Reference Gene Validation for RT-qPCR in PBMCs from Asthmatic Patients with or without Obesity

Obesity is known to impair the efficacy of glucocorticoid medications for asthma control. Glucocorticoid-induced gene expression studies may be useful to discriminate those obese asthmatic patients who present a poor response to glucocorticoids. The expression of genes of interest is normalized with respect to reference genes (RGs). Ideally, RGs have a stable expression in different samples and are not affected by experimental conditions. The objective of this work was to analyze suitable RGs to study the role of glucocorticoid-induced genes in obese asthmatic patients in further research. The gene expression of eight potential RGs (GUSB, B2M, POLR2A, PPIA, ACTB, GAPDH, HPRT1, and TBP) was assessed with reverse transcription-quantitative polymerase chain reaction in peripheral blood mononuclear cells (PBMCs) from asthmatic, obese asthmatic, and healthy individuals. Their stability was analyzed using four different algorithms-BestKeeper, ?Ct, geNorm, and NormFinder. geNorm analysis recommended the use of a minimum of three genes for normalization. Moreover, intergroup variation due to the treatment was calculated by NormFinder, which found that B2M was the gene that was least affected by different treatments. Comprehensive rankings indicated GUSB and HPRT1 as the best RGs for qPCR in PBMCs from healthy and asthmatic subjects, while B2M and PPIA were the best for obese asthmatic subjects. Finally, our results demonstrated that B2M and HPRT1 were the most stable RGs among all groups, whereas ACTB, TBP, and GAPDH were the worst shared ones

Differences in Inflammatory Cytokine Profile in Obesity-Associated Asthma: Effects of Weight Loss

Obesity and asthma are associated with systemic inflammation maintained by mediators released by adipose tissue and lung. This study investigated the inflammatory serum mediator profile in obese subjects (O) (n = 35), non-obese asthma (NOA) patients (n = 14), obese asthmatics (OA) (n = 21) and healthy controls (HC) (n = 33). The effect of weight loss after bariatric surgery (BS) was examined in 10 OA and 31 O subjects. We analyzed serum markers including leptin, adiponectin, TGF-?1, TNFR2, MCP-1, ezrin, YKL-40, ST2, IL-5, IL-9, and IL-18. Compared with HC subjects, the O group showed increased levels of leptin, TGF-?1, TNFR2, MCP-1, ezrin, YKL-40, and ST2; the OA group presented increased levels of MCP-1, ezrin, YKL-40, and IL-18, and the NOA group had increased levels of ezrin, YKL-40, IL-5, and IL-18. The higher adiponectin/leptin ratio in NOA with respect to OA subjects was the only significant difference between the two groups. IL-9 was the only cytokine with significantly higher levels in OA with respect to O subjects. TNFR2, ezrin, MCP-1, and IL-18 concentrations significantly decreased in O subjects after BS. O, OA, and NOA showed distinct patterns of systemic inflammation. Leptin and adiponectin are regulated in asthma by obesity-dependent and-independent mechanisms. Combination of asthma and obesity does not result in significant additive effects on circulating cytokine levels. © 2022 by the authors. Licensee MDPI, Basel, Switzerland