Adipocytes can be infected by SARS-CoV-2, potentially contributing to the obesity-associated severity of COVID-19. Circumstantial evidence points to angiotensin-converting enzyme 2 (ACE2) as the necessary receptor for adipocyte infection, but this has not been demonstrated experimentally. Using differentiated immortalized human preadipocyte lines that we developed, we found that visceral adipocytes express higher levels of ACE2 and are more susceptible to SARS-CoV-2 spike (S)-mediated luciferase-VSV infection than subcutaneous adipocytes. Overexpression of ACE2 significantly increased infection, whereas knockout of ACE2 significantly decreased S-mediated infection. Visceral adipocytes at baseline were susceptible to infection by SARS-CoV-2 (Delta variant); however, increased levels of viral transcript with time were not apparent. ACE2 knockout significantly decreased the susceptibility of visceral adipocytes to SARS-CoV-2, whereas overexpression of ACE2 resulted in increased SARS-CoV-2 infection and was associated with increased viral transcript levels with time, as well as induction of IL6, a marker of the proinflammatory response. Our results demonstrate that ACE2 confers susceptibility to SARS-CoV-2 infection of visceral adipocytes. Higher levels of ACE2 in these cells may play a role in establishment of infection and a proinflammatory response, potentially leading to pathogenesis.

Polychlorinated biphenyls (PCBs) are linked to cancer, learning disabilities, liver and cardiovascular disease, and diabetes. Older schools often contain high levels of PCBs, and inhalation is a major source of exposure. Technical PCB mixtures, called Aroclors, and individual dioxin-like PCBs impair adipocyte function, which can lead to type II diabetes. To determine how PCB52, a non-dioxin like PCB congener found in school air, affects adipose, adolescent male and female rats were exposed to PCB52 by nose-only inhibition for 4 h per day for 28 consecutive days. Transcriptomic analysis of white adipose revealed sex-specific differences in gene expression between PCB52- and sham-exposed males and females. Exposed females showed mitochondrial gene changes, including downregulation of the thermogenic uncoupling gene, Ucp1. Human preadipocytes/adipocytes exposed to PCB52 or its main metabolite, 4-OH-PCB52, also showed reduced norepinephrine-induced UCP1 expression. These findings suggest that PCB52 inhalation disrupts thermogenesis in adipose tissue, potentially contributing to metabolic syndrome.

Ebola virus (EBOV), the causative agent of Ebola virus disease, remains one of the World Health Organization's top 10 threats to global health. Infectious EBOV virions can be found on the surface of skin late in infection and may be transmitted to others through skin-to-skin contact. We investigate in vivo EBOV tropism and the kinetics of virus movement to and from the skin. Increasing viral loads were detected over time in the skin of EBOV-infected non-human primates and mice, with antigen detected in dermal stromal and immune cells. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a novel mechanism of virus egress to the epidermal surface. During late infection, proinflammatory responses were elevated in infected visceral organs but minimal in the skin despite significant viral loads. We observed similar viral trafficking and cell tropism in the skin of mice intraperitoneally infected with a low containment EBOV model virus, rVSV/EBOV GP, allowing more detailed mechanistic studies. Sites of virus infection in the skin were patchy, with intense focal areas of infection surrounded by uninfected areas. To investigate virus entry into the body through skin, rVSV/EBOV GP was applied to the surface of gently abraded skin to remove the stratum corneum; epidermal keratinocytes were robustly infected with subsequent systemic viral dissemination observed in some mice. Optimal levels of infection within the skin required expression of the phosphatidylserine receptor, AXL. Collectively, our data demonstrate that skin serves as an important organ targeted by EBOV, facilitating virus entry into and egress from the body. IMPORTANCE Ebola virus (EBOV) remains one of the World Health Organization's top 10 threats to global health, despite the availability of a U.S. Food and Drug Administration-approved vaccine. EBOV spreads through human-to-human contact, yet the role of skin in viral transmission remains unclear. Here, we identify skin as a site of EBOV infection, serving as a potential portal for entry into and egress from the body. In vivo, infectious virions and viral RNA increased in the skin over time, localizing to dermal myeloid and stromal cells and to cells within and surrounding hair follicles, suggesting a novel mechanism for viral shedding. Skin infection was patchy and associated with minimal inflammation, despite significant viral loads. Using a surrogate EBOV model, we demonstrate that systemic infection can occur following topical administration through abraded skin and requires phosphatidylserine receptor, AXL, for optimal infection of skin. These findings redefine the role of skin in EBOV pathogenesis, with implications for barrier-targeted interventions.

