Extended Data Fig. 5: Transcriptional analysis of gastrointestinal tissues under conditions of avoidance versus non-avoidance in BALB/c mice.

a–d, Principal component analysis considering the 500 most variable genes in (a,c) stomach- and (b,d) small intestine tissue bulk RNA-Seq data of BALB/c Cpa3^+/+ and Cpa3^Cre/+ mice undergoing the experiment outlined in Extended Data Fig. 4a. The clustering of the samples per experimental condition attests reproducibility between mice. e,f, For experimental outline, see Extended Data Fig. 4a. Stomach (e) and small intestine (f) were analyzed by RNA sequencing on the indicated days. Based on log2Foldchange-ranked gene lists, gene set enrichment analysis revealed a strong contribution of immunological Gene Ontology (GO)-terms. We annotated these GO terms according to their description into 4 subgroups: “adaptive immunity”, “innate immunity”, “mucosal immunity” and “chemotaxis” (Supplementary Table 1). The underlying most impactful “core enrichment genes” (Methods) for each GO-term were categorized according to these immunological subgroups (Supplementary Table 2). Shown are absolute log2Foldchanges of core enrichment genes contained in significantly enriched immune-related GO-pathways for stomach (e) and small (f) intestine tissues of avoiding (two-bottle test) and non-avoiding (OVA gavage) mice. Genes were annotated manually as ‘innate immunity’, ‘adaptive immunity’, ‘mucosal immunity’, and ‘chemotaxis’. g,h, Log2Foldchanges of ‘Hallmark inflammatory response’¹⁴ genes in stomach (g) and small intestine (h) tissues of avoiding (two-bottle test) and non-avoiding (OVA gavage) wild-type mice. Boxplots (e–h) show median and quantiles. Statistical analysis was performed using two-sided Wilcoxon-rank-sum tests for (e–h). Numbers of mice in a–d were (a) Cpa3^+/+ stomach naive non-challenged (n = 2), immunized non-challenged (n = 3), immunized gavage (n = 8), immunized two-bottle test (n = 12); (b) Cpa3^+/+ small intestine naive non-challenged (n = 3), immunized non-challenged (n = 3), immunized gavage (n = 7), immunized two-bottle test (n = 7), (c) Cpa3^Cre/+ stomach naive non-challenged (n = 3), immunized non-challenged (n = 2), immunized gavage (n = 9), immunized two-bottle test (n = 9); (d) Cpa3^Cre/+ small intestine naive non-challenged (n = 3), immunized non-challenged (n = 3), immunized gavage (n = 9), immunized two-bottle test (n = 8). Numbers of mice in e–h were (e,g) Cpa3^+/+ stomach immunized non-avoidance (gavage) day 7 (n = 3), day 11 (n = 2), day 16 (n = 3), immunized avoidance (two-bottle test) day 7 (n = 4), day 11 (n = 4), day 16 (n = 4) (data pooled per group and compared to reference immunized non-challenged (n = 3)), (f,h) Cpa3^+/+ small intestine immunized non-avoidance (gavage) day 7 (n = 3), day 11 (n = 2), day 16 (n = 2), immunized avoidance (two-bottle test) day 7 (n = 3), day 11 (n = 2), day 16 (n = 2) (data pooled per group and compared to reference immunized non-challenged (n = 3)). For numbers of genes in e,f, (log2FoldChange <−1 or log2FoldChange >1.5 in at least one comparison), see Supplementary Table 1. For numbers of genes in g,h, see Supplementary Table 4. e–h, Box boundaries delineate the 1st and 3rd quartiles of the data, the center line represents the median, whiskers represent the furthest points within 1.5× the interquartile range.