Adipose-Tissue-Derived Mesenchymal Stem Cells Mediate PD-L1 Overexpression in the White Adipose Tissue of Obese Individuals, Resulting in T Cell Dysfunction.

The PD-L1/PD-1 immune checkpoint axis is the strongest T cell exhaustion inducer. As immune dysfunction occurs during obesity, we analyzed the impact of obesity on PD-L1/PD-1 expression in white adipose tissue (WAT) in mice and in human white adipocytes. We found that PD-L1 was overexpressed in WAT of diet-induced obese mice and was associated with increased expression of PD-1 in visceral but not subcutaneous WAT. Human in vitro cocultures with adipose-tissue-derived mesenchymal stem cells (ASC) and mononuclear cells demonstrated that the presence of ASC harvested from obese WAT (i) enhanced PD-L1 expression as compared with ASC from lean WAT, (ii) decreased Th1 cell cytokine secretion, and (iii) resulted in decreased cytolytic activity towards adipocytes. Moreover, (iv) the implication of PD-L1 in obese ASC-mediated T cell dysfunction was demonstrated through PD-L1 blockade. Finally, (v) conditioned media gathered from these cocultures enhanced PD-L1 expression in freshly differentiated adipocytes, depending on IFNγ. Altogether, our results suggest that PD-L1 is overexpressed in the WAT of obese individuals during IFNγ secretion, leading to T cell dysfunction and notably reduced cytolytic activity. Such a mechanism could shed light on why adipose-tissue-infiltrating viruses, such as SARS-CoV-2, can worsen disease in obese individuals.

Impact of Estrogen Withdrawal and Replacement in Female Mice along the Intestinal Tract. Comparison of E2 Replacement with the Effect of a Mixture of Low Dose Pollutants.

Postmenopausal women represent a vulnerable population towards endocrine disruptors due to hormonal deficit. We previously demonstrated that chronic exposure of ovariectomized C57Bl6/J mice fed a high-fat, high-sucrose diet to a low-dose mixture of chemicals with one dioxin, one polychlorobiphenyl, one phthalate, and bisphenol A triggered metabolic alterations in the liver but the intestine was not explored. Yet, the gastrointestinal tract is the main route by which pollutants enter the body. In the present study, we investigated the metabolic consequences of ovarian withdrawal and E2 replacement on the various gut segments along with investigating the impact of the mixture of pollutants. We showed that genes encoding estrogen receptors (Esr1, Gper1 not Esr2), xenobiotic processing genes (e.g., Cyp3a11, Cyp2b10), and genes related to gut homeostasis in the jejunum (e.g., Cd36, Got2, Mmp7) and to bile acid biosynthesis in the gut (e.g., Fgf15, Slc10a2) and liver (e.g., Abcb11, Slc10a1) were under estrogen regulation. Exposure to pollutants mimicked some of the effects of E2 replacement, particularly in the ileum (e.g., Esr1, Nr1c1) suggesting that the mixture had estrogen-mimetic activities. The present findings have important implications for the understanding of estrogen-dependent metabolic alterations with regards to situations of loss of estrogens as observed after menopause.

Estrogen withdrawal and replacement differentially target liver and adipose tissues in female mice fed a high-fat high-sucrose diet: impact of a chronic exposure to a low-dose pollutant mixture☆.

Postmenopausal women may be at particular risk when exposed to chemicals especially endocrine disruptors because of hormonal deficit. To get more insight, ovariectomized C57Bl6/J mice fed a high-fat high-sucrose diet were chronically exposed from 5 to 20 weeks of age to a low-dose mixture of chemicals with one dioxin, one polychlorobiphenyl, one phthalate and bisphenol A. Part of the mice received as well E2 implants to explore the potential estrogenic dependency of the metabolic alterations. With this model, estrogen loss resulted in glucose but not lipid metabolism impairment, and E2 replacement normalized the enhanced body and fat pad weight, and the glucose intolerance and insulin resistance linked to ovariectomy. It also altered cholesterol metabolism in the liver concurrently with enhanced estrogen receptor Esr1 mRNA level. In addition, fat depots responded differently to estrogen withdrawal (e.g., selective mRNA enhancement of adipogenesis markers in subcutaneous and of inflammation in visceral fat pads) and replacement challenges. Importantly, the pollutant mixture impacted lipid deposition and mRNA expression of several genes related to lipid metabolism but not Esr1 in the liver. Adiponectin levels were altered as well. In addition, the mRNA abundance of the various estrogen receptors was regionally impacted in fat tissues. Besides, xenobiotic processing genes did not change in response to the pollutant mixture in the liver. The present findings bring new light on estrogen-dependent metabolic alterations with regards to situations of loss of estrogens as observed after menopause.

