Colorectal cancer (CRC) is one of the leading cancers worldwide, with prevalence and mortality increasing in younger people. Intestinal inflammation and sex are emerging as risk factors for this disease, with patients with inflammatory bowel disease (IBD) showing increased appearance of intestinal tumors and males showing a higher incidence compared to age-matched pre-menopausal women. Sex hormones could play a role in this difference, especially estrogens, due to the natural difference in the endogenous abundance. Estrogen signaling, mediated through nuclear estrogen receptors alpha (ERα), beta (ERβ), or transmembrane receptor GPER1, is shown to regulate intestinal homeostasis, such as cell proliferation, apoptosis, differentiation, and adherence, making it necessary for the normal functioning of the intestine. As part of its functions, it also showed involvement in regulating microbial composition, immune signaling, and even circadian rhythmicity. However, the effects of sex and estrogenic signaling, although identified, are yet to be fully understood.

Therefore, the overarching aim of this thesis was to characterize the effect of the loss of ERβ in the intestinal epithelium and its influence on gut microbiome, intestinal rhythmicity, and immune microenvironment in response to inflammation.

In paper I, we aimed to identify the effect of estrogens in low-grade inflammatory conditions. In there, we identified that male and female mice obtained distinct microbial responses to the high-fat diet-induced intestinal inflammation. We also found that estrogen was able to modestly influence microbiota, highlighting two species, Collinsella aerofaciens and Ruthenibacterium sp., which correlated with inflammation.

In paper II, we utilized the spatial proteomics platform COMET to visualize the main immune cell populations in the mouse intestine. This multiplexed method allowed us to spatially characterize the immune cell infiltration throughout the length of the colon, giving us a chance to identify possible cell-cell interactions. We found that upon inflammation, males presented greater changes in immune cell infiltration, especially B cells and anti-inflammatory-like macrophages in the distal region.

Building on these results, in paper III, we investigated whether ERβ influences the infiltration of the immune cells. We found that loss of ERβ affects macrophage infiltration, with especially strong effects in males, as well as antigen-presenting cells and natural killer cells showing sex-specific responses to colitis. Additionally, immune cell colocalization was analyzed, and we found that upon loss of ERβ male immune cell interaction pattern in colitis was affected much more and became more similar to females. Finally, by plasma cytokine assay and tissue RNA sequencing, we have shown that intestinal-specific ERβ knockout mice displayed an increased response to colitis.

In paper IV, we aimed to see whether estrogen signaling and sex affect the circadian rhythmicity in the colon. In here, we saw that intestinal inflammation disrupted the expression of core circadian genes in mice and in cell lines. This disruption was further affected by sex and, mildly, by loss of ERβ in mice. Additionally, we have validated the findings in the human IBD dataset, indicating that males had heightened responses to inflammation and a lack of ERβ. Notably, we were also able to demonstrate that ERβ participates in the regulation of circadian rhythmicity through modulation of BMAL1, a key circadian component.

Overall, this thesis demonstrates that sex and estrogen signaling, particularly through ERβ, are important modulators of intestinal inflammation, shaping the response at multiple biological levels. Taken together, these findings highlight the importance of considering sex as a variable in studies and point to ERβ as a potential target for modulation of intestinal inflammatory response, spanning microbiome composition, immune cell dynamics, and circadian regulation.