Representative flow cytometric analysis of peripheral blood CD19 positive B cells (A), IL-21R versus TLR4 on B cells (B).

<p>The numbers represent percentage of cells within the gates/quadrants. At least 10⁶ total cells were acquired followed by gating on size versus granularity followed by exclusion of dead cells and finally detection of markers described in plots.</p

Reduction of Specific Circulating Lymphocyte Populations with Metabolic Risk Factors in Patients at Risk to Develop Type 2 Diabetes

<div><p>Low-grade inflammation, characterized by increased pro-inflammatory cytokine levels, is present in patients with obesity-linked insulin resistance, hyperglycemia and hyperlipidemia and considered to play a leading role to progression into type 2 diabetes (T2D). In adipose tissue in obese patients and in pancreatic islets in T2D patients cellular inflammation is present. However, the systemic leukocyte compartment and the circulating endothelial/precursor compartment in patients at risk to develop T2D has so far not been analyzed in detail. To address this, peripheral blood cells from a cohort of 20 subjects at risk to develop diabetes with normal to impaired glucose tolerance were analyzed by flow cytometry using a wide range of cellular markers and correlated to known metabolic risk factors for T2D i.e. fasting plasma glucose (FPG), 2 h plasma glucose (2 h PG), HbA1c, body mass index (BMI), homeostasis model assessment of β-cell function (HOMA-B), homeostasis model assessment of insulin sensitivity (HOMA-IS) and fasting insulin (FI). The four highest ranked cell markers for each risk factor were identified by random forest analysis. In the cohort, a significant negative correlation between the number of TLR4⁺ CD4 T cells and increased FPG was demonstrated. Similarly, with increased BMI the frequency of TLR4⁺ B cells was significantly decreased, as was the frequency of IL-21R⁺ CD4 T cells. Unlinked to metabolic risk factors, the frequency of regulatory T cells was reduced and TLR4⁺ CD4 T cells were increased with age. Taken together, in this small cohort of subjects at risk to develop T2D, a modulation of the circulating immune cell pool was demonstrated to correlate with risk factors like FPG and BMI. This may provide novel insights into the inflammatory mechanisms involved in the progression to diabetes in subjects at risk.</p></div

Frequency of TLR4⁺ T cells in peripheral blood before and 75 min post meal (A).

<p>Cytokine production in CD4⁺ T cells in peripheral blood before and after activation of the cells for 4 h <i>ex vivo</i>: IL-4 (B), IFN-γ (C), IL-17 (D), IL-21 (E). Frequency of CECs (F) and CPCs (G) in peripheral blood before and 75 min post meal. Statistics was obtained using unpaired two-way T-test using Welsh correction.</p

Monocytes in peripheral blood. Data is displayed as mean ± standard deviation.

<p>Monocytes in peripheral blood. Data is displayed as mean ± standard deviation.</p

Representative flow cytometric analysis of peripheral blood CD3⁺CD4⁺CXCR5⁺ICOS⁺ Tfh cells (A), CD3⁺CD4⁺CD25⁺CD127⁻Foxp3⁺ Treg cells (B), CD3⁺CD4⁺IL-21R⁺ T cells (C) and CD3⁺CD4⁺TLR4⁺ T cells (D).

<p>The numbers represent percentage of cells within the gates. At least 10⁶ total cells were acquired followed by gating on size versus granularity followed by exclusion of dead cells and finally detection of markers described in plots.</p

Circulating endothelial and endothelial precursor cells.

<p>Data is displayed as mean ± standard deviation.</p><p>Circulating endothelial and endothelial precursor cells.</p

Representative flow cytometric analysis of peripheral blood CD68 positive monocytes (A), M1-like CD163^−/intCD11c^high monocytes and M2-like CD163^int/hiCD11c^int monocytes (B), IL-21R⁺ monocytes and TLR4⁺ monocytes (C).

<p>The numbers represent percentage of cells within the gates. At least 10⁶ total cells were acquired followed by gating on size versus granularity followed by exclusion of dead cells and finally detection of markers described in plots.</p

Age (years) vs after meal frequency of CD3⁺CD4⁺CD25⁺CD127⁻ T cells (A), after meal frequency of CD3⁺CD4⁺IL21R⁻TLR4⁺ T cells (B), before meal frequency of CD3⁺CD4⁺CD25⁺CD127⁻ T cells (C) and before meal frequency of CD68⁺ macrophages (D).

<p>Red dots represent women and blue dots represent men. Distribution of men and women separately vs after meal frequency of CD31⁺CD34⁺CD45^dimCD133^dim CPCs (E), before meal frequency of CD31⁺CD34⁺CD45^dimCD133^dim CPCs (F), before meal number of resting IL-21⁺ T cells (G) and after meal frequency of CD19⁺IL21R⁻TLR4⁺ B cells (H). Red bars represent women and blue dots represent men. (A and B * = significant after adjustment for multiple testing).</p

Representative flow cytometric analysis of peripheral blood, CD45 versus CD31 (A), CD31⁺CD34⁺CD45^dimCD133^dim CPC cells (B) and CD31^brightCD34⁻CD45⁻CD133⁻ CEC cells (C).

<p>The numbers represent percentage of cells within the gates. At least 10⁶ total cells were acquired followed by gating on size versus granularity followed by exclusion of dead cells and finally detection of markers described in plots.</p

FPG (mmol/l) vs before meal frequency of CD3⁺CD4⁺IL21R⁻TLR4⁺ T cells (A), before meal frequency of CD3⁺CD4⁺IL21R⁺TLR4⁻ T cells (B), before meal number of CD3⁺CD4⁺IL21R⁻TLR4⁺ T cells (C) and before meal frequency of CD19⁺IL21R⁻TLR4⁺ B cells (D).

<p>BMI vs after meal frequency of CD19⁺IL21R⁻TLR4⁺ B cells (* = significant after adjustment for multiple testing) (E), before meal frequency of CD3⁺CD4⁺IL21R⁺TLR4⁻ T cells (F), before meal frequency of activated IFN-γ⁺ T cells (G) and before meal number of CD3⁺CD4⁺CD25⁺CD127⁻ T cells (H). Red dots represent women and blue dots represent men.</p