Chronic exposure to a pollutant mixture at low doses led to tissue-specific metabolic alterations in male mice fed standard and high-fat high-sucrose diet.

Excessive consumption of industrialized food and beverages is a major etiologic factor in the epidemics of obesity and associated metabolic diseases because these products are rich in fat and sugar. In addition, they contain food contact materials and environmental pollutants identified as metabolism disrupting chemicals. To evaluate the metabolic impact of these dietary threats (individually or combined), we used a male mouse model of chronic exposure to a mixture of low-dose archetypal food-contaminating chemicals that was added in standard or high-fat, high-sucrose (HFHS) diet. Specifically, the mixture contained bisphenol A, diethylhexylphthalate, 2,3,7,8-tetrachlorodibenzo-p-dioxine and polychlorinated biphenyl 153. Exposure lasted from 5 to 20 weeks of age. Metabolic exploration was conducted setting the basis of candidate gene expression mRNA analyses in liver, jejunum and adipose tissue depots from 20 week-old mice. Strong metabolic deleterious effects of the HFHS diet were demonstrated in line with obesity-associated metabolic features and insulin resistance. Pollutant exposure resulted in significant changes on plasma triglyceride levels and on the expression levels of genes mainly encoding xenobiotic processing in jejunum; estrogen receptors, regulators of lipoprotein lipase and inflammatory markers in jejunum and adipose tissues as well as adipogenesis markers. Importantly, the impact of pollutants was principally evidenced under standard diet. In addition, depending on nutritional conditions and on the metabolic tissue considered, the impact of pollutants could mimic or oppose the HFHS effects. Collectively, the present study extends the cocktail effect concept of a low-dosed pollutant mixture and originally points to tissue-specificity responsiveness especially in jejunum and adipose tissues.

Evidence for estrogeno-mimetic effects of a mixture of low-dose pollutants in a model of ovariectomized mice.

We recently hypothesized that a mixture of low-dosed dioxin, polychlorobiphenyl, phthalate and bisphenol may induce estrogeno-mimetic activities in a model of lifelong-exposed female mice. Herein, we evaluated the impact of this mixture in estrogen deficiency conditions. Based on the protective effects of estrogens against metabolic disorders, we reasoned that exposure to pollutants should attenuate the deleterious metabolic effects induced by ovariectomy. In line with the hypothesis, exposure to pollutants was found to reduce the impact of ovariectomy on glucose intolerance and insulin resistance, to enhance the expression levels of the hepatic estrogen receptor α and to attenuate the ovariectomy-induced enhancement of the chemokine MCP-1/CCL2 considered as an indicator of estrogen signalling. Because of the very low doses of pollutants used in mixture, these findings may have strong implications in terms of understanding the potential role of environmental contaminants in the development of metabolic diseases, specifically in females during menopausal transition.

Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice.

