The R753Q polymorphism in Toll-like receptor 2 (TLR2) attenuates innate immune responses to mycobacteria and impairs MyD88 adapter recruitment to TLR2

Goutham Pattabiraman,Rahul Panchal,Andrei E Medvedev

doi:10.1074/jbc.m117.784470

Goutham Pattabiraman, Rahul Panchal + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m117.784470

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Abstract

Toll-like receptor 2 (TLR2) plays a critical role in host defenses against mycobacterial infections. The R753Q TLR2 polymorphism has been associated with increased incidence of tuberculosis and infections with non-tuberculous mycobacteria in human populations, but the mechanisms by which this polymorphism affects TLR2 signaling are unclear. In this study, we determined the impact of the R753Q TLR2 polymorphism on macrophage sensing of Mycobacterium smegmatis Upon infection with M. smegmatis, macrophages from knock-in mice harboring R753Q TLR2 expressed lower levels of TNF-α, IL-1β, IL-6, and IL-10 compared with cells from WT mice, but both R753Q TLR2- and WT-derived macrophages exhibited comparable bacterial burdens. The decreased cytokine responses in R753Q TLR2-expressing macrophages were accompanied by impaired phosphorylation of IL-1R-associated kinase 1 (IRAK-1), p38, ERK1/2 MAPKs, and p65 NF-κB, suggesting that the R753Q TLR2 polymorphism alters the functions of the myeloid differentiation primary response protein 88 (MyD88)-IRAK-dependent signaling axis. Supporting this notion, HEK293 cells stably transfected with YFP-tagged R753Q TLR2 displayed reduced recruitment of MyD88 to TLR2, decreased NF-κB activation, and impaired IL-8 expression upon exposure to M. smegmatis Collectively, our results indicate that the R753Q polymorphism alters TLR2 signaling competence, leading to impaired MyD88-TLR2 assembly, reduced phosphorylation of IRAK-1, diminished activation of MAPKs and NF-κB, and deficient induction of cytokines in macrophages infected with M. smegmatis.

Highlights

Mycobacterium tuberculosis (Mtb)2 targets over a third of the world population, causing tuberculosis or latent infection that
Because MAPKs and NF-␬B mediate the expression of inflammatory cytokines [21], we examined induction of IL-8 in HEK293 cells expressing WT or R753Q Toll-like receptor 2 (TLR2) in response to mycobacteria
Genome-wide association studies have demonstrated the association of a microsatellite polymorphism in the intron of TLR2 and the R753Q TLR2 polymorphism with increased incidence of tuberculosis in several cohorts of patients [22,23,24,25, 41]

Summary

The abbreviations used are

Mycobacterium tuberculosis; TLR, Toll-like receptor; BCG, bacillus Calmette-Guérin; TIR, Toll-IL-1R; IRAK, IL-1R-associated kinase; KI, knock-in; m.o.i., multiplicity of infection; BMDM, bone marrow-derived macrophage; MFI, mean fluorescence intensity; Ab, antibody; Pam3CSK4, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-CysSer-Lys4-OH; cDMEM, complete DMEM; Luc, luciferase; TK, thymidine kinase; PE, phycoerythrin. The impact of the R753Q polymorphism on the signaling capacities of endogenous TLR2 in macrophages during infection with live mycobacteria has not been studied. B, 293/TLR2 stable cell lines expressing WT or R753Q TLR2 species were co-transfected with pELAM-Luc and pTK-Renilla-Luc. Cells were treated for 24 h with Pam3CSK4 (1 ␮g/ml), M. smegmatis, or M. bovis BCG (all at m.o.i. 50), and cell lysates were prepared to determine firefly versus Renilla luciferase activities. We sought to determine the impact of the R753Q TLR2 polymorphism on macrophage responses to live infection with M. smegmatis. To the best of our knowledge, this is the first demonstration of deficient MyD88TLR2 assembly and activation of IRAK-1 as the mechanism by which the R753Q polymorphism impairs innate responses of macrophages during infection with live mycobacteria

Results

Discussion

Experimental procedures