Microbiota modulate immune cell populations and drive dynamic structural changes in gut- associated lymphoid tissue

Inbred mouse strains provide phenotypic homogeneity among individual mice. However, stochasticmorphogenetic events combined with epigenetic changes due to exposure to environmental factorsand ontogenic experience introduce variability to mice with virtually identical genotypes, decreasingthe reproducibility of experimental mouse models. We here used microscopic and cytometrictechniques to identify individual patterns in gut-associated lymphoid tissue (GALT) that are triggeredby microbiota exposure. By comparison of germ-free (GF), specific pathogen-free (SPF) and gnotobioticmice colonized by defined minimal mouse microbiota (oMM12) MHCII-EGFP knock-in mice wequantified antigen-presenting cells (APCs) in the lamina propria, cryptopatches (CP), isolated lymphoidfollicles (ILFs), Peyers patches (PPs) and specific sections of the mesenteric lymphoid complex. Wefound that GF mice showed a significantly larger outer intestinal surface area compared to SPF andoMM12-colonized mice, but this only partly compensated for their lower density of villi in the distalileum. GF mice also contained fewer APCs and plasma cells than oMM12 mice in the Iamina propria ofvilli and displayed a significantly smaller SILT count, area and volume. In both GF and oMM12 mice, PPfollicles were significantly smaller compared to SPF mice despite their similar counts. Furthermore, the caecal patch was dispersed into small units in GF mice but compact in SPF mice.Taken together, we here bring further evidence that microbiota regulate SILT differentiation, PPs sizeand morphology, MLNs cellular composition and caecal patch morphology, directly affecting not onlythe functional configuration of the immune system but also the differentiation of lymphoid structures.These findings may enable researchers to conduct microbiologically controlled experiments acrosslaboratories, thereby promoting reproducibility in animal studies.