Environmental contaminants are suspected to be involved in the epidemic incidence of metabolic disorders, food ingestion being a primarily route of exposure. We hypothesized that life-long consumption of a high-fat diet that contains low doses of pollutants will aggravate metabolic disorders induced by obesity itself. Mice were challenged from preconception throughout life with a high-fat diet containing pollutants commonly present in food (2,3,7,8-tetrachlorodibenzo-p-dioxin, polychlorinated biphenyl 153, diethylhexyl phthalate, and bisphenol A), added at low doses in the tolerable daily intake range. We measured several blood parameters, glucose and insulin tolerance, hepatic lipid accumulation, and gene expression in adult mice. Pollutant-exposed mice exhibited significant sex-dependent metabolic disorders in the absence of toxicity and weight gain. In males, pollutants increased the expression of hepatic genes (from 36 to 88%) encoding proteins related to cholesterol biosynthesis and decreased (40%) hepatic total cholesterol levels. In females, there was a marked deterioration of glucose tolerance, which may be related to the 2-fold induction of estrogen sulfotransferase and reduced expression of estrogen receptor α (25%) and estrogen target genes (>34%). Because of the very low doses of pollutants used in the mixture, these findings may have strong implications in terms of understanding the potential role of environmental contaminants in food in the development of metabolic diseases.

Direct and indirect impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on adult mouse Leydig cells: an in vitro study.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants that exert adverse effects on reproductive processes. In testis, Leydig cells which produce testosterone are under hormonal and local control exerted by cytokines including TNFα. Using mouse Leydig primary cell cultures as a model, we studied the effects of TCDD on the steroidogenic outcome of Leydig cells and the gene expression levels of Ccl5 and Cxcl4, previously shown to be target genes of TCDD in testis. We found that TCDD did not alter the steroidogenic outcome of Leydig cells but that it up-regulated Cxcl4 gene expression levels. TCDD also impacted Ccl5 gene expression when cells had been co-treated with TNFα. TCDD action probably initiated with binding to the aryl hydrocarbon receptor (AhR) present on Leydig cells. TCDD regulated the gene expression levels of AhR (transient down-regulation) and its repressor AhRR and Cyp1b1 (up-regulation). The trophic human chorionic gonadotropin (hCG) hormone did not impact AhR, its repressor AhRR or Cyp1b1 but it opposed the TCDD-enhanced AhRR mRNA levels. Conversely, TNFα stimulated AhR gene expression levels. Collectively, it is suggested that the impact of TCDD on expression of target genes in Leydig cells may operate under the complex network of hormones and cytokines.

A comprehensive survey of the laminins and collagens type IV expressed in mouse Leydig cells and their regulation by LH/hCG.

Extracellular matrix (ECM) proteins have been shown to alter Leydig cell steroidogenesis in vitro, substantiating the hypothesis that Leydig cell steroidogenic activity and matrix environment are interdependent events. However, the nature of the ECM components synthesized by Leydig cells and their regulation by LH/human chorionic gonadotropin (hCG) remain unknown. Here, we examine the occurrence of the 11 laminin subunits and the 6 alpha chains of collagen IV (COL4A1-6) by RT-PCR in Leydig cells cultured with or without LH/hCG. Leydig cells were a tumor Leydig cell line (mLTC-1) or 8-week-old mice Leydig cells. Based on PCR data, it is suggested that normal Leydig cells may synthesize a maximum of 11 laminin heterotrimers and the 6 alpha chains of collagen IV. They also may synthesize various proteases and inhibitors of the metzincin family. The mLTC-1 cells have a limited repertoire as compared with normal Leydig cells. Interestingly, none of the ten proteases and inhibitors monitored is under LH-hCG regulation whereas every protease and inhibitor of the serine protease family yet identified in Leydig cells is under gonadotropin regulation. In addition, a few laminin and collagen subunit genes are regulated by LH/hCG. These are laminins alpha3 and gamma3 (Lama3 and Lamc3), Col4a3, and Col4a6, which are negatively regulated by LH/hCG in both Leydig cell types, and Col4a4, which was downregulated in primary cultures but not in mLTC-1 cells. Collectively, the present study suggests that Leydig cells modulate in a selective fashion their matrix environment in response to their trophic hormone. This may alter the steroidogenic outcome of Leydig cells.

The mouse testis is the source of various serine proteases and serine proteinase inhibitors (SERPINs): Serine proteases and SERPINs identified in Leydig cells are under gonadotropin regulation.

