Chronic inflammation contributes to the development of colorectal cancer, partly through its regulation of the microenvironment and antitumor immunity. Interestingly, women have a lower incidence of colorectal cancer, and estrogen treatment has been shown to reduce the occurrence of colorectal tumors. While intestinal estrogen receptor beta (ERβ, Esr2) can protect against colitis and colitis-induced cancer in mice, its role in shaping the tumor microenvironment remains unknown. In this study, we performed RNA sequencing to analyze the transcriptome of colonic epithelia and tumors from azoxymethane/dextran sulfate sodium-treated wild-type and intestinal ERβ knockout (ERβKOVil) mice and vehicle-treated controls. This revealed significant differences in gene expression and enriched biological processes influenced by sex and genotype, with immune-related responses being overrepresented. Deconvolution supported differential immune cell abundance and immunostaining showed that tumors from ERβKOVil mice displayed significantly increased macrophage infiltration, decreased T cell infiltration, and impaired natural killer cell infiltration. Further, ERβ mRNA levels in clinical colorectal tumors correlated with immune signaling profiles and better survival. Our findings indicate that intestinal ERβ promotes an antitumor microenvironment and could potentially affect the effectiveness of immunotherapy. These insights highlight the importance of ERβ in modulating antitumor immunity and underscore its therapeutic potential in colorectal cancer.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

Inflammatory intestinal conditions are a major disease burden. Numerous factors shape the distribution of immune cells in the colon, but a spatial characterization of the homeostatic and inflamed colonic immune microenvironment is lacking. Here, we use the COMET platform for multiplex immunofluorescence to profile the infiltration of nine immune cell populations in mice of both sexes (N = 16) with full spatial context, including in regions of squamous metaplasia. Unsupervised clustering, neighborhood analysis, and manual quantification along the proximal-distal axis characterized the colonic immune landscape, quantified cell-cell interactions, and revealed sex differences. The distal colon was the most affected region during colitis, which was pronounced in males, who exhibited a sex-dependent increase of B cells and reduction of M2-like macrophages. Regions of squamous metaplasia exhibited strong infiltration of numerous immune cell populations, especially in males. Females exhibited more helper T cells and neutrophils at homeostasis and increased M2-like macrophage infiltration in the mid-colon upon colitis. Sex differences were corroborated by plasma cytokine profiles. Our results provide a foundation for future studies of inflammatory intestinal conditions. (Figure presented.)

Granulosa cell tumors (GCTs) are rare ovarian tumors comprising an adult and a juvenile subtype. They have a generally good prognosis, but the survival rate drastically declines in patients with late-stage or recurring tumors. Due to the rarity of GCTs, the tumor type is largely understudied and lacks a specific treatment strategy. Estrogen receptor beta (ERβ/ESR2) has been found to be highly expressed in GCTs, which could be of therapeutic importance since it can be targeted with small molecules. However, its role in GCTs is not known. In this review, we summarize the current knowledge about the action of ERβ in the ovary and discuss its prospective role in GCTs.

A high-fat diet can lead to gut microbiota dysbiosis, chronic intestinal inflammation, and metabolic syndrome. Notably, resulting phenotypes, such as glucose and insulin levels, colonic crypt cell proliferation, and macrophage infiltration, exhibit sex differences, and females are less affected. This is, in part, attributed to sex hormones. To investigate if there are sex differences in the microbiota and if estrogenic ligands can attenuate high-fat diet-induced dysbiosis, we used whole-genome shotgun sequencing to characterize the impact of diet, sex, and estrogenic ligands on the microbial composition of the cecal content of mice. We here report clear host sex differences along with remarkably sex-dependent responses to high-fat diet. Females, specifically, exhibited increased abundance of Blautia hansenii, and its levels correlated negatively with insulin levels in both sexes. Estrogen treatment had a modest impact on the microbiota diversity but altered a few important species in males. This included Collinsella aerofaciens F, which we show correlated with colonic macrophage infiltration. In conclusion, male and female mice exhibit clear differences in their cecal microbial composition and in how diet and estrogens impact the composition. Further, specific microbial strains are significantly correlated with metabolic parameters.

