Regulatory T cell-mediated control mechanisms of inflammatory and immune responses to critical injury/sepsis in Diabetic vs. non-diabetic ICU patients

Project Summary:

Background: The incidence of Type-2 diabetes (T2D) is increasing all over the world as well as in the Kingdom of Saudi Arabia. T2D has emerged as an important risk factor for Intensive Care Unit (ICU) admissions. We previously showed that the immune response to acute illness in diabetics differed from that of non-diabetic critically-ill patients as plasma levels of sRAGE/NF-кB were significantly higher in diabetics than in non-diabetics whereas, plasma IL-6 levels were lower in diabetic than in non-diabetic patients. We speculate that a distinct subpopulation of CD4+ T cells called T regulatory cells (Tregs) may be involved in this suppression.

Objectives: The main objectives will be to: (i) determine T-helper versus Treg cell ratios; (ii) identify Treg subpopulations; (iii) characterize Th1/Th2/Regulatory phenotypes/cytokines in plasma/PBMC; (iv) assess Treg suppressive function; and (iv) elucidate the mechanistic role of TLR4-8 innate immune receptors.

Methodology: Peripheral blood will be collected from diabetic/non-diabetic septic shock patients at days 1, 3, 5, 7, and 14 of the ICU admission for separation of plasma and PBMC. RNA will also be collected from whole blood/PBMCs. T-helper subsets will be identified by surface phenotyping using flow cytometry and signature cytokines measured by multiplex bead array assays. Quantitative real-time RT-PCR will be used to determine cytokine mRNA expression. CD25+high/CD25+int Tregs will be isolated and purity will be assessed by FoxP3 staining. Resting and activated Tregs will be sorted using high-throughput multiplex Luminex platform. MDSC precursors will be expanded by in-vitro culturing and confirmed by staining for selection markers. Treg suppressive activity will be demonstrated by coculture assays. Commercial human apoptosis RT2 Profiler PCR array-based high-throughput assay will be used to measure the relative expression of 84 apoptosis-related genes in CD4+T effector cells. The data (mean±SE) will be analyzed using STATA version-12.1 software.

Conclusion/Significance: This study will help understand the differential inflammatory and immune mechanisms elicited against critical injury in diabetic and non-diabetic patients. The findings will also guide care/management of the two distinct types of septic shock patients in the ICU.