The occurrence of various serine proteinases and serine proteinases inhibitors (SERPINs) was investigated by RT-PCR in whole testes of 1-, 3-, and 8-wk-old mice in crude and enriched germ cell fractions, mouse Leydig tumor cells (mLTC-1), and primary cultures of 3- and 8-wk-old enriched fractions of Leydig cells and 3-wk-old Sertoli cells. New members were identified in the testis protease repertoire. Within the Leydig repertoire, a PCR product was found for plasminogen activators urokinase plasminogen activator (uPA) and tissue plasminogen activator (8-wk-old cells), matriptase-2 (mLTC-1), kallikrein-21, SERPINA5, SERPINB2 (primary cultures), and serine peptidase inhibitor Kunitz type 2 (SPINT2). The gonadotropin regulation was explored by semiquantitative RT-PCR, using steroidogenic acute regulatory protein (StAR) as a positive control. Matriptase-2, kallikrein-21, SPINT2, and SERPINA5 were down-regulated, whereas uPA and its receptor were up-regulated by human chorionic gonadotropin (hCG) via cAMP in the mLTC-1 cells. Positive effects were observed transiently after 1-8 h of hCG exposure, and negative effects, first evidenced after 6 h, lasted 48 h. The hCG-induced effects were confirmed in primary cultures. In addition, SERPINB2 was augmented by hCG in primary cultures. Addition of either trypsin or protease inhibitors did not alter the hCG-induced surge of StAR. Because hCG regulated proteases and SERPINs (whereas testosterone did not), it could alter the proteolytic balance of Leydig cells and consequently the metabolism of extracellular matrix components. Therefore, even though a direct interplay between the early hCG-induced surge of uPA and StAR is unlikely, our data together with the literature suggest that extracellular matrix proteins alter Leydig cell steroidogenesis.

Diethylstilbestrol inhibits the expression of the steroidogenic acute regulatory protein in mouse fetal testis.

This study investigated the early deleterious effects of an in-utero exposure to diethylstilbestrol (DES) on mouse testicular development. To that purpose, pregnant mice were injected daily with up to 100 microg/kg DES from 10.5 to 17.5 days postcoitum (dpc). At 18.5 dpc, testes were removed from fetuses for RNA (RT-PCR) and protein (Western blot, immunohistochemistry) analysis. Twenty-two genes were selected among which transcription factors, markers of differentiation of the different testicular cell lineages, steroidogenic enzymes and hormone receptors. The Steroidogenic Acute Regulatory (StAR) protein produced by the fetal Leydig cells was dramatically reduced in the DES-exposed testes. The P450c17 was the other gene modified following DES exposure. The alteration of these two genes is consistent with the decrease observed in the intratesticular testosterone levels, in the DES-exposed testes. Collectively, we demonstrated that DES did not alter testicular cell lineage specification but that it strongly inhibited the major function of the fetal Leydig cells.

Evidence that MMP-2 and TIMP-2 are at play in the FSH-induced changes in Sertoli cells.

FSH has been shown to elicit in vitro changes in the Sertoli cell cytoskeleton through involving proteases, and cytochalasin-D mimics FSH. Testis extracts were screened (RT-PCR) for various metalloproteinases (MMPs), 20-day-old rat Sertoli cells were purified, cultured and treated with FSH, cytochalasin D and TNFalpha (to antagonize FSH action). Cell shape (phase-contrast microscopy) and levels for MMP-2 (gelatin zymography) and its inhibitor TIMP-2 (Northern and Western blot) were monitored. TNFalpha-treated cells spread readily and grew larger than FSH-treated cells. Cytochalasin-D mimicked FSH, and MMP-2 production and TIMP-2 gene expression were augmented. Interestingly, TNFalpha reversed FSH- and cytochalasin D-induced effects both on cell shape and on MMP-2 and TIMP-2. These effects occurred during the first 48 h of culture, when Sertoli cells migrated from the freshly dispersed aggregates, but not once cells were organized in monolayers. MMP-2 and TIMP-2 are likely involved in the FSH-induced changes in Sertoli cells.