MHC class II molecules enhance Toll-like receptor mediated innate immune responses

Abstract

Background: Major histocompatibility complex (MHC) class II molecules play crucial roles in immune activation by presenting foreign peptides to antigen-specific T helper cells and thereby inducing adaptive immune responses. Although adaptive immunity is a highly effective defense system, it takes several days to become fully operational and needs to be triggered by danger-signals generated during the preceding innate immune response. Here we show that MHC class II molecules synergize with Toll-like receptor (TLR) 2 and TLR4 in inducing an innate immune response.

Methodology/principal findings: We found that co-expression of MHC class II molecules and TLR2 or TLR4 in human embryonic kidney (HEK) cells 293 leads to enhanced production of the anti-microbial peptide human-beta-defensin (hBD) 2 after treatment with TLR2 stimulus bacterial lipoprotein (BLP) or TLR4 ligand lipopolysaccharide (LPS), respectively. Furthermore, we found that peritoneal macrophages of MHC class II knock-out mice show a decreased responsiveness to TLR2 and TLR4 stimuli compared to macrophages of wild-type mice. Finally, we show that MHC class II molecules are physically and functionally associated with TLR2 in lipid raft domains of the cell membrane.

Conclusions/significance: These results demonstrate that MHC class II molecules are, in addition to their central role in adaptive immunity, also implicated in generating optimal innate immune responses.

Figure 1. Expression of HLA-DR enhances TLR2 and TLR4 dependent activation of HEK293 cells.

(A, B) Co-expression of HLA-DR in TLR2⁺ or TLR4⁺ HEK293 cells leads to enhanced hBD-2 expression (n≥3) after stimulation with BLP and LPS, respectively (black bars) compared to untreated cells (grey bars). (C) Co-expression of HLA-DR in TLR7⁺ cells did not increase the TNF expression (n = 3) after treatment with Loxoribine (black bars) compared to untreated cells (grey bars). (D) Co-expression of HLA-DR lacking the intracellular part in TLR2⁺ HEK293 cells failed to enhance hBD-2 expression after stimulation with BLP (n = 3). Error bars represent standard errors; * p<0.05, ** p = 0.055.

Figure 2. Peritoneal macrophages from MHC class II knock-out mice show impaired responsiveness to BLP and LPS.

(A–E) Cytokine secretion of peritoneal macrophages from MHC class II knock-out mice (grey bars; n≥4) is reduced compared to wild-type control mice (black bars; n≥3) after ex vivo stimulation with BLP and LPS. Error bars represent standard errors; ** p = 0.053, * p<0.05.

Figure 3. TLR2 and HLA-DR interact physically in lipid raft domains.

(A) Co-precipitation of labeled recombinant TLR2 (lane 1, 3, 4) or a control protein (lane 2, luciferase) with HLA-DR molecules purified by immunoprecipitation with anti HLA-DR antibodies (lane 1, 2, 4) or control antibodies (lane 3) from cell lysates of HLA-DR positive (lane 1, 2, 3) or negative (lane 4) HEK293 cells. A ¹⁴C-labeled molecular weight marker (in kD) is shown in the left lane. (B) Proximity ligation assays showed that TLR2 and HLA-DR are close together (red signal), while TLR2 and HLA-ABC do not co-localize (no red signal). Nuclei are stained by Hoechst 33342 (blue). (C) Treatment of TLR2⁺ (grey bars; n = 3) or HLA-DR⁺/TLR2⁺ (black bars; n = 3) HEK293 cells with MCD, a lipid raft-destroying agent, prior to stimulation with BLP leads to impaired hBD-2 gene expression. The effect is restored via adding MCD saturated with cholesterol acting as cholesterol donor. Error bars represent standard errors. (D) Immunofluorescence microscopy showed that TLR2 (αTLR2-FITC; green) and HLA-DR (αHLA-DR-APC-Cy9; blue) co-localize in lipid raft domains (CT-rhodamine; red) of HLA-DR⁺/TLR2⁺ HEK293 cells (indicated by the white arrows). Pictures are representatives of at least five experiments.