopesii culture supernatant with and without 10μM serine protease inhibitor AEBSF. m, In vitro cleavage assay by incubating recombinant IL-33 protein with K. pintolopesii culture supernatant and different serine protease inhibitors. Western blots with both short exposure (exp.) and long exposure were shown for IL-33 cleavage fragment. Unless otherwise specified, all data were generated by using K. pintolopesii W26G strain and repeated at least twice with similar results. P value was calculated by two-sided t test. Data were presented as mean ± SD. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 8.. K. pintolopesii induces type 2 immunity in BALB/c mice and protects from helminth infection in C57BL/6 mice.

a, Frequency of CD11b⁺ Siglec-F⁺ eosinophils within Lin⁻ CD45⁺ cells in BALB/c mice colonized with and without K. pintolopesii under FFD condition. Two-sided t test was used for statistical analysis. N=5 mice per group. Lin: CD3, CD5, CD19, NK1.1. b, Frequency of Gata3⁺ T cell within CD4⁺ T cell in mice treated as in panel a. c, Western blots and quantification of IL-33 expression level in mouse stomach tissue treated as indicated. Full length IL-33 (35kD) and fragments below 25kD were all used for the quantification (n=5 mice per group). d, Western blots and the quantification of IL-33 expression in mouse stomach tissue colonized or not with K. pintolopesii under RC condition (n=5 mice per group). e, Western blots and its quantification of mouse stomach IL-33 expression between RC-fed and FFD-fed mice that were all colonized with K. pintolopesii (n=5 mice per group). f, Schematic for N. brasiliensis infection, generated by BioRender. Mice were fed on FFD diet as indicated. g, Percentage for CD11b⁺ Siglec-F⁺ cells among CD45⁺ total cells from ssLN of mice infected or not with N. brasiliensis and colonized or not with K. pintolopesii (Related to Fig. 4j, k. Ctrl, n=3, N.b, n=5 and N.b+K.p, n=5). h, PAS staining for goblet cells in naïve control and K. pintolopesii colonized mice (n=5 per group). Mice were fed on FFD. Scale bar, 50μm. i, K. pintolopesii fecal CFU count of mice infected with H. polygyrus (Hpb+PBS, n=11; Hpb+Kp, n=17, Related to Fig. 4n, o). All experiments were repeated at least twice with similar results. Unless otherwise specified, all data were generated by using K. pintolopesii W26G strain and P value was calculated by using two-sided t test. Data were presented as mean ± SD. For gel source data, see Supplementary Figure 1.

Extended Data Fig. 9.. Bioinformatics analysis of Kazachstania species in human.

Raw ITS sequencing data from five published studies was retrieved from the NCBI SRA database and reprocessed, yielding 708 samples from different individuals. The per-sample relative abundances of all Kazachstania species-level taxa were extracted. Each column of cells in the figure corresponds to one sample and each row corresponds to one Kazachstania species-level taxon. Cells are filled in where the corresponding taxon is present at least 0.001 relative abundance and are otherwise left uncolored. Colored bands at the top of the figure indicate which study was the source of the samples.

Figure 1.. K. pintolopesii is present across mouse colonies and stably colonizes laboratory mice.

a, ITS mycobiota sequencing and Kazachstania 18S rRNA qPCR (black circle, pooled from 5 samples) was performed on fecal samples from different institutions. CA, California; NJ, New Jersey; NY, New York; QC, Quebec. D.L, detection limit. b, c, ITS seq results for lab mice (n=10), wildling (n=10) and wild mice from New York City (NYC, n = 6) and Los Angeles (LA, n = 5) (b) and its 18S qPCR results (c). d, Phylogenetic tree of K. pintolopesii strains (KPIN-4417 as reference) and fecal CFUs after two weeks of colonization in mice (n=3). e, W26G fecal CFUs after colonization of adults (n=10, or less for constipated mice) or their progeny (F1, n=10). f, Growth curves for K. pintolopesii at various temperatures (n=3 replicates). g, Kaplan-Meier survival analysis for cyclophosphamide-induced systemic dissemination in mice by K. pintolopesii (n=7, K.p), C. albicans (n=5, C.a), and non-colonized control (n=3). h-j, Fecal CFUs for the indicated fungal species with or without K. pintolopesii colonization (h, n=5; i, n=3; j, n=4). k, Mouse fecal C.a-RFP abundance among total fungi (CFW⁺) for each group (n=5). l, m, PCoA plot of bacteria community structure (l), Bray-Curtis dissimilarities calculated by one-sided Wilcoxon rank sum test (m, left) and Weighted Unifrac Dissimilarity (“Distance”) analysis (m, right, shaded area represents 95% confidence interval) for bacterial composition changes among the indicated groups (n=10). n, K. pintolopesii longitudinal fecal CFU analysis in GF mice (n = 3) or in SPF mice treated with and without (Ctrl) indicated antibiotics (n = 4). Data represent at least two independent experiments with similar results. Two-sided t test was used for c, h, i, k, m (right), One-way ANOVA for d, j and Two-way ANOVA for n with multiple comparisons, log-rank test for g. Data are presented as mean ± SD.

