Decreased leukocyte exhaustion is associated with decreased IFN-ß and increased α-defensin-1 levels in type-2 diabetes.

Pan-leukocyte exhaustion is a physiological process associated with immune homeostasis. Too much of immune exhaustion can lead to immune impairment and increased susceptibility to infections and cancer; too little can lead to chronic inflammation. Since type-2 diabetes subjects have both impaired immunity and metainflammation, we looked at TLR induced pan-immune exhaustion and its regulation, in these subjects. TLR induced expression of PD-1 and CTLA-4 in T cells, B cells, monocytes and neutrophils, in whole blood cultures, were quantified by flowcytometry. Circulating levels and in vitro secretion of IFN-ß and α-Defensin-1 (α-DEF-1) were quantified by ELISA. TLR induced expression of PU.1, IRF-3,-4 and -5 in whole blood cultures was quantified by qRT-PCR. Systemic lipid and protein peroxidation was quantified by spectrophotometry. TLR induced expression of PD-1 (in monocytes) and CTLA-4 expression (in B cells and neutrophils) were decreased in drug naive newly diagnosed diabetes patients. This was associated with decreased secretion and circulating levels of IFN-ß and increased secretion and circulating levels of α-DEF-1 in these subjects. No major derangement in the transcriptional network was seen. Many of these defects were only partially rectified in those under treatment. Together, we hypothesize that the high defensin levels could inhibit TLR signalling leading to reduced IFN-ß levels, which in turn could lead to reduced expression of PD-1 and CTLA-4 in the immune cells. This effect along with systemic redox stress could fuel metainflammation in type-2 diabetes.

Increased serum levels of novel T cell cytokines IL-33, IL-9 and IL-17 in subjects with type-1 diabetes.

The role of adaptive immune cytokines in the pathogenesis of type-1 Diabetes Mellitus (T1DM) is well known. Even though reports on the serum levels of both Th-1 and Th-2 cytokines are available, those on newly described T cell cytokines such as IL-17, IL-33 and IL-9 in T1DM are scarce. We therefore measured the serum levels of both T cell polarizing (IL-33 and IL-12) and T cell effector (IFN-γ, IL-4, IL-10, IL-17 and IL-9) cytokines in T1DM subjects with and without microvascular (retinopathy and nephropathy) complications (MVC). All the tested cytokines were significantly elevated in T1DM subjects except for IFN-γ (which failed to attain statistical significance) with no significant difference between those with and without MVC. From the serum cytokine analysis, no apparent Th polarization could be determined for the T1DM subjects.

In silico identification and wet lab validation of novel cryptic B cell epitopes in ZnT8 zinc transporter autoantigen.

Zinc transporter 8 (ZnT8) is a novel immunodominant autoantigen, associated with Type-1 diabetes. A non-synonymous polymorphism (R325W) in its gene is associated with Type-2 diabetes. In this study, we performed an in silico B cell epitope prediction followed by wetlab validation of ZnT8. Apart from the previously characterized polymorphic epitope (BE-5 TAASR*DS), two novel epitopes BE-2 (N-terminus) and BE-6 (C-terminus) were identified. Wet lab validation of these epitopes was carried out by measuring ZnT8 specific isotypes (IgG, IgM and IgA) in the sera of Normal Glucose Tolerant (NGT), Type-1 diabetic (T1DM) and Type-2 diabetic (T2DM) patients by indirect ELISA. Unexpectedly, compared to NGT, significantly decreased levels of IgG and IgA isotypes was seen in T1DM subjects without complications. IgM levels were reduced in T1DM subjects with retinopathy. Newly diagnosed T1DM subjects initiated on insulin therapy showed an increase in IgA and decrease in IgM titre. Like T1DM, significantly reduced level of IgG, IgM and IgA was seen in T2DM subjects. For the first time, we have identified novel cryptic B cell epitopes in ZnT8 autoantigen against which the naturally occurring autoantibody levels were found to be reduced in diabetes.