Ageing is an ill-defined process involving changes in various body systems, which converts a mature, fit person into an increasingly infirm one. With the passage of time, individuals show decreasing cell-protection mechanisms and detrimental physiological changes in metabolic processes and physiological functions of various tissues including the heart, brain, and skeletal muscles1. This leads to increased morbidity and mortality due to autoimmune diseases, cancer and infectious disease2,3, as well as a decline of mental health, well-being, and cognitive abilities4,5.

One of the most important effects of the ageing process is a significant decline of the efficacy of both the adaptive and innate immune systems, which has been described for several species6,7. Furthermore, one study on oral and parenteral vaccination in naturally ageing mice showed that age-associated decrease in antigen-specific immune responses occurs earlier in the mucosal immune system than in systemic immune system8.

Aging significantly increases the vulnerability to gastrointestinal (GI) disorders with approximately 40% of geriatric patients reporting at least one GI complaint during routine physical examination9. Despite the need to further understand age-associated factors that increase the susceptibility to GI dysfunction, there is a paucity of studies investigating the key factors in aging that affect the GI tract. To date, studies in rodents have demonstrated that aging alters intestinal smooth muscle contractility10, as well as the neural innervations of the GI tract musculature11. Several studies in rodents have also reported an increase in intestinal permeability to macromolecules with age12,13. Specifically, advancing age was shown to correlate with an enhanced transepithelial permeability of D-mannitol, indicating that there may be an age-associated decline in barrier function14. In humans, the decreased intestinal motility results in a slower intestinal transit that affects defecation and leads to constipation15. The elderly are at a higher risk for infections, especially severe infections, as well as for certain autoimmune diseases and cancer, and their immune responses to vaccination are diminished16. It is considered that aged humans exhibit a loss of naive T cells and a more restricted T cell repertoire17. Furthermore, aging results in decreased human CD8+ cytotoxic T lymphocyte responses, restricted B cell clonal diversity, failure to produce high-affinity Abs, and an increase in memory T cells18,19. It has been suggested that although certain dendritic cell (DC) populations are fully functional in ageing20,21, both foreign and self-antigens induce enhanced pro-inflammatory cytokines22. Very old individuals with a more balanced pro- and anti-inflammatory phenotype may be the most fortunate23,24. The association of inflammation in ageing has been termed ‘inflammageing’25.

Human microbiome analyses have revealed significant changes in the intestinal microflora specifically with an increase of Bacteroides ssp in the elderly (<65 years)26,27. However, other authors have concluded that the change in the microbiota was seen only in centenarians with increased inflammatory cytokine responses, but not in elderly with an average age 70 ± 3 years)28. In centenarians, the microbiota differs significantly from the adult-like pattern, by having a low diversity in terms of species composition. Bacteroidetes and Firmicutes still dominate the gut microbiota of extremely old people (representing over 93% of the total bacteria). However, in comparison to the younger adults, specific changes in the relative proportion of Firmicutes subgroups were observed, with a decrease in the contributing Clostridium cluster XIVa, an increase in Bacilli, and a rearrangement of the Clostridium cluster IV composition28. Moreover, the gut microbiota of centenarians is enriched in Proteobacteria, a group containing “pathobionts”, shown to cause harm in a compromised or susceptible host29,30.

For maintaining the mammalian intestinal homeostasis with the microbiota, a key element is to minimize and regulate contact between luminal microorganisms and the intestinal epithelial cell surface. In the small intestine, physical separation of bacteria and the epithelium is largely accomplished by secretion of mucus, antimicrobial proteins, and IgA into the lumen31,32. Intestinal mucus is primarily composed of the highly O-glycosylated mucin 2 (Muc2), which is secreted by goblet cells in the epithelium. In the mouse colon, less antimicrobial peptides are secreted, therefore, a thick stratified inner layer is needed to separate the commensal microbes from the epithelium33. Both mucus layers have essentially the same composition, suggesting the outer mucus layer arises from limited proteolytic cleavage and volumetric expansion of the inner layer. The density and stratified organization of the inner mucus layer is proposed to prevent penetration by bacteria thereby minimizing and regulating contact between bacteria and the epithelium33.

Muc2 is the major secreted intestinal mucin and its absence in Muc2−/− mice leads to colitis, which starts in the distal colon and spreads to the proximal colon34,35. Colitis is associated with increased microbiota diversity and an early colonization with pathobionts such as Bacteroides fragilis36. Moreover, it has been shown that even decreased Muc2 production, as observed in Muc2+/− mice perturbs intestinal homeostasis and microbiota composition. Decreased mucus production is also observed in other mouse models of colitis leading to increased epithelial contact with bacteria as observed in Muc2+/− mice37,38.

Several studies have shown age-associated effects on various components of the intestinal barrier and immune system (9). A recent study in accelerated Aging Ercc1−/Δ7 mice showed that a decline in the mucus barrier occurs by 16 -weeks of age39. Knowledge of the impact of ageing on the GI tract mucus layer of naturally aged mice is incomplete and limited to reports of altered gastric mucus layer40 and colonic mucus in 38-week old rats41. Moreover, none of the above-mentioned studies in naturally aged rodents have deeply investigated the genome-wide effects of ageing in the physiology of the small and large intestine using transcriptomics combined with other techniques such as histology and microbiota profiling. Such knowledge might provide new insights into the dynamics of the interplay between the host and microbiota in elderly and have implications for future interventions, for example by manipulation of the microbiota. To address this knowledge gap, we took advantage of 10-week- and 19-month-old litter-mate mice, which provides an opportunity to identify microbiota and host gene expression changes in association with ageing. Although mice have a lifespan of about 28 months, we hypothesise that 19-month-old mouse will develop significant changes related to age in their intestinal physiology, which might lead to altered microbiota-host interactions and altered intestinal physiology. Microbiota composition was determined and transcriptomics data was obtained from colonic tissue. Furthermore, (immuno)-histology and FISH techniques were performed on mouse colonic tissue to obtain temporal data on morphological changes, mucus production, and compartmentalization of bacteria in the lumen.