Polymorphic Immune Mechanisms Regulate Commensal Repertoire

Abstract

Environmental influences (infections and diet) strongly affect a host's microbiota. However, host genetics may influence commensal communities, as suggested by the greater similarity between the microbiomes of identical twins compared to non-identical twins. Variability of human genomes and microbiomes complicates the understanding of polymorphic mechanisms regulating the commensal communities. Whereas animal studies allow genetic modifications, they are sensitive to influences known as "cage" or "legacy" effects. Here, we analyze ex-germ-free mice of various genetic backgrounds, including immunodeficient and major histocompatibility complex (MHC) congenic strains, receiving identical input microbiota. The host's polymorphic mechanisms affect the gut microbiome, and both innate (anti-microbial peptides, complement, pentraxins, and enzymes affecting microbial survival) and adaptive (MHC-dependent and MHC-independent) pathways influence the microbiota. In our experiments, polymorphic mechanisms regulate only a limited number of microbial lineages (independently of their abundance). Our comparative analyses suggest that some microbes may benefit from the specific immune responses that they elicit.

Keywords: IgA-seq; MHC and microbiota; SFB; commensal repertoire and host genetics; defensins; microbiome; microbiota composition.

Figure 1.. Input Microbiome Defines the Output Microbiota Composition in Genetically Identical Gnotobiotic Mice

(A) Experimental scheme. Generation 0 (G0) mice were transferred with cecal contents from C57BL/6J mice (input microbiota). Generation 1 (G1) progeny were used to isolate cecal microbial DNA at 8 weeks of age. DNA was analyzed by targeted amplicon sequencing of 16S rRNA encoding genes. Experiments were repeated multiple times with variable input. (B) Principal-component analysis (PCA) of the microbiomes of ex-germ-free C57BL/6J female mice transferred with C57BL/6J microbiota in four independent experiments (left panel) and relative abundance of common taxonomic families (right panel) in the microbiomes of individual mice from the same experiments. Colored dots, individual mice. The numbers of animals from all of the cohorts used in experiments are shown in Table S1.

Figure 2.. Mouse Strains Shape Intestinal Microbiota Composition in a Genetic Background- and an Input-Dependent Manner

(A) Experimental scheme. Within the same cohort, GF mice of different genetic backgrounds were repopulated with cecal microbiota from the same input source. The cecal microbiota of their progeny was obtained at 8 weeks of age and analyzed by sequencing 16S rRNA gene amplicons. (B and C) Microbiomes of mice from three ex-GF mouse strains (C57BL/6J [red], C3H/HeN [blue], and BALB/cJ [orange]) repopulated with the microbiota from SPF C57BL/6 mice in two independent experiments (cohorts 1, B, and 2, C) were compared by PCA analysis using a relative abundance of bacterial genera. Individual mice are represented by rectangles (males) and circles (females). (D) Comparison of the microbiomes of ex-GF C57BL/6J males and females from cohort 1. Males are represented by red rectangles and females by black circles. (E–H) Comparison of the microbiomes of two ex-GF strains (C57BL/6J and BALB/cJ) colonized with the same microbiotas shown as PCA (E), or as heatmaps for lineages with statistically significant differences (p < 0.05, FDR-adjusted t test) in cohorts 1 (F), 3 (G), and (H). Non-specific genera identified at family, order, or class level are marked with an asterisk. Lineages that are commonly present in mice of the same genetic background in more than one experiment are in bold. (I) Relative operational taxonomic unit (OTU) abundance of the 3 lineages that were found enhanced in C57BL/6J mice compared to BALB/cJ mice in cohorts 1 and 3, but not in cohort 2 (see Figure S2). (J and K) Comparison of the microbiomes of ex-GF BALB/cJ and C3H/HeN mice repopulated with the same microbiota from a C57BL/6J donor (J) and heatmaps for statistically significant lineage differences (p < 0.05, FDR-adjusted t test) (K).

Figure 3.. MHC-Dependent and -Independent Adaptive Mechanisms Contribute Moderately to the Shaping of the Intestinal Microbiota

(A) Mutual positioning of the microbiomes of MHC congenic strains BALB/cJ (H-2^d) and BALB/c (H-2^j) compared to positions of the microbiomes of C57BL/6J and C3H/HeN mice in PCA space (see also Figure S3). (B and C) Pairwise comparisons of the microbiomes of mice from MHC congenic strains BALB/c versus BALB/c.H-2^j (B) and BALB/c versus BALB.B (C). (D) Microbiomes of ex-GF C57BL/6J mice compared to the microbiomes of a pair of MHC congenic strains (I/LnJ [H-2] and I/LnJ-H-2^k) transferred with the same microbiota. (E) Pairwise analysis of I/LnJ and I/LnJ-H-2^k microbiomes from the same experiment. (F) Statistically significant bacterial lineage differences between BALB.B (H-2^b) and MHC congenic BALB/c (H-2^d) mice (p < 0.05, FDR-adjusted t test) shown as a contiguous heatmap (left) and their similarity with lineages observed in C57BL/6J mice (H-2^b) (separate heatmap at right). Mice were from the same cohort 2. Genera significantly differentially abundant between BALB/cJ and C57BL/6J (p < 0.05, FDR-adjusted t test) are marked in bold. (G) Heatmap showing abundances of bacterial lineages significantly (p < 0.05, FDR-adjusted t test) different between MHC-sharing ex-GF C57BL/6J and BALB.B mice. (H) Bacterial lineages with significantly (p < 0.05, FDR-adjusted t test) different abundances in BALB/cJ and BALB/c.Rag1^−/−mice. Dehalobacterium (bold) is also present in (G). (I) qPCR comparison of the SFB loads in three BALB/c-based strains (BALB.B, BALB/cJ, and BALB/c.Rag1^−/−). Log2 (fold change) compared to B6 was calculated as described in Experimental Model and Subject Details. Horizontal lines indicate the means of values obtained for each individual data point ± SD. Significance for real-time PCR was calculated by ordinary one-way ANOVA. (J and K) Comparison of IgA⁺ and IgA^- bacteria found in ex-GF MHC congenic BALB/cJ (J) and BALB.B (K) mice transferred with the same input microbiota. Shared genera are marked in bold. Heatmaps show lineages with statistically significant differences (p < 0.05, FDR-adjusted t test). Error bars indicate SD.

Figure 4.. Polymorphic Gene Expression in the Intestinal Compartments of Ex-GF C57BL/6J and BALB/cJ Mice Colonized with the Same Microbiota (Cohort 2)

(A) PCA of RNA-seq data generated from different intestinal compartments from the two mouse strains. A total of 6 BALB/c and 7 C57BL/6 mice were used. (B) Heatmaps showing significant (p < 0.05, FDR-adjusted t test) differences in gene expression identified between the two strains using log2 fold change as a readout. Gene names in bold reflect statistical significance in a specific compartment. (C) Differences in the expression of defensins alpha between mice from the two strains. BLAST sequence similarity of BALB/c sequence and C57BL/6J reference gene sequence is indicated at right.