Plasmacytoid dendritic cell number and responses to Toll-like receptor 7 and 9 agonists vary in HIV Type 1-infected individuals in relation to clinical state

Abstract

In HIV-1 infection, plasmacytoid dendritic cell (PDC) numbers and function are decreased. No detailed comparisons of PDC responses to various stimuli in HIV-1-infected patients are available. Using for the first time purified PDCs, we compared PDC responses [interferon (IFN)-α production/cell] to various stimuli in a large number (n=48) of HIV-1-infected patients and healthy volunteers (n=19). Toll-like receptor (TLR)7- and TLR9-induced expression of PDC surface activation and maturation markers was also compared in the two populations. We have confirmed that PDC number coincides with CD4(+) T cell counts and clinical state. Notably, we have shown that a direct association of PDC function in terms of IFN-α production/cell exists with PDC numbers and CD4(+) cell counts when PDCs are exposed to a TLR9 ligand and HIV-infected cells, but not with a TLR7 ligand. Moreover, in the HIV-infected subjects but not the healthy controls, the magnitude of IFN-α release per PDC in response to the TLR7 ligand is significantly (p<0.01) lower than that to the TLR9 ligand. However, in both study populations, the TLR7 stimulation in comparison to TLR9 stimulation induced higher expression of PDC surface activation and maturation markers and significantly (p<0.05) decreased the expression of BDCA-2, a negative regulator of interferon. Furthermore, the cross-ligation of BDCA-2 significantly (p<0.05) inhibited TLR9- but not TLR7-induced IFN-α production by PDCs from both clinical groups. These findings suggest that differences exist in TLR7- and TLR9-induced IFN-α production by PDCs in HIV-infected individuals that are not directly related to BDCA-2 down-modulation.

FIG. 1.

Correlation of CD4⁺ T cells with plasmacytoid dendritic cell (PDC) numbers. (A) CD4⁺ T cell counts as well as percentages correlate positively with PDC numbers in HIV-infected individuals (n=48). The trend in these associations remained true whether (B) the study group (n=24) did not receive antiretroviral treatment or (C) the study group (n=24) received antiretroviral treatment. Spearman's rank correlation coefficients (r) are shown where an asterisk (*) denotes level of significance (p<0.05).

FIG. 2.

Correlation of viral load with PDC numbers. (A) HIV-1 RNA (copies/ml) correlates inversely with PDC numbers in HIV-infected individuals (n=48). The trend in this association remained true whether (B) the study group (n=24) did not receive antiretroviral treatment or (C) the study group (n=24) received antiretroviral treatment. Spearman's rank correlation coefficients (r) are shown where an asterisk (*) denotes level of significance (p<0.05).

FIG. 3.

Correlation of PDC function with PDC numbers. (A) Interferon (IFN)-α (pg/ml) per 10⁴ PDC induced by Toll-like receptor (TLR)9 agonist (CpG-A) but not by TLR7 agonist (S-27609) correlated positively with PDC counts in HIV-1-infected individuals (n=48). The trend in these associations remained the same whether (B) the study group (n=24) did not receive antiretroviral treatment or (C) the study group (n=24) received antiretroviral treatment. Spearman's rank correlation coefficients (r) are shown where an asterisk (*) denotes level of significance (p<0.05).

FIG. 4.

PDCs from HIV-infected individuals differ in their responses to TLR7 and TLR9 agonists. In HIV-1-infected individuals but not the healthy controls, IFN-α (pg/ml) production per 10⁴ PDCs induced by the TLR9 agonist (CpG-A) was significantly (p<0.001) higher than that observed with both the TLR7 agonist (S-27609) and HIV-infected CD4⁺ T cells. The Wilcoxon signed-rank test was used to determine the differences between IFN-α (pg/ml) induced by two agonists within each study population where an asterisk (*) denotes level of significance (p<0.01). The bars represent the median values. In six healthy controls, the IFN-α (pg/ml) levels in response to TLR7 stimulation were low with two controls having no detectable IFN-α (pg/ml) production and four controls having less than 30 pg/ml IFN-α levels.

FIG. 5.

Clinical state does not influence PDC responses. Differences in IFN-α (pg/ml) induced in 10⁴ PDC by TLR7 and TLR9 agonists remained the same within the groups of HIV-infected individuals based on median CD4⁺ T cell counts (515 cells/μl of blood) or median PDC numbers (5.9 cells/μl of blood). The Wilcoxon signed-rank test was used to determine the differences between IFN-α (pg/ml) induced by two agonists where an asterisk (*) denotes level of significance (p<0.05).

FIG. 6.

Differences in TLR7- and TLR9-induced IFN-α is not limited to a particular IFN-α subtype. The ratio of TLR7 (S-27609)-induced to TLR9 (CpG-A)-induced IFN-α in HIV-1-infected individuals is not significantly different (p=0.55) when measured by single or multisubtype IFN-α ELISA.

FIG. 7.

Expression of PDC surface markers in HIV-infected individuals and healthy controls. No significant differences were observed among HIV-infected individuals (n=10) and healthy controls (n=8) in the expression of surface markers on PDC stimulated with the (A) TLR9 agonist and (B) TLR7 agonist or (C) left unstimulated. The Mann–Whitney test was used to compare the medians of the expression of surface markers between both the study populations.

FIG. 8.

Differences in expression of surface markers on PDC stimulated with TLR9 and TLR7 agonists. Significant differences were observed in the expression of PDC surface markers for activation (CD80, CD86), chemokine receptor (CCR7), but not maturation marker (CD83) on PDC stimulated with TLR9 and TLR7 agonists in both (A) healthy controls and (B) HIV-infected individuals. The Wilcoxon signed-rank test was used to compare the medians where an asterisk (*) denotes level of significance (p<0.05).

FIG. 9.

BDCA-2 down-modulation and IFN-α production by TLR9 and TLR7 agonists. Anti-BDCA-2 monoclonal antibodies (mAbs) significantly inhibit IFN-α production by the (A) TLR9 agonist but not the (B) TLR7 agonist in both HIV-infected individuals and healthy controls. The observations remained the same when single IFN-αA or multisubtype ELISA was used to measure IFN-α production. A one-way ANOVA test was used to compare the means of three groups within each study population where an asterisk (*) denotes level of significance (p<0.05).