Investigation of SARS-CoV-2 individual proteins reveals the in vitro and in vivo immunogenicity of membrane protein.

Evidence in SARS-CoV-2 patients have identified that viral infection is accompanied by the expression of inflammatory mediators by both immune and stromal cells within the pulmonary system. However, the immunogenicity of individual SARS-CoV-2 proteins has yet to be evaluated. The SARS-CoV-2 virus consists of 29 proteins, categorized either as nonstructural proteins (NSP's), structural proteins (SP's) or accessory proteins. Here we sought to evaluate the immunogenicity of NSP 1, 7, 8, 9, 10, 12, 14, 16 and the SP's spike protein (full length, S1, S2 and receptor binding domain subunits), nucleocapsid and membrane SARS-CoV-2 proteins against THP-1 and human peripheral blood mononuclear cells (PBMCs). Our results indicate that various SARS-CoV-2 proteins elicit a proinflammatory immune response indicated by increases in cytokines TNF, IL-6 and IL-1ß. Our results support that SARS-CoV-2 membrane protein induced a robust increase of TNF, IL-6, IL-1ß and IL-10 expression in both THP-1 and human PBMC's. Further evaluation of intranasal membrane protein challenge in male and female BALB/c mice show increases in BALF (bronchalveolar lavage fluid) proinflammatory cytokine expression, elevated cellularity, predominantly neutrophilic, and concomitant peribronchiolar and perivascular lymphomononuclear and neutrophilic inflammation. Our results suggest that individual membrane associated SARS-CoV-2 proteins induce a robust immune response that may contribute to viral induced cytokine release syndrome (CRS) in the lungs of moderate to severe COVID-19 patients. We posit that SARS-CoV-2 membrane challenges in immune-competent mice can serve as an adequate surrogate for the development of novel treatments for SARS-CoV-2 induced pulmonary inflammation, thereby avoiding expensive live virus studies under BSL-3 conditions.

High-fat diet-induced dysregulation of ovarian gene expression is restored with chronic omega-3 fatty acid supplementation.

Chronic high-fat diet (HFD) consumption causes ovarian dysfunction in rodents. Acute dietary treatment with docosahexaenoic acid (DHA) increases oocyte quality and ovarian reserve at advanced reproductive age. We hypothesized that DHA supplementation after HFD exposure reverses HFD-induced ovarian defects. We conducted a dietary intervention with reversal to chow, DHA-supplemented chow, or DHA-supplemented HFD after HFD consumption. After 10 weeks, HFD-fed mice had impaired estrous cyclicity, decreased primordial follicles, and altered ovarian expression of 24 genes compared to chow controls. Diet reversal to either chow or chow + DHA restored estrous cyclicity, however only chow + DHA appeared to mitigated the impact of HFD on ovarian reserve. All dietary interventions restored HFD-dysregulated gene expression to chow levels. We found no association between follicular fluid DHA levels and ovarian reserve. In conclusion our data suggest some benefit of DHA supplementation after HFD, particularly in regards to ovarian gene expression, however complete restoration of ovarian function was not achieved.

High-fat diet exposure, regardless of induction of obesity, is associated with altered expression of genes critical to normal ovulatory function.

We evaluated the impact of high-fat diet (HFD) on ovarian gene expression. Female 5-week-old C57BL/6J mice were fed a 60% HFD or standard chow for 10 weeks. HFD-fed mice were then separated into obese (HF-Ob) and lean (HF-Ln) based on body weight. HFD exposure led to impairment of the estrous cycle, changes in hormones affecting reproduction, and decreased primordial follicles regardless of the development of obesity. RNA-sequencing of whole ovaries identified multiple genes with altered expression after HFD, with 25 genes displaying decreased expression in both HF-Ln and HF-Ob mice compared to the chow-fed controls (q < 0.05). Several of these 25 genes are involved in normal ovarian functions, including ovulation (Edn2, Tnfaip6, Errfi1, Prkg2, and Nfil3), luteinization (Edn2), and luteolysis (Nr4a1). Taken together, elevated dietary fat intake, regardless of obesity, is associated with impaired estrous cycle, depletion of the ovarian reserve, and altered expression of genes critical to normal ovulatory function.

Fat-1 Transgene Is Associated With Improved Reproductive Outcomes.

High intake of ω-3 polyunsaturated fatty acids (PUFAs) has been associated with a variety of health benefits. However, the role of ω-3 PUFAs in female reproductive function is unclear, with studies showing both positive and negative effects. The type of diet that ω-3 fatty acids are consumed with, for example, a balanced diet vs a high-fat diet (HFD), may influence how ω-3 fatty acids affect female reproductive function. To address the role of ω-3 PUFAs in female reproduction, we used the fat-1 mouse both with and without HFD exposure. Fat-1 mice constitutively express the fat-1 transgene, allowing the conversion of ω-6 to ω-3 fatty acids to yield an optimal tissue ratio of ω-6 to ω-3 fatty acids (∼1:1). In our study, at 15 weeks of age, fat-1 mice had elevated primordial follicles compared with wild-type controls with both standard chow and HFD feeding. Higher serum levels of the ω-3 docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA) were positively associated with primordial follicle numbers, whereas the ratio of the ω-6 arachidonic acid to EPA + DPA + DHA had the opposite effect. Furthermore, fat-1 mice had increased pregnancy rates and shorter time to pregnancy when fed an HFD compared with wild-type mice. In conclusion, our novel preclinical model suggests that high tissue levels of long-chain ω-3 PUFAs are associated with an improved ovarian reserve and improved reproductive outcomes. Further studies are needed to evaluate ω-3 PUFAs as a potential intervention strategy in women with diminished ovarian reserve.