Aged Gut Microbiota Contributes to Systemical Inflammaging after Transfer to Germ-Free Mice

Abstract

Advanced age is associated with chronic low-grade inflammation, which is usually referred to as inflammaging. Elderly are also known to have an altered gut microbiota composition. However, whether inflammaging is a cause or consequence of an altered gut microbiota composition is not clear. In this study, gut microbiota from young or old conventional mice was transferred to young germ-free (GF) mice. Four weeks after gut microbiota transfer immune cell populations in spleen, Peyer's patches, and mesenteric lymph nodes from conventionalized GF mice were analyzed by flow cytometry. In addition, whole-genome gene expression in the ileum was analyzed by microarray. Gut microbiota composition of donor and recipient mice was analyzed with 16S rDNA sequencing. Here, we show by transferring aged microbiota to young GF mice that certain bacterial species within the aged microbiota promote inflammaging. This effect was associated with lower levels of Akkermansia and higher levels of TM7 bacteria and Proteobacteria in the aged microbiota after transfer. The aged microbiota promoted inflammation in the small intestine in the GF mice and enhanced leakage of inflammatory bacterial components into the circulation was observed. Moreover, the aged microbiota promoted increased T cell activation in the systemic compartment. In conclusion, these data indicate that the gut microbiota from old mice contributes to inflammaging after transfer to young GF mice.

Keywords: aging; germ-free mice; gut microbiome; immune system; inflammaging.

Figure 1

Old microbiota induces higher frequencies of T helper (Th) subsets in the spleen. Spleen, mesenteric lymph node, and Peyer’s patch (PP) CD4⁺ T cell populations were analyzed with flow cytometry after isolation from young or old conventional (conv) mice (n = 10), germ-free (GF) recipient mice of young or old microbiota (n = 10), and GF control mice (n = 5). (A) Percentage of splenic CD4⁺ T cells expressing GATA-3 (Th2). (B) Percentage among total live cells of splenic CD4⁺ T cells expressing CD25 and Foxp3 (T_reg). (C) Percentage among total live cells of splenic CD4⁺ T cells expressing T-bet (Th1). (D) Percentage among total live PP cells of CD4⁺ T cells expressing T-bet (Th1). All data are expressed as means. *P < 0.05, **P < 0.01.

Figure 2

Old microbiota upregulates several immune pathways in the ileum. Whole-genome gene expression in the ileum of mice (n = 5 per group) was assessed with Affymetrix GeneChip Mouse Gene 1.1 ST arrays. The most highly upregulated genes were analyzed with the STRING database. Only genes with at least one interaction are shown. The following interactions are indicated: from curated databases (blue), experimentally determined (pink), textmining (yellow), co-expression (black), and protein homology (purple). (A) Genes that were upregulated (P < 0.05, fold-change >1.3) in the ileum of old conventional mice compared with young conventional mice. (B) Genes that were upregulated (P < 0.05, fold-change >1.2) in the ileum of germ-free (GF) mice that received old microbiota compared with GF mice that received the young microbiota.

Figure 2

Old microbiota upregulates several immune pathways in the ileum. Whole-genome gene expression in the ileum of mice (n = 5 per group) was assessed with Affymetrix GeneChip Mouse Gene 1.1 ST arrays. The most highly upregulated genes were analyzed with the STRING database. Only genes with at least one interaction are shown. The following interactions are indicated: from curated databases (blue), experimentally determined (pink), textmining (yellow), co-expression (black), and protein homology (purple). (A) Genes that were upregulated (P < 0.05, fold-change >1.3) in the ileum of old conventional mice compared with young conventional mice. (B) Genes that were upregulated (P < 0.05, fold-change >1.2) in the ileum of germ-free (GF) mice that received old microbiota compared with GF mice that received the young microbiota.

Figure 3

Markers of inflammation are upregulated in the presence of old microbiota. Whole-genome gene expression in the ileum of mice (n = 5 per group) was assessed with Affymetrix GeneChip Mouse Gene 1.1 ST arrays. Genes that were significantly differentially expressed (P < 0.05 and fold-change >1.2) between young and old conventional mice, or germ-free (GF) recipients of young or old microbiota were analyzed with Ingenuity Pathway Analysis. (A) Canonical pathways that were most significantly affected by age or after transfer of aged microbiota. (B) Most significantly predicted upstream regulators by comparing young and old conventional mice, or GF recipients of young or old microbiota. (C) Venn diagram of differentially expressed genes between young and old conventional mice, and GF recipients of young or old microbiota.

Figure 4

Transfer of old microbiota enhances inflammatory bacterial components in serum. Human Embryonic Kidney 293 cells transfected with mouse toll-like receptor (TLR)2/CD14 (A) or mouse TLR4/MD-2/CD14 (B) were stimulated with 2.5% serum from young or old conventional (conv) mice (n = 10), germ-free (GF) recipient mice of young or old microbiota (n = 10), and GF control mice (n = 5). Activation of these receptors was measured with a secreted embryonic alkaline phosphatase reporter coupled to the NF-kB/AP-1 promoter. All data are expressed as means. **P < 0.01.

Figure 5

Gut microbiota composition in conventional and conventionalized mice. Fecal samples were collected from conventional (conv) mice (n = 10) at the time of transfer to the germ-free (GF) recipient mice, or from the GF recipient mice (n = 10) 1 and 4 weeks after the transfer. Gut microbiota composition was analyzed with 16S rDNA sequencing and data are presented as the relative abundance of the different bacterial groups for each individual mouse. The most highly abundant bacterial groups are indicated.

Figure 6

Transfer of aged microbiota to germ-free (GF) mice leads to altered gut microbiota composition. Fecal samples were collected from conventional (conv) mice (n = 10) at the time of transfer (inoculum) to the GF recipient mice, or from the GF recipient mice (n = 10) 1 and 4 weeks after the transfer. Gut microbiota composition was analyzed with 16S rDNA sequencing. (A) Redundancy analysis of gut microbiota composition of the different experimental groups at the genus level. (B) Bacterial phyla that were significantly different in abundance in young or old conventional mice, or in GF mice conventionalized with young or old microbiota after 1 or 4 weeks *P < 0.05, **P < 0.01, ***P < 0.01.