The R753Q polymorphism in Toll-like receptor 2 (TLR2) attenuates innate immune responses to mycobacteria and impairs MyD88 adapter recruitment to TLR2.
Pattabiraman G1, Panchal R1, Medvedev AE2.
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.To examine the impact of the R753Q TLR2 SNP on cell responses to live mycobacteria, we first used HEK293 cells stably expressing YFP-tagged human WT (293/TLR2WT) or R753Q TLR2 (293/TLR2R753Q). Cell activation was judged by phosphorylation of p38 MAPK and p65 NF-κB, activation of the NF-κB-driven luciferase reporter, and induction of IL-8 expression. Exposure of 293/TLR2WT cells to M. smegmatis (multiplicity of infection (m.o.i.) = 50) markedly enhanced phosphorylation of p38 MAPK and p65 NF-κB (Fig. 1A) and led to a 6.1-fold increase in NF-κB reporter activation (Fig. 1B). In contrast, 293/TLR2R753Q cells showed deficient phosphorylation of p38 MAPK and p65 NF-κB and impaired activation of the NF-κB reporter (78% decrease) in response to M. smegmatis and M. bovis BCG (Fig. 1).Because MAPKs and NF-κB mediate the expression of inflammatory cytokines (21), we next examined induction of IL-8 in HEK293 cells expressing WT or R753Q TLR2 in response to mycobacteria. Stimulation of 293/TLR2WT cells with Pam3CSK4, M. smegmatis, or M. bovis BCG resulted in ∼10-, ∼43-, and ∼2.5-fold up-regulation in the levels of IL-8 mRNA, whereas 293/TLR2R753Q transfectants showed 74%, 64%, and 58% inhibition in the activation of these responses, respectively (Fig. 1C). Similarly, in contrast to a marked up-regulation of IL-8 secretion observed in 293/TLR2WT cells exposed to Pam3CSK4, M. smegmatis, or M. bovis BCG, a significantly lower induction of IL-8 secretion was observed in 293/TLR2R753Q in response to stimulation with Pam3CSK4 or mycobacteria (Fig. 1D). Immunoblot analyses revealed a similar expression of WT versus R753Q TLR2 in HEK293 stable transfectants (Fig. 1A and data not shown), indicating that the observed differences in cell responses to live M. smegmatis or M. bovis BCG were not due to altered expression of the mutant TLR2. These data indicate that the R753Q SNP attenuates the ability of TLR2 to activate p38 MAPK- and p65 NF-κB-driven pathways in response to M. smegmatis and M. bovis BCG infection, leading to deficient induction of IL-8.The following Abs were used in this study: anti-GFP (Thermo Fisher Scientific, Waltham, MA); anti-FLAG (Sigma-Aldrich, St. Louis, MO); anti-MyD88, anti-p65 NF-κB, anti-TLR2, and anti-β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA); and anti-p-p38, anti-p-p65, anti-p-ERK1/2, anti-ERK1/2, anti-p38, anti-p-Akt, anti-Akt, and anti-β-tubulin (Cell Signaling Technology, Inc., Danvers, MA.). S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4-OH (Pam3CSK4) was obtained from InvivoGen (San Diego, CA). Zombie® Aqua cell viability dye, PE-labeled anti-mouse CD282 (TLR2) Ab, and PE-labeled rat IgG2a κ isotype control Ab were from BioLegend (San Diego, CA). HEK293T and HEK293 cells were obtained from the ATCC and maintained in complete DMEM supplemented with 10% FBS (HyClone, Logan, UT), 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (Thermo Fisher Scientific) (cDMEM).We thank Drs. Volker Briken (University of Maryland College Park, College Park, MD) and Matyas Sandor (University of Wisconsin, Madison, WI) for kindly providing us with GFP-expressing M. smegmatis and M. bovis BCG, respectively. We also thank Sarah Ahlbrand (University of Maryland College Park) for help and advice regarding M. smegmatis protocols.