Table 1.
Patient cohorts and characteristics.
Figure 1.
Distribution of antigen presenting cells in peripheral blood during chronic HIV infection.
(A) Multicolor flow cytometric gating strategy for the identification of APC populations in peripheral blood. APCs (CD3-CD19-CD56-HLADR+CD11c+CD123-), monocytes (CD3-CD19-CD56-HLADR+CD123-CD11c+CD14+), and mDCs (CD3-CD19-CD56-HLADR+CD123-CD11c+CD14+) were detected following the exclusion of dead cells and CD3+, CD19+ and CD56+ cells. The HLA-DR-positive cells were gated for CD11c and CD123. Data were analyzed using FlowJo. (B) Percentages of circulating APCs (HIV− n = 37, HIV+ n = 94). (C) Monocyte percentage of the peripheral blood APC population. (D) Myeloid dendritic cell population of the peripheral blood APC population. (E) Plasma sCD14 concentrations as determined by ELISA (HIV− n = 25; HIV+ n = 66). Each dot represents an individual patient. In the HIV+ group, open circles represent therapy-naïve patients and closed circles represent patients on HAART. Bars indicate median value. P values determined using Mann Whitney U test. P values as indicated.
Figure 2.
Enhanced inflammatory response by APCs from HIV-infected patients to commensal lactobacilli.
(A) Representative flow cytometry plots of APCs producing proinflammatory cytokines in response to L. plantarum WCFS1. (B) Frequencies of APCs from HIV-negative controls (n = 29) and HIV-infected patients (n = 62) producing IL-6, IL-12/IL-23p40, and TNFα in response to L. plantarum WCFS1 determined by multicolor flow cytometry. (C) Concentrations of IL-6, IL-12/IL-23p40, and TNFα following stimulation with L. plantarum WCFS1 as determined by ELISA (HIV− n = 4; HIV+ n = 10). Frequencies of APCs from HIV-negative controls (n = 14) and HIV-infected patients (n = 23) producing IL-6, IL-12/IL-23p40, and TNFα in response to (D) L. gasseri 1SL4 and (E) L. casei BL23 as measured by multicolor flow cytometry. Each dot represents an individual subject. In the HIV+ group, open circles represent therapy-naïve patients, closed circles represent patients on HAART. Bars indicate median value. Bar graphs represent mean +/− SEM. P values determined using Mann Whitney U test, P values as indicated.
Figure 3.
Increased expression of pattern recognition receptors on APCs from HIV-infected patients.
(A) Heatmap and (B) fold changes of PRR gene expression in isolated CD11c+ APCs from HIV-infected patients were compared to HIV-negative controls (HIV− n = 4, HIV+ n = 4) using DNA microarray analysis. P values determined by unpaired t test (*P<0.05, **P<0.01, ***P<0.001). (C) Representative flow cytometry histograms of TLR2 expression of APCs from HIV-negative controls (black) and HIV+ patients (gray). (D) Median fluorescence intensity of TLR2 expression of APCs from HIV-negative controls (n = 20) and HIV-infected patients (n = 46). (E) Representative flow cytometry histograms of CD36 expression of APCs from HIV-negative controls (black) and HIV+ patients (gray). (F) Median fluorescence intensity of CD36 expression of APCs from HIV-negative controls (n = 20) and HIV-infected patients (n = 46). (G) Frequencies of APCs from HIV-infected patients (n = 7) producing IL-6, IL-12/IL23p40, and TNFα in response to L. plantarum WCFS1 with or without TLR2 blocking antibody. (H) Concentrations of IL-6, IL-12/IL-23p40, and TNFα following stimulation with L. plantarum WCFS1 with or without TLR2 blocking antibody as determined by ELISA (HIV+ n = 3–6). Each dot represents an individual subject. In the HIV+ group, open circles represent therapy-naïve patients, closed circles represent patients on HAART. Bars indicate median value. Bar graphs represent mean +/− SEM. P values determined using Mann Whitney U test or paired t test.
Figure 4.
Increased phosphorylation of p38-MAPK in mDCs and monocytes from HIV-infected patients following stimulation with L. plantarum.
(A) Representative flow cytometric gating strategy detecting phosphorylation of p38 in mDCs following stimulation with L. plantarum WCFS1. Myeloid dendritic cells were gated based on forward and side scatter followed by lineage-negative (CD3-CD14-CD16-CD19-CD20-CD56-), and were positive for both HLA-DR and CD11c. (B) Frequencies of mDCs phosphorylated p38 following stimulation with L. plantarum WCFS1 as determined by phosflow (HIV− n = 7; HIV+ n = 14). (C) Representative flow cytometric gating strategy detecting phosphorylation of p38 in monocytes following stimulation with L. plantarum WCFS1. Monocytes were gated based on forward and side scatter and were positive for the lineage marker, HLA-DR, and CD11c. (D) Frequencies of monocytes phosphorylated p38 following stimulation with L. plantarum WCFS1 as determined by phosflow (HIV− n = 7; HIV+ n = 14). Each dot represents an individual subject and bars indicate median. P values determined using Mann Whitney U test.
Figure 5.
Enhanced APC inflammatory response to commensal lactobacilli signals predominantly through p38-MAPK.
(A) Representative flow cytometry plot and frequencies of APCs from HIV-infected patients producing proinflammatory cytokines IL-6, IL-12/IL-23p40, and TNFα in response to (B) L. plantarum WCFS1 (n = 16), (C) L. gasseri 1SL4 (n = 12), and (D) L. casei BL23 (n = 11) with or without BIRB796 pretreatment. (E) Relative expression of MKP-1 in unstimulated and bacterial stimulated APCs as determined by real-time PCR (HIV− n = 3–9, HIV+ n = 5–16). Bar graphs represent mean +/− SEM. P values determined using paired t or Mann Whitney U test (*P<0.05, **P<0.01, ***P<0.001).