Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands - PubMed (original) (raw)
Dendritic cells from HIV-1 infected individuals are less responsive to toll-like receptor (TLR) ligands
Jeffrey A Martinson et al. Cell Immunol. 2007 Nov-Dec.
Abstract
We compared TLR responsiveness in PBMC from HIV-1-infected and uninfected individuals using the TLR agonists: TLR7 (3M-001), TLR8 (3M-002), and TLR7/8 (3M-011). Activation and maturation of plasmacytoid dendritic cells (pDC) were measured by evaluating CD86, CD40, and CD83 expression and myeloid dendritic cell (mDC) activation was measured by evaluating CD40 expression. All agonists tested induced activation and maturation of pDC in PBMC cultures of cells from HIV+ and HIV- individuals. The TLR7 agonist induced significantly less pDC maturation in cells from HIV+ individuals. Quantitative assessment of secreted IFN-alpha and pro-inflammatory cytokines at the single cell level showed that pDC from HIV+ individuals stimulated with TLR7 and TLR7/8 induced IFN-alpha. TLR8 and TLR7/8 agonists induced IL-12 and COX-2 expression in mDC from HIV+ and HIV- individuals. Understanding pDC and mDC activation and maturation in HIV-1 infection could lead to more rational development of immunotherapeutic strategies to stimulate the adaptive immune response to HIV-1.
Figures
Figure 1. pDC from HIV infected individuals are activated by TLR agonists
PBMC from 5 HIV+ and 5 HIV− individuals were incubated for 24 hours with TLR7, TLR8, TLR7/8, control cTLR7/8 agonists, or media alone. Flow cytometry was used to determine pDC expression of (a) CD86, (b) CD40 and (c) CD83. Results are expressed as the mean ± SEM of the percentage of pDC that express each cell surface marker. Statistical comparisons were made between the media, control and the corresponding TLR7, TLR8 or TLR7/8 agonist stimulated cultures using a Student’s t-test; values were considered significant if p<0.05. *Significantly different from HIV− control; **Significantly different from HIV+ control; •–• Significant difference between HIV− and HIV+ cultures.
Figure 2. TLR 8 agonist activates mDC from HIV infected individuals
PBMC from 5 HIV+ and 5 HIV− individuals were incubated for 24 hours with TLR7, TLR8, TLR7/8, control cTLR7/8 agonists, or media alone. Flow cytometry was used to determine the (a) percentage of CD40 positive mDC and (b) CD40 mean fluorescence intensity (MFI) on mDC. Results are expressed as the mean ± SEM of the percentage of mDC that express CD40 or mDC-CD40 MFI. Statistical comparisons were made between the media control and the corresponding TLR7, TLR8 or TLR7/8 agonist stimulated cultures using a Student’s t-test; values were considered significant if p<0.05. *Significantly different from HIV− control; **significantly different from HIV+ control; •–• significant difference between HIV− and HIV+ cultures.
Figure 3. TLR7, TLR8 and TLR7/8 agonists induce pro-inflammatory cytokine secretion
PBMC from 5 HIV+ and 5 HIV− individuals were incubated for 24 hours with a TLR7, TLR8, TLR7/8, control cTLR7/8 agonists or media alone. Culture supernatants were collected and the concentration of (a) IFN-α was measured by ELISA, while (b) IL-1β, TNF-α, IL-10 and IL-12p70 were measured by CBA. Results are expressed as the mean ± SEM concentration (pg/ml). Statistical comparisons were made between the media control and the corresponding TLR7, TLR8 or TLR7/8 agonist stimulated cultures using a Student’s t-test; values were considered significant if p<0.05. *Significantly different from HIV− control; **Significantly different from HIV+ control; •–• Significant difference between HIV− and HIV+ cultures.
Figure 4. HIV-1 and TLR agonists activate IFN-α production in pDC in the absence of costimulatory cytokines
PBMC were incubated for 20 hours with inactivated HIV-1 or synthetic TLR agonists in the presence of 5 ug/ml the protein transport inhibitor Brefeldin A (BFA). Intracellular accumulation of IFN-α was measured. (a) Representative flow cytometric analysis of the selective detection of intracellular IFN-α in pDC [logical gating was used to identify the pDC population from 1 HIV-1 infected individual (Lin1−/HLA DR+/CD123+)] following stimulation. (b) 12 HIV+ and 10 HIV− individuals were evaluated for intracellular accumulation of IFN-α following stimulation with TLR4, TLR7, TLR8, TLR7/8 and TLR9 agonists in the presence BFA. TLR4 was included as a negative control and TLR9 was included as a positive control. (c) 4 HIV+ and 10 HIV− individuals were evaluated for intracellular accumulation of IFN-α following stimulation with AT-2 inactivated HIV-1Ada (R5-tropic), AT-2 inactivated HIV-1MN (X4-tropic) and matched control microvesicles (MV-CCR5, MV-CEMX) in the presence of BFA. Results are expressed as the mean ± SEM of the percentage of pDC that expresses intracellular IFN-α. Statistical comparisons were made between the media control and the corresponding TLR agonist or AT-2 inactivated HIV-1 virus using a Student’s t-test; values were considered significant if p<0.05. *Significantly different from HIV− control; **significantly different from HIV+ control; •–• significant difference between HIV− and HIV+ cultures.
Figure 5. TLR agonists induce IL-12 and COX-2 in mDC in the absence of costimulatory cytokines
PBMC were incubated for 20 hours with inactivated HIV-1 or synthetic TLR agonists in the presence of 5 ug/ml the protein transport inhibitor Brefeldin A (BFA). Intracellular accumulation of IFN-α was measured. (a) Representative flow cytometric analysis of the selective detection of intracellular IL-12p40/p70 or COX-2 in mDC; [logical gating was used to identify the mDC population from 1 HIV infected individual (Lin1-/HLA DR+/CD11c+)] following stimulation. (b) 12 HIV+ and 10 uninfected individuals were evaluated for intracellular accumulation of IL-12 in mDC following stimulation with TLR4, TLR7, TLR8, TLR7/8 and TLR9 agonists in the presence BFA. TLR4 was included as a negative control and TLR9 was included as a positive control. (c) The same individuals were evaluated for intracellular accumulation of COX-2 in mDC. Results in b and c are expressed as the mean ± SEM percentage of mDC expressing the intracellular marker. Statistical comparisons were made between the media control and the corresponding TLR agonist using a Student’s t-test; values were considered significant if p<0.05. *Significantly different from HIV− control; **significantly different from HIV+ control; •–• significant difference between HIV− and HIV+ cultures.
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