Epigenetic Modulation of GPER Expression in Gastric and Colonic Smooth Muscle of Male and Female Non-Obese Diabetic (NOD) Mice: Insights into H3K4me3 and H3K27ac Modifications.

Type 1 diabetes (T1D) affects gastrointestinal (GI) motility, favoring gastroparesis, constipation, and fecal incontinence, which are more prevalent in women. The mechanisms are unknown. Given the G-protein-coupled estrogen receptor's (GPER) role in GI motility, we investigated sex-related diabetes-induced epigenetic changes in GPER. We assessed GPER mRNA and protein expression levels using qPCR and Western blot analyses, and quantified the changes in nuclear DNA methyltransferases and histone modifications (H3K4me3, H3Ac, and H3K27Ac) by ELISA kits. Targeted bisulfite and chromatin immunoprecipitation assays were used to evaluate DNA methylation and histone modifications around the GPER promoter by chromatin immunoprecipitation assays in gastric and colonic smooth muscle tissues of male and female control (CTR) and non-obese diabetic (NOD) mice. GPER expression was downregulated in NOD, with sex-dependent variations. In the gastric smooth muscle, not in colonic smooth muscle, downregulation coincided with differences in methylation ratios between regions 1 and 2 of the GPER promoter of NOD. DNA methylation was higher in NOD male colonic smooth muscle than in NOD females. H3K4me3 and H3ac enrichment decreased in NOD gastric smooth muscle. H3K4me3 levels diminished in the colonic smooth muscle of NOD. H3K27ac levels were unaffected, but enrichment decreased in NOD male gastric smooth muscle; however, it increased in the NOD male colonic smooth muscle and decreased in the female NOD colonic smooth muscle. Male NOD colonic smooth muscle exhibited decreased H3K27ac levels, not female, whereas female NOD colonic smooth muscle demonstrated diminished enrichment of H3ac at the GPER promoter, contrary to male NOD. Sex-specific epigenetic mechanisms contribute to T1D-mediated suppression of GPER expression in the GI tract. These insights advance our understanding of T1D complications and suggest promising avenues for targeted therapeutic interventions.

Sex-specific epigenetics drive low GPER expression in gastrointestinal smooth muscles in type 2 diabetic mice.

Type 2 diabetes mellitus (T2D) causes gastroparesis, delayed intestinal transit, and constipation, for unknown reasons. Complications are predominant in women than men (particularly pregnant and postmenopausal women), suggesting a female hormone-mediated mechanism. Low G-protein coupled estrogen receptor (GPER) expression from epigenetic modifications may explain it. We explored sexually differentiated GPER expression and gastrointestinal symptoms related to GPER alterations in wild-type (WT) and T2D mice (db/db). We also created smooth muscle-specific GPER knockout (GPER KO) mice to phenotypically explore the effect of GPER deficiency on gastrointestinal motility. GPER mRNA and protein expression, DNA methylation and histone modifications were measured from stomach and colon samples of db/db and WT mice. Changes in gut motility were also evaluated as daily fecal pellet production patterns. We found that WT female tissues have the highest GPER mRNA and protein expressions. The expression is lowest in all db/db. GPER downregulation is associated with promoter hypermethylation and reduced enrichment of H3K4me3 and H3K27ac marks around the GPER promoter. We also observed sex-specific disparities in fecal pellet production patterns of the GPER KO mice compared to WT. We thus, conclude that T2D impairs gut GPER expression, and epigenetic sex-specific mechanisms matter in the downregulation.

Immune Profile of Exosomes in African American Breast Cancer Patients Is Mediated by Kaiso/THBS1/CD47 Signaling.

African American (AA) women with breast cancer are more likely to have higher inflammation and a stronger overall immune response, which correlate with poorer outcomes. In this report, we applied the nanostring immune panel to identify differences in inflammatory and immune gene expression by race. We observed a higher expression of multiple cytokines in AA patients compared to EA patients, with high expression of CD47, TGFB1, and NFKB1 associated with the transcriptional repressor Kaiso. To investigate the mechanism associated with this expression pattern, we observed that Kaiso depletion results in decreased expression of CD47, and its ligand SIRPA. Furthermore, Kaiso appears to directly bind to the methylated sequences of the THBS1 promotor and repress gene expression. Similarly, Kaiso depletion attenuated tumor formation in athymic nude mice, and these Kaiso-depleted xenograft tissues showed significantly higher phagocytosis and increased infiltration of M1 macrophages. In vitro validation using MCF7 and THP1 macrophages treated with Kaiso-depleted exosomes showed a reduced expression of immune-related markers (CD47 and SIRPA) and macrophage polarization towards the M1 phenotype compared to MCF7 cells treated with exosomes isolated from high-Kaiso cells. Lastly, analysis of TCGA breast cancer patient data demonstrates that this gene signature is most prominent in the basal-like subtype, which is more frequently observed in AA breast cancer patients.

A Novel CD206 Targeting Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice.

Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.

A Novel CD206 Targeting Peptide Inhibits Bleomycin Induced Pulmonary Fibrosis in Mice.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.