TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

Abstract

Toll-like receptors (TLRs) play a crucial role in innate immunity and provide a first line of host defense against invading pathogens. Of the identified human TLRs, TLR10 remains an orphan receptor whose ligands and functions are poorly understood. In the present study, we sought to evaluate the level of TLR10 expression in breast milk (BM) and explore its potential function in the context of HIV-1 infection. We evaluated HIV-1-infected (Nigerian: n = 40) and uninfected (Nigerian: n = 27; Canadian: n = 15) BM samples for TLR expression (i.e., TLR10, TLR2, and TLR1) and report here that HIV-1-infected BM from Nigerian women showed significantly higher levels of TLR10, TLR1, and TLR2 expression. Moreover, the level of TLR10 expression in HIV-1-infected BM was upregulated by over 100-fold compared to that from uninfected control women. In vitro studies using TZMbl cells demonstrated that TLR10 overexpression contributes to higher HIV-1 infection and proviral DNA integration. Conversely, TLR10 inhibition significantly decreased HIV-1 infection. Notably, HIV-1 gp41 was recognized as a TLR10 ligand, leading to the induction of IL-8 and NF-κBα activation. The identification of a TLR10 ligand and its involvement in HIV-1 infection enhances our current understanding of HIV-1 replication and may assist in the development of improved future therapeutic strategies.

Keywords: HIV-1; IL-8; NF-κBα; TLR10; gp41; human breast milk; p17; p24.

Figure 1

Flow cytometry analyses of TLR1, TLR2, and TLR10 expression in the two major types of primary BM cells obtained from HIV-1 uninfected women at 3 months post-partum. Leucocytes were gated with CD45-PerCP-Cy5.5 and CD14-V450 whereas the epithelial cells were gated with MUC1-PE-Cy7 and CD45-. The antibodies TLR1-APC TLR2-APC and TLR10-PE were separately used to detect TLR1, TLR2, and TLR10 in CD14⁺-CD45⁺-MUC1⁻ and MUC1⁺-CD45⁻ cell types to differentiate the two major constituents of BM. Representative images are shown with TLR1, TLR2, and TLR10 expression depicted as percentages on right corner of each image.

Figure 2

Significantly elevated expression level of TLR1 and TLR10 in HIV-1 infected human primary BM cells. Expression of TLR1 and TLR10 as measured by qRT-PCR with the mRNA extracted from primary BM cells collected from HIV-1 negative Hamilton, Canada women (HIV-N Hamilton) and Nigerian HIV-1 negative (HIV-N) and HIV-1 positive (HIV-P) women. TLR1 expression is shown on right (p = 0.0006) whereas TLR10 expression is shown on left (p < 0.0001).

Figure 3

Overexpression or siRNA mediated knockdown of TLR10 significantly alters HIV-1 infection and integration (A) HIV-1 infection was significantly enhanced in HIV-1 reporter TZMbl cells transiently overexpressing TLR10 alone and co-transfected with TLR2 or TLR1 expression plasmids by measuring luciferase activity in relative light units (RLU). (B) HIV-1 integration was significantly increased in stable TZMbl reporter cells overexpressing TLR10, TLR2, and TLR1. TZMbl, TLR2- stable, and TLR10-stable cells were used for co-transfection with plasmids: empty vector, TLR2, TLR10, and TLR1 vector, TLR10 and TLR1 vector, and TLR2 and TLR1 vector. Proviral DNA (DNA pol) was detected by PCR and normalized to the 18S rRNA gene. (C) Proviral DNA was obviously decreased in macrophages with TLR10 knocked down prior to HIV-1 infection. T20: Enfuvirtide, an HIV-1 fusion inhibitor used as a negative control. Data set is representative of three different experiments completed in triplicate (Statistic marks in the plots: ^*p < 0.05, ^**p < 0.01 for Mann Whitney t-tests, each group compared to the vector group in A and B, or to the control group in C; pair comparison ∧, # in B, respectively).

Figure 4

Effects of PAMPs and HIV-1 on the level of TLR expression in cell cultures. (A) qRT-PCR of TLR10, TLR2, and TLR1 mRNA from MCF-10A cells treated with TLR2 PAMP Pam3CSK4, TLR4 PAMP LPS, TLR7/TLR8 PAMP ssRNA40, and cell-free HIV-1 BAL. (B) q-RT-PCR in THP-1 cells. (C) qRT-PCR in primary BM cells. Data set is representative of three different experiments completed in triplicate. Statistic marks: ^*p < 0.05, ^**p < 0.01 for the Mann-Whitney t-tests, each group compared to medium-treated group (Medium).

Figure 5

Effects of HIV-1 proteins on TLR expression and cellular responses. (A) MCF-10A cells were treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, gp41, and gp120. Cellular TLR10, TLR2, and TLR1 mRNAs were analyzed by qRT-PCR. (B) IL-8 production in cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins were analyzed by ELISA. (C) The expression of TLR mRNAs analyzed by qRT-PCR in THP-1 cells treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, and gp41. (D) IL-8 production in THP-1 cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins was analyzed by ELISA. Data set is representative of three different experiments completed in triplicate. Statistic marks: ^*p < 0.05, ^**p < 0.01 for the Mann-Whitney t-tests, each group was compared to the medium-treated group (Medium).

Figure 6

HIV-1 proteins, p17 and gp41 elicit cellular responses through sensing TLR10, TLR2, and TLR1. IL-8 production stimulated by Pam3CSK4, p17, p24, and gp41 was greatly suppressed in THP-1 cells knocked-down of TLR10 with specific siRNA (A) and THP-1 cells neutralized with anti-TLR10 antibody (B). (C) IL-8 stimulated by Pam3CSK4 and gp41 was reduced in MCF-10A cells neutralized with an anti-TLR10 antibody. (D) Primary BM cells displayed a significant decrease in IL-8 production induced by HIV-1 gp41 following neutralization with an anti-TLR10 antibody. Data set is representative of three different experiments completed in triplicate. Statistic marks: ^*p < 0.05; ^**p < 0.01 for Mann Whitney t-tests, with each group compared to the medium treated group (Medium).

Figure 7

HIV-1 proteins lead to NF-κBα activation (A) Western blot analysis of the cell lysates extracted from MCF-10A, THP-1, and primary BM cells treated with HIV-1 proteins and detected with an antibody against the active NF-κBα subunit, phospho(P)-IκBα, with anti β-actin used as the loading control on the same membranes (B) Confocal microscopy of HIV-treated MCF-10A and THP-1 cells fixed and stained with an anti- NF-κBα subunit p65 antibody and visualized with a second antibody, anti-rabbit-IgG-alexa488 (GFP; green), p65 was translocated upon activation into the nuclei (RFP; red with propidium iodide staining) displayed in yellow (Merged).

Figure 8

siRNA mediated knockdown or antibody mediated blocking of TLR10 inhibits NF-κBα activation induced by HIV-1 proteins (A) anti-TLR10 antibody decreased gp41 induced P-IκBα by half in MCF-10A cells. (B) TLR10 siRNA ablated the induction of P-IκBα by gp41 in THP-1 cells.