Human Visceral Adipose Tissue (VAT) Inflammation Is Not Associated With Perforin Deficiency in Type 2 Diabetes Mellitus (DM) Subjects as Compared to Health Controls

Abstract

Abstract Introduction: Role of T cells in VAT inflammation is poorly characterised. Perforin deficiency is associated with autoimmune inflammatory diseases like Hemophagocytosis Lymphohistiocytosis,as also in context of type 1 DM pathogenesis. Data from animal models suggest that perforin deficiency leads to VAT inflammation1Objective: We hypothesized CD8+/perf+ and CD56+/perf+ cells to be decreased in type 2 DM as compared to healthy controls. The present study also explored the difference in activation of T/NK cells between two groups Methods: 2×2 cm omental tissue was obtained from subjects undergoing elective abdominal surgery. The sample was transported in RPMI solution and stored in -80 ℃. Processing involved thawing, incubation at 37.6 ℃ for 24 hours with type IV Collagenase (1 mg/ml, Sigma Aldrich) 1ml/g of tissue,centrifuge (32g for 10 min at 10℃). The resultant Stromal Vascular fraction (SVF) was suspended in phosphate buffer saline (PBS), passed through cell strainer to make single cell suspension. It was again centrifuged and tagged with CD markers of interest. Fc block was added and single cell solution with FACS fluid prepared. It was run in BD CANTO-2 flow cytometer as described2Results: Of seventeen samples analysed, twelve samples of type 2 diabetes subjects were compared with five healthy controls. All results are presented in median. The diabetics had higher HbA1c (8.1 % vs 6%), higher BMI (28 kg/m2 vs 24 kg/m2), hsCRP(2.1 mg/dl Vs 0.9 mg/dl) but there was no difference in HOMA-Ir (5 vs 5.2 mU/L/mg/dl). The percentage of CD4+ + CD8+ cells/g of VAT was similar in both cases and control (20 × 103 Vs 23× 103). CD8+/CD45+/perf+ and CD56+/CD45+/perf+ could not be identified in any of the samples. Although CD8+/CD45+/perf-- and CD56+/CD45+/perf-- cells were identified. Next, we analysed the same cells for cytotoxic activation by 107a. The percentage of 107a positivity was low in CD 8+ (7% and 4 % respectively in cases and control) and CD 56+ cells (10% and 9 % respectively in cases and control),Although clinically type 2 DM subjects were obese and had inflammation (i.e higher hsCRP), there was no difference in VAT activation of immune cells studied. Also, we could not delineate perforin in any of the samples. Conclusion: Taken together this work suggests VAT T cell immune milieu in human Type 2 DM is different from mouse model. It is neither characterised by perforin deficiency nor activation of T cell/NK cell. This study points towards the probability that, the role of T cell/NK cells in human VAT infiltration could be fundamentally different from mice models. Further studies should be focussed on functional characteristics of these cells and interaction with VAT macrophages.