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. 2013 Oct 23;27(16):2505-17.
doi: 10.1097/01.aids.0000432455.06476.bc.

HIV-1 infection induces strong production of IP-10 through TLR7/9-dependent pathways

Rachel P Simmons  1 , Eileen P Scully, Erin E Groden, Kelly B Arnold, J Judy Chang, Kim Lane, Jeff Lifson, Eric Rosenberg, Douglas A Lauffenburger, Marcus Altfeld
Affiliations

HIV-1 infection induces strong production of IP-10 through TLR7/9-dependent pathways

Rachel P Simmons et al. AIDS. .

Abstract

Objective: To study the cytokine/chemokine profiles in response to HIV-1 viremia, and elucidate the pathways leading to HIV-1-induced inflammation.

Design/methods: Plasma levels of 19 cytokines in individuals with early HIV-1 infection and individuals undergoing treatment interruptions were evaluated via multiplex assay. To investigate the cellular sources of relevant cytokines, sorted cells from HIV-1 infected individuals were assessed for mRNA expression. Relevant signaling pathways were assessed by comparing cytokine production patterns of peripheral blood mononuclear cells stimulated with intact HIV-1 or specific Toll-like receptor (TLR) stimulants with and without a TLR7/9 antagonist.

Results: IP-10 plasma concentration was most significantly associated with HIV-1 viral load and was the most significant contributor in a multivariate model. IP-10 mRNA was highly expressed in monocytes and mDCs and these cells were the dominant producers after in-vitro stimulation with TLR7/8 ligands (CL097 and ssRNAGag1166), AT-2 HIV-1, and HIV-1NL43 virus. Partial least square discriminant analysis of culture supernatants revealed distinct cytokine/chemokine secretion profiles associated with intact viruses compared with TLR7/8 ligands alone, with IP-10 production linked to the former. A TLR7/9 antagonist blocked IP-10 production following whole virus stimulation, suggesting the involvement of TLR7/9 in the recognition of HIV-1 by these cells.

Conclusion: Monocytes and mDCs produce significant amounts of IP-10 in response to HIV-1 viremia and after in-vitro stimulation with HIV-1. Stimulation with HIV-1-derived TLR7/8-ligands versus HIV-1 resulted in distinct cytokine/chemokine profiles, indicating additional pathways other than TLR7/8 that lead to the activation of innate immune cells by HIV-1.

