Monocyte production of pro-inflammatory cytokines in the basal state and upon stimulation.

<div><p>HIV-infected subjects are shown in red circles and HIV-uninfected subjects are shown in blue triangles. In the no stimulation condition (basal state), HIV-infected subjects showed higher levels of IL-1β (upper left panel) and IL-8 (lower left panel). Upon stimulation with either oxLDL or LPS, HIV-infected subjects exhibited higher levels of IL-1β, IL-8 and IL-6 (upper right panel) compared to the HIV-uninfected subjects. While HIV-1 infected subjects did tend to have higher TNF (lower right panel) responses upon stimulation, these differences were not significant.</p> <p>**** p < 0.0001, *** p < 0.001, ** p < 0.01.</p></div

Per cell expression levels of IL-1β and TNF by each monocyte subset before and after stimulation.

<div><p>Geometric mean fluorescence (GMF) intensities are shown by monocyte subset for IL-1β (left column) and TNF (right column) in the basal (top row) state, and after oxLDL (middle row) or LPS (bottom row) stimulation. In HIV-infected subjects, IL-1β was expressed at highest levels by the Mono1 (CD14++CD16-) and Mono2 (CD14++CD16+) populations. TNF expression was observed in all subsets after stimuli, with the brightest monocyte subsets being Mono2 (CD14++CD16+) and Mono3 (CD14+ CD16+). Statistical significance was adjusted for multiple comparisons (p = 0.05/4 comparisons per stimulation condition: p = 0.0125).</p> <p>**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.0125.</p></div

Gating strategy for identification of total monocytes, monocyte subsets and detection of cytokine expression.

<p>Gating Strategy. Panel A. Identification of total monocytes from peripheral blood mononuclear cells by exclusion of doublets, dead cells, CD3, CD56, CD19, CD20 and low HLA-DR expressing cells. Double-negative CD14-CD16- cells were also excluded. Forward scatter (FSC) vs. side scatter (SSC) plots are shown comparing total PBMC (doublet and dead cells excluded) and total monocytes. Panel B. Monocyte subsets were identified based on the expression of CD14 and CD16 (left column). The diagram at the bottom of the left column is a visual guide for the terminology of monocyte subsets employed in this report. Intracellular cytokines (IL-1β, IL-8/CXCL8, IL-6 and TNF) produced in total monocytes were detected in response to no stimulus, oxidized low density lipoprotein (oxLDL) or lipopolysaccharide (LPS). Fluorescence minus one control condition, in which the antibody conjugate in question is omitted to guide creation of the gate that defines positive expression of that target, is shown on the bottom row. The subject presented is HIV-infected and displays high but representative responses to stimuli.</p

HIV-1 infected subjects with well-controlled viremia have a greater proportion of classical (Mono1, CD14++CD16-) monocytes and a lower frequency of intermediate (Mono2, CD14++CD16+) monocytes, which correlates with production of IL-1β.

<p>HIV-infected subjects are shown in red circles, and HIV-uninfected subjects are shown in blue triangles. Panel A. HIV-infected subjects had a greater fraction of monocytes that fall into the Mono1 (CD14++CD16- classical) subset, and a lower fraction that fall into the Mono2 (CD14++CD16+ intermediate) subsets. Upper right diagram represents monocyte gating scheme (Mono 1-4). Panel B. A lower proportion of monocytes in the Mono2 subset was associated with higher basal IL-1β production. **** p < 0.0001, *** p < 0.001,.</p