Human TOLLIP regulates TLR2 and TLR4 signaling and its polymorphisms are associated with susceptibility to tuberculosis

Abstract

Tuberculosis, one of the leading causes of death worldwide, stimulates inflammatory responses with beneficial and pathologic consequences. The regulation and nature of an optimal inflammatory response to Mycobacterium tuberculosis remains poorly understood in humans. Insight into mechanisms of negative regulation of the TLR-mediated innate immune response to M. tuberculosis could provide significant breakthroughs in the design of new vaccines and drugs. We hypothesized that TOLLIP and its common variants negatively regulate TLR signaling in human monocytes and are associated with susceptibility to tuberculosis. Using short hairpin RNA knockdown of TOLLIP in peripheral blood human monocytes, we found that TOLLIP suppresses TNF and IL-6 production after stimulation with TLR2 and TLR4 ligands. In contrast, secretion of the anti-inflammatory cytokine IL-10 was induced by TOLLIP. We also discovered two common polymorphisms that are associated with either decreased levels of mRNA expression (rs3750920) or increased IL-6 production (rs5743899) in a sample of 56 healthy volunteers. Furthermore, in a case-population study in Vietnam with 760 cord blood samples and 671 TB case patients, we found that SNPs rs3750920 and rs5743899 were associated with susceptibility to tuberculosis (p = 7.03 × 10(-16) and 6.97 × 10(-7), respectively). These data demonstrate that TOLLIP has an anti-inflammatory effect on TLR signaling in humans and that TOLLIP deficiency is associated with an increased risk of tuberculosis. To our knowledge, these data also show the first associations of TOLLIP polymorphisms with any infectious disease. These data also implicate an unexpected mechanism of negative regulation of TLR signaling in human tuberculosis pathogenesis.

Figure 1. Lentiviral shRNA knockdown of TOLLIP in primary human monocytes

Peripheral blood monocytes were isolated and incubated with media, lentiviral particles encoding scrambled shRNA, or lentiviral particles encoding shRNA for 3 areas with in the TOLLIP gene for 24 hours. These cells were then incubated with TLR ligands for 24 hours and secreted cytokine responses were measured in the supernatant. A) mRNA levels of expression after lentiviral knockdown of peripheral blood monocytes. This data shows one representative experiment (out of a total of 2). B) IL-6, C) TNF, and D) IL-10, cytokine response after media, PAM2, PAM3, or LPS stimulation in primary monocytes incubated with nothing, control shRNA, or TOLLIP shRNA-encoding lentiviral particles. * p<0.01, ANOVA with Mann-Whitney test. MTb stimulation is from an independent experiment.

Figure 2. TOLLIP polymorphisms and variation in mRNA expression

Genomic DNA and mRNA were isolated from monocytes from 84 healthy individuals. TOLLIP mRNA expression was measured and normalized to GAPDH. Genotypes of six TOLLIP polymorphisms were examined for associations with TOLLIP mRNA expression. (rs5743899, AA = 49, AG = 31, GG = 4, Fig 2a; rs5743942, CC = 17, CT = 31, TT = 20, Fig 2b; ; rs3793964; CC = 36, CT = 24, TT = 16, Fig. 2c; rs3750920, CC = 26, CT = 32, TT = 16, Fig. 2d; rs3829223, CC = 19, CC = 32, TT = 27, Fig 2e; and rs3168046, CC = 15, CT = 22, TT = 14, Fig 2f). * p<0.01; # p=0.15, ANOVA analysis with Mann-Whitney test.

Figure 3. TOLLIP SNP rs5743899 and cytokine responses after TLR and MTb stimulation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells were isolated from 64 healthy volunteers in Seattle and stimulated with media or TLR ligands (LPS at 10 ng/ml, PAM2 at 250 ng/ml, PAM3 at 250 ng/ml, MTb whole cell lysate at 1 µg/ml) for 24 hours. Secreted A) IL-6 and B) IL-10 levels were measured in supernatants via ELISA. C–F) IL-6 responses after TLR stimulation, stratified by rs5743899 genotype. AA individuals = 35, AG = 21, GG = 7. G–J) IL-10 responses after TLR stimulation, stratified by rs5743899 genotype. * p<0.01; ** p=0.03 by Mann-Whitney test in a recessive model.

Figure 4. TOLLIP polymorphism rs3750920 and cytokine responses after TLR and MTb stimulation of peripheral blood mononuclear cells

Identical experimental details as Figure 3, except data was stratified by genotype rs3750920, CC = 18 individuals, CT = 24, TT 12. A–D) IL-6 responses and E–H) IL-10 responses after stimulation with media or TLR ligands (LPS at 10 ng/ml, PAM2 250 ng/ml, PAM3 250ng/ml, MTb whole cell lysate at 1 µg/ml) for 24 hours.

Figure 5

Chromosomal map and linkage disequilibrium plots of TOLLIP polymorphisms in Vietnamese cohort. (A) Chromosomal map shows genomic location of polymorphisms. Boxes show exons within gene on chromosome 11. rs3168046 was located on the 3’ UTR for the TOLLIP gene. (B, C): Linkage Disequilibrium plots with R² and D’ values for controls in Vietnam. White boxes on top of plot show frequency of polymorphisms. Degree of shading proportionate to D’ or R² value.