Wastewater-based epidemiology (WBE) can be used to track the spread of SARS-CoV-2 in a population. This study pre-sents the learning outcomes from over two-year long monitoring of SARS-CoV-2 in Stockholm, Sweden. The three main wastewater treatment plants in Stockholm, with a total of six inlets, were monitored from April 2020 until June 2022 (in total 600 samples). This spans five major SARS-CoV-2 waves, where WBE data provided early warning signals for each wave. Further, the measured SARS-CoV-2 content in the wastewater correlated significantly with the level of positive COVID-19 tests (r = 0.86; p << 0.0001) measured by widespread testing of the population. Moreover, as a proof-of-concept, six SARS-CoV-2 variants of concern were monitored using hpPCR assay, demonstrating that var-iants can be traced through wastewater monitoring.During this long-term surveillance, two sampling protocols, two RNA concentration/extraction methods, two calcula-tion approaches, and normalization to the RNA virus Pepper mild mottle virus (PMMoV) were evaluated. In addition, a study of storage conditions was performed, demonstrating that the decay of viral RNA was significantly reduced upon the addition of glycerol to the wastewater before storage at -80 degrees C. Our results provide valuable information that can facilitate the incorporation of WBE as a prediction tool for possible future outbreaks of SARS-CoV-2 and preparations for future pandemics.

Background: Estrogen receptor beta (ERβ, Esr2) plays a pivotal role in folliculogenesis and ovulation, yet its exact mechanism of action is mainly uncharacterized.

Results: We here performed ERβ ChIP-sequencing of mouse ovaries followed by complementary RNA-sequencing of wild-type and ERβ knockout ovaries. By integrating the ERβ cistrome and transcriptome, we identified its direct target genes and enriched biological functions in the ovary. This demonstrated its strong impact on genes regulating organism development, cell migration, lipid metabolism, response to hypoxia, and response to estrogen. Cell-type deconvolution analysis of the bulk RNA-seq data revealed a decrease in luteal cells and an increased proportion of theca cells and a specific type of cumulus cells upon ERβ loss. Moreover, we identified a significant overlap with the gene regulatory network of liver receptor homolog 1 (LRH-1, Nr5a2) and showed that ERβ and LRH-1 extensively bound to the same chromatin locations in granulosa cells. Using ChIP-reChIP, we corroborated simultaneous ERβ and LRH-1 co-binding at the ERβ-repressed gene Greb1 but not at the ERβ-upregulated genes Cyp11a1 and Fkbp5. Transactivation assay experimentation further showed that ERβ and LRH-1 can inhibit their respective transcriptional activity at classical response elements.

Conclusions: By characterizing the genome-wide endogenous ERβ chromatin binding, gene regulations, and extensive crosstalk between ERβ and LRH-1, along with experimental corroborations, our data offer genome-wide mechanistic underpinnings of ovarian physiology and fertility.

Estrogen regulates a variety of important physiological functions in both males and females, where the regulation of female reproduction and the development of sexual organs are typical examples. The effects of estrogen are predominantly exerted via signaling through the two nuclear receptors estrogen receptor α (ERα) and β (ERβ), or the membrane G protein-coupled estrogen receptor 1 (GPER1). While estrogen signaling is important for human health, dysregulation of signaling can have adverse effects and impact the development and progression of a wide range of diseases including reproductive disorders and cancer.

ERβ has been shown to be highly important for ovarian function by regulating folliculogenesis and ovulation but has also been implied to protect against the development of colorectal cancer (CRC) by mediating the effects of estrogen. Despite the known role of ERβ, there is a lack of mechanistic understanding regarding how ERβ acts under both normal conditions and during disease. The overall aim of this thesis was to characterize the function and molecular mechanism of endogenous ERβ and to decipher its role in the normal ovary as well as its impact on colitis and CRC development. To further understand the role of estrogen signaling in the colon, we also aimed to identify sex differences during CRC development. 

In paper I we characterized the full cistrome of endogenous ovarian ERβ in the mouse and explored its transcriptional impact. We confirmed a direct role for ERβ in the regulation of essential ovarian functions and identified a novel crosstalk with the nuclear receptor LRH-1.  

In paper II we induced colitis-associated CRC (CAC) in intestinal epithelial-specific ERβ knockout mice and identified a protective effect by intestinal ERβ against tumor development in both male and female mice. We further characterized sex-dependent effects and proposed an underlying mechanism involving the regulation of TNFα/NFκB signaling. 

In paper III we expanded the investigation of sex-dependent changes during chemically-induced colitis in wildtype mice and identified a sex-specific response related to inflammatory response. We further found that male mice have an enhanced response to induced colitis.

In paper IV the transcriptome of colitis-induced tumors and their immune cell infiltration was explored in wildtype and intestinal epithelial-specific ERβ knockout mice of both sexes. This showed that sex differences in the transcriptome appear to be dependent on the expression of ERβ. Also, the identified ERβ-dependent changes in the tumor transcriptome of female mice were specifically related to immune response. We corroborated an impact of ERβ on the infiltration of immune cells, especially a reduction of regulatory T cell and NK cell recruitment. 

