HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-κB Pathway

Abstract

We recently reported that the human immunodeficiency virus type-1 (HIV-1) Tat protein induced the expression of programmed death ligand-1 (PD-L1) on dendritic cells (DCs) through a TLR4 pathway. However, the underlying mechanisms by which HIV-1 Tat protein induces the abnormal hyper-activation of the immune system seen in HIV-1 infected patients remain to be fully elucidated. In the present study, we report that HIV-1 Tat protein induced the production of significant amounts of the pro-inflammatory IL-6 and IL-8 cytokines by DCs and monocytes from both healthy and HIV-1 infected patients. Such production was abrogated in the presence of anti-TLR4 blocking antibodies or soluble recombinant TLR4-MD2 as a decoy receptor, suggesting TLR4 was recruited by Tat protein. Tat-induced murine IL-6 and CXCL1/KC a functional homologue of human IL-8 was abolished in peritoneal macrophages derived from TLR4 KO but not from Wt mice, confirming the involvement of the TLR4 pathway. Furthermore, the recruitment of TLR4-MD2-CD14 complex by Tat protein was demonstrated by the activation of TLR4 downstream pathways including NF-κB and SOCS-1 and by down-modulation of cell surface TLR4 by endocytosis in dynamin and lipid-raft-dependent manners. Collectively, these findings demonstrate, for the first time, that HIV-1 Tat interacts with TLR4-MD2-CD14 complex and activates the NF-κB pathway, leading to overproduction of IL-6 and IL-8 pro-inflammatory cytokines by myeloid cells from both healthy and HIV-1 infected patients. This study reveals a novel mechanism by which HIV-1, via its early expressed Tat protein, hijacks the TLR4 pathway, hence establishing abnormal hyper-activation of the immune system.

HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes

“…4B). The latter result is in agreement with our previous data showing that the Tat 1-45 mutant was also able to stimulate the TLR4 pathway (21,22). The degradation of MAL/ TIRAP following Tat stimulation seems to be related to its proteasomal degradation.…”

“…In previous work, we showed that Tat activates NF-B in a TLR4-dependent manner (22). Downstream of TLR4 activation, MyD88 and MAL/TIRAP work together to mediate the early activation of NF-B, while the TRIF pathway is involved in the late phase of NF-B activation.…”

“…TLR4 knockout (KO), MyD88 KO, TRIF KO, and TIRAP/MAL KO mice with the C57BL/6 strain background (TLR4 Ϫ/Ϫ , MyD88 Ϫ/Ϫ , TRIF Ϫ/Ϫ , and TIRAP Ϫ/Ϫ mice) were a generous gift from B. Ryffel (Laboratory of Molecular Immunology and Embryology, CNRS, Orléans, France). Primary macrophages were isolated as previously described (22). Briefly, mice were injected intraperitoneally with 1 ml of 3% thioglycolate medium (bioMérieux).…”

“…HEK TLR4/ MD2-CD14 cells were seeded into 24-well plates at 4 ϫ 10 5 cells/well the day before transfection. After 24 h, the cells (60 to 70% confluence) were cotransfected with pORF-LacZ (Invivogen) and an NF-B reporter plasmid (p-Nifty2-Seap; Invivogen) together with the indicated amounts of plasmid encoding small interfering RNA (siRNA) targeting a specific gene of interest (control, TLR4, TLR2, MyD88, or TIRAP) by use of a calcium phosphate transfection system as described previously (22). After 24 h of transfection, cells were washed with PBS and stimulated with Tat or LPS or left untreated.…”

“…In addition to RNA and viral proteins, HIV-1 DNA produced by reverse transcription can also be sensed, at least by cGAS (19) and IFI16 (20), which, like TLRs, can initiate NLRP inflammasome signaling, thus underlying the cross talk and the redundancy of innate immunity sensing. More recently, our group has shown that by interacting with high affinity with TLR4/MD2, the HIV-1 Tat protein stimulates the production of TNF-␣ and IL-10 (21) in addition to other inflammatory cytokines (22)(23)(24) and the positive modulation of immunosuppressive factors, including IDO (23) and PD-L1 (24), by dendritic cells.…”