Metabolic alterations unravel the maternofetal immune responses with disease severity in pregnant women infected with SARS-CoV-2.

Pregnancy being an immune compromised state, coronavirus disease of 2019 (COVID-19) disease poses high risk of premature delivery and threat to fetus. Plasma metabolome regulates immune cellular responses, therefore we aimed to analyze the change in plasma secretome, metabolome, and immune cells with disease severity in COVID-19 positive pregnant females and their cord blood. COVID-19 reverse transcriptase-polymerase chain reaction positive pregnant females (n = 112) with asymptomatic (Asy) (n = 82), mild (n = 21), or moderate (n = 9) disease, healthy pregnant (n = 18), COVID-19 positive nonpregnant females (n = 7) were included. Eighty-two cord blood from COVID-19 positive and seven healthy cord blood were also analyzed. Mother's peripheral blood and cord blood were analyzed for untargeted metabolome profiling and cytokines by using high-resolution mass spectrometry and cytokine bead array. Immune scan was performed only in mothers' blood by flow cytometry. In Asy severe acute respiratory syndrome coronavirus 2 infection, the amino acid metabolic pathways such as glycine, serine, l-lactate, and threonine metabolism were upregulated with downregulation of riboflavin and tyrosine metabolism. However, with mild-to-moderate disease, the pyruvate and nicotinamide adenine dinucleotide (NAD+ ) metabolism were mostly altered. Cord blood mimicked the mother's metabolomic profiles by showing altered valine, leucine, isoleucine, glycine, serine, threonine in Asy and NAD+ , riboflavin metabolism in mild and moderate. Additionally, with disease severity tumor necrosis factor-α, interferon (IFN)-α, IFN-γ, interleukin (IL)-6 cytokine storm, IL-9 was raised in both mothers and neonates. Pyruvate, NAD metabolism and increase in IL-9 and IFN-γ had an impact on nonclassical monocytes, exhausted T and B cells. Our results demonstrated that immune-metabolic interplay in mother and fetus is influenced with increase in IL-9 and IFN-γ regulated pyruvate, lactate tricarboxylic acid, and riboflavin metabolism with context to disease severity.

Palmitic acid causes hepatocyte inflammation by suppressing the BMAL1-NAD+-SIRT2 axis.

Palmitic acid is the most abundant saturated fatty acid in circulation and causes hepatocyte toxicity and inflammation. As saturated fatty acid can also disrupt the circadian rhythm, the present work evaluated the connection between clock genes and NAD+ dependent Sirtuins in protecting hepatocytes from lipid-induced damage. Hepatocytes (immortal cells PH5CH8, hepatoma cells HepG2) treated with higher doses of palmitic acid (400-600µM) showed typical features of steatosis accompanied with growth inhibition and increased level of inflammatory markers (IL-6 IL-8, IL-1α and IL-1ß) together with decline in NAD+ levels. Palmitic acid treated hepatocytes showed significant decline in not only the protein levels of SIRT2 but also its activity as revealed by the acetylation status of its downstream targets (Tubulin and NF-ƙB). Additionally, the circadian expression of both SIRT2 and BMAL1 was inhibited in presence of palmitic acid in only the non-cancerous hepatocytes, PH5CH8 cells. Clinical specimens obtained from subjects with NASH-associated fibrosis, ranging from absent (F0) to cirrhosis (F4), showed a significant decline in levels of SIRT2 and BMAL1, especially in the cirrhotic liver. Ectopic expression of BMAL1 or activating SIRT2 by supplementation with nicotinamide riboside (precursor of NAD+) dampened the palmitic acid induced lipoinflammation and lipotoxicity more effectively in PH5CH8 cells as compared to HepG2 cells. Mechanistically, palmitic acid caused transcriptional suppression of SIRT2 by disrupting the chromatin occupancy of BMAL1 at its promoter site. Overall, the work suggested that SIRT2 is a clock-controlled gene that is transcriptionally regulated by BMAL1. In conclusion the activation of the BMAL1-NAD+-SIRT2 axis shows hepatoprotective effects by preventing lipotoxicity and dampening inflammation.

Immune drivers of HBsAg loss in HBeAg-negative CHB patients after stopping nucleotide analog and administration of Peg-IFN.

BACKGROUND: The stoppage of nucleoside analog (NA) can lead to immune flare and loss of HBsAg in a proportion of HBeAg-negative chronic hepatitis B (CHB) patients. HBsAg loss could be improved by instituting Peg-Interferon therapy in those who show an immune flare after the stoppage of NA. We investigated the immune drivers of HBsAg loss in NA-treated HBeAg-negative CHB patients after stopping NAs and administration of Peg-IFN-α2b therapy. METHODS: Fifty-five NA-treated eAg-ve, HBV DNA not detected CHB patients were subjected to stopping NA therapy. Twenty-two (40%) patients relapsed (REL-CHBV) within 6 months (HBV DNA ≥2000 IU/mL, ALT ≥2XULN) and were started on Peg-IFN-α2b (1.5 mcg/kg) for 48 weeks (PEG-CHBV). Cytokine levels, immune responses, and T-cell functionality were assessed. RESULTS: Only 22 (40%) of 55 patients clinically relapsed, of which 6 (27%) cleared HBsAg. None of the 33 (60%) nonrelapsers cleared HBsAg. REL-CHBV patients had significantly increased IL-6 (p=0.035), IFN-γ (p=0.049), Th1/17 (p=0.005), CD4 effector memory (EM) (p=0.01), Tfh1/17 (p=0.005), and mature B cells (p=0.04) compared with CHBV. Six months after Peg-IFN therapy, immune resetting with a significant increase in CXCL10 (p=0.042), CD8 (p=0.01), CD19 (p=0.001), and mature B cells (p=0.001) was observed. HBV-specific T-cell functionality showed increased Tfh-secreting IFN-γ (p=0.001), IL-21 (p=0.001), and TNF-α (p=0.005) in relapsers and IFN-γ-secreting CD4 T cell (p=0.03) in PEG-CHBV. CONCLUSIONS: Stopping NA therapy induces flare in about 40% of HBeAg-negative patients. Peg-IFN therapy given to such patients causes immune restoration with HBsAg loss in one fourth of them.