Ebola virus (EBOV) causes severe human disease. During late infection, EBOV virions are on the skin's surface; however, the permissive skin cell types and the route of virus translocation to the epidermal surface are unknown. We describe a human skin explant model and demonstrate that EBOV infection of human skin via basal media increases in a time-dependent and dose-dependent manner. In the dermis, cells of myeloid, endothelial, and fibroblast origin were EBOV antigen-positive whereas keratinocytes harbored virus in the epidermis. Infectious virus was detected on the apical epidermal surface within 3 days, indicating that virus propagates and traffics through the explants. Purified human fibroblasts and keratinocytes supported EBOV infection ex vivo and both cell types required the phosphatidylserine receptor, AXL, and the endosomal protein, NPC1, for virus entry. This platform identified susceptible cell types and demonstrated dynamic trafficking of EBOV virions. These findings may explain person-to-person transmission via skin contact.

The omentum is the primary site of metastasis for ovarian cancer (OC). Interactions between cancer cells and adipocytes drive an invasive and prometastatic phenotype. Here we studied cancer cell- adipocyte crosstalk by using a direct coculture model with immortalized human visceral nondiabetic pre-adipocytes (VNPADs) and OC cells. We demonstrated increased proliferation, invasiveness, and resistance to cisplatin of cocultured compared with monocultured OC cells. RNA sequencing of OC cells from coculture versus monoculture revealed significant transcriptomic changes, identifying over 200 differentially expressed genes common to OVCAR5 and OVCAR8 cell lines. Enriched pathways included PI3K/AKT and complement activation. Lipid transfer into OC cells from adipocytes induced upregulation of complement C3 and C5 proteins. Inhibiting C3 or C5 reversed the invasive phenotype and C3 knockdown reduced tumor progression in vivo. Increased C3 expression was observed in omental implants compared with primary ovarian tumors and C3 secretion was higher in OC ascites from high-BMI versus low-BMI patients. C3 upregulation in OC cells involved activation of the ATF4-mediated integrated stress response (ISR). Overall, adipocyte-cancer cell interactions promoted invasiveness and tumorigenesis via lipid transfer, activating the ISR, and upregulating complement proteins C3 and C5.

Ebola virus (EBOV), the causative agent of Ebola virus disease (EVD), remains one of WHO’s top ten threats to global health. Infectious EBOV virions can be found on the surface of skin late during systemic infection and passed from the deceased through skin-to-skin contact. Here, we assess viral load and antigen expression in the skin of EBOV-infected non-human primates (NHP) and mouse adapted-EBOV (ma-EBOV) - infected mice and use the low containment viral model, rVSV/EBOV GP, to mechanistically define skin infection in mice. Viral RNA peaked within the skin proximal to the site of injection in EBOV-infected NHPs on day 6. In contrast, mouse skin sites distal to the site of ma-EBOV injection achieved maximal viral loads by day 3. At late times of infection, viral antigen-positive cells co-localized with markers for endothelial, stromal, and immune cells in the dermis. Epidermal cells within and surrounding hair follicles also harbored viral antigen, suggesting a potential mechanism of virus trafficking to the epidermal surface. Despite robust viral infection, distal skin sites of ma-EBOV-infected mice had low expression of proinflammatory stimulated genes. A similar cellular tropism was observed in the skin of mice infected with rVSV/EBOV GP, with discrete focal areas of intense infection. When virus was applied to the surface of gently abraded skin to remove the stratum corneum, epidermal keratinocytes were robustly infected, followed by systemic viral dissemination. To define cell surface receptors critical for virus trafficking to and replication within the skin, mice lacking the phosphatidylserine receptors were infected intraperitoneally with rVSV/EBOV GP. At day 3 of infection, skin distal to the site of infection of TIM-1 knock out (KO) mice had significantly lower levels of infectious virus than the control mice, suggesting that TIM-1 is essential for efficient distribution of virus to the skin. Our findings reveal that EBOV targets specific skin cell populations at late times of viral infection and that the host receptor TIM-1 is required for optimal viral dissemination. ### Competing Interest Statement The authors have declared no competing interest.