In summary, this thesis provides new mechanistic understanding of the transcriptional role of ERβ in the normal ovary and in the colon microenvironment. This includes the discovery of crosstalk with LRH-1 in the ovary and NFκB in the colon. Our characterization provides a foundation to develop targeted therapies for improved fertility and chemoprevention in CRC. This thesis also highlights the importance of including both sexes in colitis and CRC research to advance our knowledge and improve treatment development.

Östrogen reglerar en mängd viktiga fysiologiska funktioner hos både män och kvinnor, där regleringen av kvinnlig reproduktion och utveckling av genitalier är typexempel. Effekterna av östrogen sker till övervägande del via signalering genom de två nukleära receptorerna östrogenreceptor α (ERα) och β (ERβ), eller den membranbundna receptorn G-proteinkopplad östrogenreceptor 1 (GPER1). Även om östrogensignaleringen är viktig för människors hälsa, kan en dysreglering ha negativa effekter och påverka utvecklingen och progressionen av ett brett spektrum av sjukdomar, inklusive reproduktionsstörningar och cancer.

ERβ har visat sig vara mycket viktig för äggstockarnas funktion via reglering av follikulogenes och ägglossning, men har även kopplats till en skyddande effekt mot utvecklingen av kolorektalcancer (CRC). Trots den kända rollen för ERβ, finns det en brist på mekanistisk förståelse för hur ERβ verkar under både normala förhållanden och vid sjukdom. Det övergripande syftet med denna avhandling var att karakterisera funktionen och den molekylära mekanismen för endogent ERβ och att förstå dess roll i den normala äggstocken samt dess inverkan på kolit och CRC-utveckling. För att ytterligare förstå rollen av östrogensignalering i tjocktarmen, syftade vi även till att identifiera könsskillnader under CRC-utveckling.

I artikel I kartlade vi alla regulatoriska DNA-sekvenser som binds av endogent ERβ i äggstock och  utforskade ERβs effekt på transkription. Vi bekräftade att ERβ har en direkt roll i regleringen av essentiella funktioner i äggstocken och identifierade en ny interaktion med den nukleära receptorn LRH-1.

I artikel II inducerade vi kolit-associerad CRC (CAC) i tarmepitelspecifika ERβ knockoutmöss och identifierade en skyddande effekt av ERb i tarmen mot tumörutveckling i både hanar och honor. Vi karakteriserade även könsberoende effekter och proponerade en underliggande mekanism involverande reglering av TNFα/NFκB-signalering.

I artikel III expanderade vi vår studie av könsberoende förändringar under kemiskt inducerad kolit hos vildtypsmöss och identifierade ett könsspecifikt svar relaterat till inflammation. Vi fann vidare att responsen på inducerad kolit var starkare i hanar. 

I artikel IV studerade vi transkriptomet i kolit-inducerade tumörer och dess immuncellsinfiltration i vildtyp och tarmepitelspecifika ERβ knockout-möss av båda könen. Detta visade att könsskillnader i transkriptomet verkar vara kopplat till uttrycket av ERβ. Dessutom var de identifierade ERβ-beroende förändringarna i tumörtranskriptomet hos honmöss specifikt relaterade till immunsvar. Vi konfirmerade en inverkan av ERβ på infiltrationen av immunceller, specifikt en minskad rekrytering av regulatoriska T-celler och NK-celler.

Sammanfattningsvis ger denna avhandling ny mekanistisk förståelse av ERβs transkriptionella roll i den normala äggstocken och i kolonmikromiljön. Detta inkluderar upptäckten av interaktionen med LRH-1 i äggstocken och NFκB i tjocktarmen. Vår karakterisering ger en grund för utvecklingen av nya riktade terapier för förbättrad fertilitet och kemoprevention av CRC. Detta arbete belyser även vikten av att inkludera båda könen i kolit- och CRC-forskning för att främja vår kunskap och förbättra behandlingsutvecklingen.

Antibodies can cross-react with proteins other than their intended targets, and antibody-based applications can, if not properly validated, lead to flawed interpretations. When evaluating 13 anti-estrogen receptor beta (ERβ) antibodies in 2017, we concluded that only one of them was specific. Applying this antibody in immunohistochemistry of over 44 different normal human tissues and 20 types of cancers revealed ERβ expression in only a few selected tissues. This aligned with mRNA evidence but contradicted a large set of published literature. ERβ protein expression continues to be reported in tissues without clear support by mRNA expression. In this chapter, we describe how ERβ antibodies can be thoroughly validated and discuss selection of well-characterized positive and negative controls. The validation scheme presented is applicable for immunohistochemistry and Western blotting. The protocol includes evaluation of mRNA evidence, use of public databases, assessment of on- and off-target binding, and an optional step for corroboration with immunoprecipitation and mass spectrometry.