Figure 2.. K. pintolopesii induces type 2 immune responses under mucosal perturbation.

a, Relative K. pintolopesii abundance in mucosa samples as indicated (n=4). Data are presented as mean ± SEM, One-Way ANOVA with multiple comparisons. b, Representative fluorescence in-situ hybridization (FISH) images of K. pintolopesii in stomach, duodenum and colon of colonized GF mice. c, FISH images for stomach mucus layer (left) and its thickness quantification (right) of K. pintolopesii colonized RC and FFD treated mice (n=9). d, Fecal K. pintolopesii CFU from mice fed on RC and FFD (n=10) two weeks after colonization. e, f, The total number of CD45⁺ cells (e) and quantification of CD4⁺ T cell subsets frequency (f) in stomach and small intestine-draining lymph nodes (ssLN) from mice treated as indicated (n=5). g-i, Representative flow plots for duodenal lamina propria eosinophils (Siglec-F⁺CD11b⁺) (g), cell frequency (h) and counts (i) in each treated group (n=5). j, k, Total ssLN eosinophils (j) and Gata3⁺CD4⁺ T cell counts (k) from mice colonized with different K. pintolopesii strains (n=5). l, Representative FISH images and thickness quantification of stomach mucus layer from SPF and GF mice colonized with W26G. m, Representative flow cytometry plots of eosinophil frequences in duodenal lamina propria of GF mice colonized or not with K. pintolopesii. n, o, Total eosinophils (n) and GATA3⁺ CD4⁺ T cell counts (o) in ssLN from indicated treatment groups. GF, n = 5; GF/K.p, n = 7. p, Total eosinophil cell counts in ssLN of mice pretreated with antibiotics (Abx) and colonized or not with K. pintolopesii (n=7). Unless otherwise specified, all data are presented as mean ± SD. P values were calculated by two-sided t test. Data represent at least two independent experiments with similar results. b, c, l, scale bar, 50 μm.

Figure 3.. Fungal activation of epithelial cell-derived IL-33 triggers type 2 immunity.

a-d, Transcriptomic analysis of gastric tissues (a), heatmap of upregulated type 2 immunity-related genes (b), GSEA pathway analysis (c) and qPCR gene expression assessment (d) of mice colonized (or not, Ctrl) with K. pintolopesii for two weeks (n = 5). e, f, Experimental schematic (e) and IL-33 analysis and relative intensity quantification (f) in bulk stomach tissues upon colonization with K. pintolopesii W26G (K.p W26G, n=5) and Ctrl (n=4) (left); K. pintolopesii ID1LA, QC1, C89-2 and Ctrl (n=3, right); bar graphs show corresponding IL-33 quantification. g, h, Experimental schematic (g) and IL-33 analysis (h) in the stomach mucosa of mice colonized or not (Ctrl) with W26G (n=5). i, T-distributed stochastic neighbor embedding (TSNE) map for IL-33 expression level in cell populations of the small intestine. j, k, Representative flow plots (j) and eosinophil numbers (k) in ssLN of mice colonized (IL-33^ΔIEC /K.p, n=6 and IL-33^litt/K.p, n=9) or not (IL-33^ΔIEC, n=3 and IL-33^litt, n=3) with W26G for two weeks. l-n, Representative flow plots (l), eosinophil numbers (m) and Th2 cells (n) in ssLN of mice colonized with W26G and treated with anti-ST2 (n=5) or IgG isotype control (n=5). All experiments were performed on FFD and repeated at least twice with similar results. All data are presented as mean ± SD. DESeq2 Wald test, adjusted for multiple comparisons was used for a. Two-sided t test was used for panel d, f (left), m, n and one-way ANOVA with multiple comparisons for f (right), k. For gel source data, see Supplementary Figure 1.

Figure 4.. K. pintolopesii aggravates food allergy and protects from helminth infection.

a, Schematic of ovalbumin (OVA)-induced food allergy model. b-i, Kaplan-Meier survival analysis (b), disease onset time after the first OVA challenge (c), rectal temperature for surviving mice 60 minutes after the last OVA challenge or sham gavage (d), total serum IgE (e) and OVA-specific IgE titers (f) before OVA challenge, representative flow cytometry plot (g) and quantification (h) of CD4⁺GATA3⁺Foxp3⁻ cells in ssLN at endpoint and serum MCPT-1 concentration (i) for ctrl (n = 4, Ctrl) and OVA-challenged mice colonized (OVA/K.p) or not (OVA) with K. pintolopesii (c, OVA, n = 14; OVA/K.p, n = 18; d, OVA, n = 14; OVA/K.p, n = 14; e-f, OVA, n = 15; OVA/K.p, n = 19; g-h, OVA, n = 14; OVA/K.p, n = 10; i, OVA, n = 13; OVA/K.p, n = 13). j-l, Representative flow plot for CD11b⁺Siglec-F⁺ population in ssLN (j), cell number quantification (k), representative periodic acid-Schiff (PAS) staining images on duodenal tissue and goblet cell quantification (l) for control (n=3, Ctrl), N brasiliensis infected group with (n=5, N.b/K.p) or without (n=5, N.b) K. pintolopesii colonization. Scale bar, 50μm. m, N. brasiliensis worm counts for each group as indicated (n=6). n, o, Hpb worm counts of the entire small intestine (n) and fecal egg counts (o) after 28 days infection with Hpb with or without K. pintolopesii colonization (Hpb, n=11; Hpb/K.p, n=16). All experiments were repeated at least twice with similar results. All data are presented as mean ± SD. Log-rank test was used for b. Two-sided t test was used for c, f, m, n and o. One-way ANOVA with multiple comparisons were used for d, e, h, i, k, I. ROUT analysis (Q=1%) was used for outlier exclusion.