Context Obesity is a disease with deleterious effects on the female reproductive tract, including the endometrium.Objective We sought to understand the effects of excess adipose on the benign endometrium.Methods A physiologic in vitro coculture system was developed, consisting of multicellular human endometrial organoids, adipose spheroids, and menstrual cycle hormones. Native human endometrial tissue samples from women with and without obesity were also analyzed. Benign endometrial tissues from premenopausal women ages 33 to 53 undergoing hysterectomy were obtained following written consent at Northwestern University Prentice Women's Hospital, Chicago, Illinois. Gene expression, protein expression, chromatin binding, and expression of DNA damage and oxidative damage markers were measured.Results Under high adiposity conditions, endometrial organoids downregulated endometrial secretory phase genes, suggestive of an altered progesterone response. Progesterone specifically upregulated the metallothionein (MT) gene family in the epithelial cells of endometrial organoids, while high adiposity significantly downregulated the MT genes. Silencing MT genes in endometrial epithelial cells resulted in increased DNA damage, illustrating the protective role of MTs. Native endometrium from women with obesity displayed increased MT expression and oxidative damage in the stroma and not in the epithelium, indicating the cell-specific impact of obesity on MT genes.Conclusion Taken together, the in vitro and in vivo systems used here revealed that high adiposity or obesity can alter MT expression by decreasing progesterone response in the epithelial cells and increasing oxidative stress in the stroma.

Influenza A viruses (IAV) utilize sialic acid (Sia) containing cell surface glycoconjugates for host cell infection, and IAV strains from different host species show preferences for structurally distinct Sia at the termini of glycoconjugates. Various types of cell surface glycoconjugates (N-glycans, O-glycans, glycolipids) display significant diversity in both structure and carbohydrate composition. To define the types of sialylglycoconjugates that facilitate IAV infection, we utilized the CRISPR/Cas9 technique to truncate the three major types of glycoconjugates, either individually or in combination, by targeting glycosyltransferases essential to glycan biosynthesis in a human lung epithelial cell line. Our studies show that both human and avian IAV strains do not display strict preferences for a specific type of glycoconjugate. Interestingly, truncation of the three major types of glycoconjugates significantly decreased replication of human IAV strains, yet did not impact replication of avian IAV strains. Unlike human IAV strains, avian IAV strains were able to efficiently utilize other less prevalent shorter glycoconjugates such as sialyl Tn and sialyl T antigens. Taken together, our studies demonstrate that avian IAV strains utilize a broader repertoire of glycoconjugates for host cell infection as compared to human IAV strains. IMPORTANCE It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Polychlorinated biphenyls (PCBs) accumulate in adipose tissue and are linked to obesity and diabetes. The congener, PCB52 (2,2′,5,5′-tetrachorobiphenyl), is found at high levels in school air. Hydroxylation of PCB52 to 4-OH-PCB52 (4-hydroxy-2,2′,5,5′-tetrachorobiphenyl) may increase its toxicity. To understand PCB52's role in causing adipose dysfunction, we exposed human preadipocytes to PCB52 or 4-OH-PCB52 across a time course and assessed transcript changes using RNAseq. 4-OH-PCB52 caused considerably more changes in the number of differentially expressed genes as compared to PCB52. Both PCB52 and 4-OH-PCB52 upregulated transcript levels of the sulfotransferase SULT1E1 at early time points, but cytochrome P450 genes were generally not affected. A set of genes known to be transcriptionally regulated by PPARα were consistently downregulated by PCB52 at all time points. In contrast, 4-OH-PCB52 affected a variety of pathways, including those involving cytokine responses, hormone responses, focal adhesion, Hippo, and Wnt signaling. Sets of genes known to be transcriptionally regulated by IL17A or parathyroid hormone (PTH) were found to be consistently downregulated by 4-OH-PCB52. Most of the genes affected by PCB52 and 4-OH-PCB52 were different and, of those that were the same, many were changed in an opposite direction. These studies provide insight into how PCB52 or its metabolites may cause adipose dysfunction to cause disease.

Figure S2. Representative IPA mechanistic network for signaling regulators predicted to contribute to the RABL6A knockdown phenotype.

