Human TLR1 deficiency is associated with impaired mycobacterial signaling and protection from leprosy reversal reaction

Abstract

Toll-like receptors (TLRs) are important regulators of the innate immune response to pathogens, including Mycobacterium leprae, which is recognized by TLR1/2 heterodimers. We previously identified a transmembrane domain polymorphism, TLR1_T1805G, that encodes an isoleucine to serine substitution and is associated with impaired signaling. We hypothesized that this TLR1 SNP regulates the innate immune response and susceptibility to leprosy. In HEK293 cells transfected with the 1805T or 1805G variant and stimulated with extracts of M. leprae, NF-kappaB activity was impaired in cells with the 1805G polymorphism. We next stimulated PBMCs from individuals with different genotypes for this SNP and found that 1805GG individuals had significantly reduced cytokine responses to both whole irradiated M. leprae and cell wall extracts. To investigate whether TLR1 variation is associated with clinical presentations of leprosy or leprosy immune reactions, we examined 933 Nepalese leprosy patients, including 238 with reversal reaction (RR), an immune reaction characterized by a Th1 T cell cytokine response. We found that the 1805G allele was associated with protection from RR with an odds ratio (OR) of 0.51 (95% CI 0.29-0.87, p = 0.01). Individuals with 1805 genotypes GG or TG also had a reduced risk of RR in comparison to genotype TT with an OR of 0.55 (95% CI 0.31-0.97, p = 0.04). To our knowledge, this is the first association of TLR1 with a Th1-mediated immune response. Our findings suggest that TLR1 deficiency influences adaptive immunity during leprosy infection to affect clinical manifestations such as nerve damage and disability.

Figure 1. NF-κB activity in response to M. leprae is diminished in the 1805T variant.

HEK293 cells were transfected with an NF-κB luciferase reporter, a Renilla luciferase construct to control for transfection efficiency (pRL-TK), and CD14. Additional transfectants varied by condition and included: empty plasmid vector (EV, clear), TLR2 alone (T2, gray), or TLR2 with one of two TLR1 constructs, 1805T (black) or 1805G (stippled). Luciferase activity represents basal (media stimulation) or stimulated activity of transfected cells. Mean values (+/−standard deviation) are depicted for two representative experiments, each performed in triplicate. RLU, relative luciferase units; P3 300, PAM3 at a dose of 300 ng/ml; M2 100, Malp-2, at 100 ng/ml; ML, whole, irradiated ML at 5, 50, or 250 µg/ml; MLcw, M. leprae cell wall at a dose of 1 or 10 µg/ml. * = P≤0.01, by Student's t-test when comparing the 1805T and 1805G variants (both with T2); # = P≤0.01, by Student's t-test when comparing T2+1805T and T2; ˆ = P≤0.01, by Student's t-test when comparing T2+1805G and T2.

Figure 2. IL-6 production by human primary cells following stimulation with M. leprae.

Peripheral blood mononuclear cells were stimulated for 18 hours and supernatants were assayed for cytokine production by ELISA. PBMCs were derived from whole blood taken from 15 individuals with the genotype 1805TTor 1805TG (TT/TG: dark circles) and 13 individuals with the 1805GG genotypes (GG: open circles). (A): PBMCs stimulated in triplicate with media, PAM2 at 75 ng/ml (P2 75), PAM3 at 75 ng/ml (P3 75), or LPS at 10 ng/ml (LPS 10). (B): PBMCs stimulated in triplicate with whole irradiated ML at 20 or 100 µg/ml (ML20 or ML100) or MLcw at 2 or 10 µg/ml (MLcw 2 or MLcw10). The mean level and standard error of the mean are depicted and were derived from averaging the responses of individuals stimulated in triplicate. The median level is depicted by a bar. *P≤0.01, **P≤0.001 by Mann–Whitney U-test.

Figure 3. IL-β and TNF-α production in human mononuclear cells following stimulation with M. leprae.

Peripheral blood mononuclear cells were stimulated for 18 hours and supernatants were assayed for cytokine production by luminex multiplex bead assay. PBMCs were obtained from 11 individuals with the genotype 1805TT or 1805TG (TT/TG: dark circles) and 10 individuals with the 1805GG genotypes (GG: open circles). PBMCs stimulated with media, PAM2 at 75 ng/mL (P2), PAM3 at 75 ng/mL (P3), or LPS at 10 ng/ml (LPS), whole irradiated ML at 20 µg/ml (ML) or MLcw at 10 µg/ml (MLcw). IL-1β production following stimulation is shown in (A), and TNF-α production in (B). The mean level and standard error of the mean are depicted and were derived from averaging the responses of individual within each genotype group. The median level is depicted by a bar; *p≤0.05, **p≤0.001 by Mann–Whitney U-test.