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Figures

Figure 1
Figure 1. Relationship between plasma cytokine and chemokine levels and viral load
A) Relationship between plasma cytokine and chemokine levels and viral load following all STIs. The relationship between fold change in cytokine or chemokine level and log viral load was determined by linear regression for each analyte. Data from all treatment interruptions was combined. Baseline cytokine or chemokine level was defined as the average of 3 levels just prior to stopping ART. P values were considered significant (*) if less than 0.0026 using a Bonferroni correction for multiple comparisons. B) PLSR analysis of multivariate relationships between cytokine levels and plasma viral load. Analysis of all variables tested resulted in a model predictive of viral load, with relative contributions of various cytokines as indicated by a higher VIP score. In both early infection (left panel) and the STI groups, IP-10 contributed significantly to the model predicting viral load. C) Plasma IP-10 levels are significantly related to CD8+ T cell activation. Matched PBMC samples (identical or within one week of plasma samples) were analyzed for levels of CD3+CD8+ T cell activation as indicated by expression of CD38 and HLADR. P value shown for linear regression analysis.
Figure 1
Figure 1. Relationship between plasma cytokine and chemokine levels and viral load
A) Relationship between plasma cytokine and chemokine levels and viral load following all STIs. The relationship between fold change in cytokine or chemokine level and log viral load was determined by linear regression for each analyte. Data from all treatment interruptions was combined. Baseline cytokine or chemokine level was defined as the average of 3 levels just prior to stopping ART. P values were considered significant (*) if less than 0.0026 using a Bonferroni correction for multiple comparisons. B) PLSR analysis of multivariate relationships between cytokine levels and plasma viral load. Analysis of all variables tested resulted in a model predictive of viral load, with relative contributions of various cytokines as indicated by a higher VIP score. In both early infection (left panel) and the STI groups, IP-10 contributed significantly to the model predicting viral load. C) Plasma IP-10 levels are significantly related to CD8+ T cell activation. Matched PBMC samples (identical or within one week of plasma samples) were analyzed for levels of CD3+CD8+ T cell activation as indicated by expression of CD38 and HLADR. P value shown for linear regression analysis.
Figure 2
Figure 2. Cellular sources of IP-10
A) IP-10 mRNA expression in sorted cell populations from individuals with early HIV-1 infection. IP-10 mRNA expression relative to beta actin was determined for each cell population. Box plots show 25th to 75th percentile with whiskers to the minimum and maximum and bisecting line at the median. Data was analyzed with nonparametric analysis of variance using the Friedman’s test with a p<0.0001 across the groups. When controlled for multiple comparisons with the Dunn’s test, the levels differed significantly between T cells and mDCs and monocytes and B cells and Monocytes. B) Monocyte IP-10 mRNA levels from patient PBMCs are related to both plasma IP-10 level and log viral load. Relative expression of IP-10 mRNA was compared by linear regression to plasma IP-10 levels (left panel) and log viral load (right panel) from matched samples. C) Assessment of monocyte and mDC production of IP-10 in response to HIV-1. Proportion of monocytes (top row,) and mDCs (second row) that produce IP-10 quantified by intracellular cytokine staining following PBMC stimulation with media (negative, gray shaded histogram) or AT-2 HIV-1 or HIV-1NL43.(blue open histograms). Gating strategies as detailed in the methods section, these plots are representative experiments. D) In)vitro IP-10 production from different cell types and geometric mean fluorescence intensity (MFI) of IP-10 producing cells. Proportion of monocytes, mDCs, pDCs, B cells, CD4+ T cells, CD8+ T cells, and NK cells that produce IP-10 quantified by intracellular cytokine staining following PBMC stimulation with AT-2 HIV-1 or HIV-1NL43. (left panel). MFI of the IP-10 producing cells from the 3 populations with the most significant production (right panel). Values are reported following background subtraction and represent the average of 5 experiments.
Figure 2
Figure 2. Cellular sources of IP-10
A) IP-10 mRNA expression in sorted cell populations from individuals with early HIV-1 infection. IP-10 mRNA expression relative to beta actin was determined for each cell population. Box plots show 25th to 75th percentile with whiskers to the minimum and maximum and bisecting line at the median. Data was analyzed with nonparametric analysis of variance using the Friedman’s test with a p<0.0001 across the groups. When controlled for multiple comparisons with the Dunn’s test, the levels differed significantly between T cells and mDCs and monocytes and B cells and Monocytes. B) Monocyte IP-10 mRNA levels from patient PBMCs are related to both plasma IP-10 level and log viral load. Relative expression of IP-10 mRNA was compared by linear regression to plasma IP-10 levels (left panel) and log viral load (right panel) from matched samples. C) Assessment of monocyte and mDC production of IP-10 in response to HIV-1. Proportion of monocytes (top row,) and mDCs (second row) that produce IP-10 quantified by intracellular cytokine staining following PBMC stimulation with media (negative, gray shaded histogram) or AT-2 HIV-1 or HIV-1NL43.(blue open histograms). Gating strategies as detailed in the methods section, these plots are representative experiments. D) In)vitro IP-10 production from different cell types and geometric mean fluorescence intensity (MFI) of IP-10 producing cells. Proportion of monocytes, mDCs, pDCs, B cells, CD4+ T cells, CD8+ T cells, and NK cells that produce IP-10 quantified by intracellular cytokine staining following PBMC stimulation with AT-2 HIV-1 or HIV-1NL43. (left panel). MFI of the IP-10 producing cells from the 3 populations with the most significant production (right panel). Values are reported following background subtraction and represent the average of 5 experiments.
Figure 3
Figure 3. Partial least square discriminant analysis of cytokine profiles following stimulation with TLR Ligands and HIV-1
A PLSDA score plot (left) indicates 19 cytokine secretion measurements differentiate between TLR (CL097 and ssRNAGag1166) and HIV-1(AT-2 HIV-1 or HIV-1NL43) stimuli (separated by LV1) and also between AT-2 HIV-1 and HIV-1 strain NL-43 stimuli (separated by LV2). A loadings plot (right) illustrates that HIV stimuli (positive scores on LV1) are associated with a profile involving IP-10, IL-2, IL-4, IL-13, and IL-5 compared to TLR stimuli. LV2 (not shown) distinguishes between AT-2 and NL-43 HIV strains.
Figure 4
Figure 4. Assessment of monocyte, mDC, and pDC production of TNFα or IP-10 with and without pre-treatment with a TLR7/9 antagonist
Proportion of monocytes, mDCs, and pDCs that produce TNFα or IP-10 quantified by intracellular cytokine staining in response to CL097, AT-2 HIV-1 or JRCSF with (closed squares) or without (open circles) pre-treatment with a TLR7/9 antagonist. The horizontal lines are the medians. Data are reported following background subtraction. The stated p values were calculated using Mann-Whitney test for non-parametric data. P values were considered significant (*) if less than 0.05.
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