Figure S3. Quantitative RT-PCR showing RABL6A depletion does not affect p21 mRNA expression

Figure S6. Down-regulation of p27 in arrested RABL6A knockdown cells promotes S phase entry.

Table S1. List of differentially regulated genes caused by RABL6A knockdown (KD), relative to control (CON), in BON-1 PNET cells.

Polychlorinated biphenyls (PCBs) were used extensively in building materials, including those used in schools. PCBs ac-cumulate in fat, and exposure to PCBs is associated with the development of cancer, neurodevelopmental disorders, car-diovascular disease, obesity, and diabetes. The non-dioxin -like PCB congener, PCB52 (2,2 ' ,5,5 '-tetrachlorobiphenyl), is found at one of the highest levels of any congener in school air. PCB52 is oxidized in the liver to hydroxylated forms, mainly 4-OH-PCB52 (2,2',5,5'-tetrachlorobiphenyl-4-ol). In a previous study, we reported on RNAseq data generated from exposure of human preadipocytes to the dioxin-like PCB con -gener, PCB126. In this new dataset, we used identical tech-niques to examine alterations in gene transcript levels in hu-man preadipocytes exposed to PCB52 or 4-OH-PCB52 over a time course. This updated set of data provides a com-prehensive transcriptional profile of changes that occur in preadipocytes exposed to PCB52 or 4-OH-PCB52 over time and allows for comparison of these changes between the par-ent compound and its hydroxy metabolite. The datasets will allow others to explore how PCB52 and 4-OH-PCB52 impact biological pathways in preadipocytes. Further studies can be performed to determine how these changes might lead to disease. & COPY; 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Figure S1. Quantitative PCR measurement of human RABL6A DNA copy number in human PNET patient samples.

Figure S3. Quantitative RT-PCR showing RABL6A depletion does not affect p21 mRNA expression

Figure S4. Quantitative RT-PCR showing effective silencing of Rb1 mRNA by Rb1 shRNAs in RABL6A knockdown cells.

Figure S5. Effect of RABL6A overexpression on Rb1 and p21 mRNA and protein expression

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in adipose tissue and have been associated with cardiometabolic disease. We have previously demonstrated that exposure of human preadipocytes to the dioxin-like PCB126 disrupts adipogenesis via the aryl hydrocarbon receptor (AhR). To further understand how PCB126 disrupts adipose tissue cells, we performed RNAseq analysis of PCB126-treated human preadipocytes over a 3-day time course. The most significant predicted upstream regulator affected by PCB126 exposure at the early time point of 9 h was the AhR. Progressive changes occurred in the number and magnitude of transcript levels of genes associated with inflammation, most closely fitting the pathways of cytokine-cytokine-receptor signaling and the AGE-RAGE diabetic complications pathway. Transcript levels of genes involved in the IL-17A, IL-1β, MAP kinase, and NF-κB signaling pathways were increasingly dysregulated by PCB126 over time. Our results illustrate the progressive time-dependent nature of transcriptional changes caused by toxicants such as PCB126, point to important pathways affected by PCB126 exposure, and provide a rich dataset for further studies to address how PCB126 and other AhR agonists disrupt preadipocyte function. These findings have implications for understanding how dioxin-like PCBs and other dioxin-like compounds are involved in the development of obesity and diabetes.

Adipose tissue is an endocrine organ with strong proinflammatory capacity; however, the role of this tissue in highly pathogenic virus infections has not been extensively examined. We show that mice infected with a mouse-adapted Ebola Virus (EBOV) exhibit increasing levels of viral transcript in visceral and subcutaneous adipose tissue over the course of infection. Human adipocytes were found to be susceptible to EBOV. Endocytosis and macropinocytosis inhibitors effectively blocked infection of adipocytes by a replication competent recombinant VSV virus that expresses EBOV glycoprotein (EBOV-GP/rVSV). While EBOV-GP/rVSV infection of adipocytes caused a robust induction of interferon responsive genes, EBOV infection resulted in modest upregulation of these genes. However, both EBOV-GP/rVSVand EBOV induced comparable and significant induction of the proinflammatory genes CXCL8, IL6, CCL2, and F3 (Tissue Factor). Our results suggest that adipocytes in adipose tissue may contribute to the inflammatory response and coagulopathy that occur during EBOV pathogenesis.