A Functional Toll-Interacting Protein Variant Is Associated with Bacillus Calmette-Guérin-Specific Immune Responses and Tuberculosis

Abstract

Rationale: The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood.

Objectives: To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis.

Methods: We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection.

Measurements and main results: We identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2⁺ CD4⁺ T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection.

Conclusions: TOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

Keywords: Toll-interacting protein; adaptive immunity; bacillus Calmette-Guérin; genetics; tuberculosis.

Figure 1.

Single-nucleotide polymorphism (SNP) rs5743854 regulates Toll-interacting protein (TOLLIP) mRNA expression in South African infant monocytes. (A) Genomic location of TOLLIP SNPs. The TOLLIP coding region ± 10 kb flanking regions among South African case–control genetic study population are shown. TOLLIP spans 36 kb on chromosome 11p15.5 and consists of six exons (dark gray regions). Four candidate functional SNPs are shown. Curved arrow = transcriptional start site. (B) Linkage disequilibrium of four potential regulatory TOLLIP SNPs associated with pediatric tuberculosis in the Cape Mixed Ancestry population. Minor allele frequencies of TOLLIP SNPs of interest (gray squares) and R² linkage disequilibrium values (white squares) are indicated. (C–E) Assessment of candidate functional SNPs with luciferase signaling assay. HEK293T cells were transfected with firefly luciferase-expressing plasmids (pGL4) under the control of various promoter fragment sequences. Cells were cotransfected with the Renilla luciferase gene under an SV40 promoter; data are reported as relative luciferase units, which are calculated by dividing the firefly luciferase signal by the Renilla luciferase signal. Firefly luciferase–expressing pGL4 plasmids under the control of 1-kb promoter sequence fragment containing (C) rs5743854 allele G or allele C, (D) rs7481967 allele G or allele A, or (E) rs4963035 allele A or allele G are shown. All data are representative of at least three independent experiments. ***P < 0.001 by Student’s two-sided t test. (F) Correlation between TOLLIP SNP rs5743854 genotype and TOLLIP mRNA expression in peripheral blood monocytes from 156 South African 10-week-old infants. mRNA was isolated from plate adherent monocytes derived from cryopreserved peripheral blood mononuclear cells. The line indicates the median value for each genotype. ***P = 0.008 by Kruskal-Wallis test. MAF = minor allele frequency; RLU = relative luciferase units.

Figure 2.

Toll-interacting protein rs5743854 deficiency allele is associated with diminished bacillus Calmette-Guérin (BCG)-specific T-cell cytokine responses and proliferation. Whole blood was incubated with media or 10⁵ cfu/ml Danish BCG, combined with anti-CD28/CD49d for 7 hours before adding Brefeldin A for an additional 5 hours, then fixed and permeabilized. (A) Gating strategy used for identifying CD4⁺ and CD8⁺ T cells. Representative dot plots from a single study participant are shown. From left to right, leukocytes from whole blood were acquired and cell doublets excluded. CD14⁺ cells were excluded, then leukocytes were further identified. T cells were selected and then separated into CD4⁺ and CD8⁺ T cells. (B–D) Frequencies of IL-2, tumor necrosis factor, IFN-γ, IL-17, and IL-22 cytokine-expressing T cells (background-subtracted), stratified by rs5743854 genotype. Median with intraquartile range shown. CD4⁺ T cells (B) and CD8⁺ T cells (C) in discovery cohort (n: C/C = 45, G/C = 34, G/G = 10). No CD8⁺ IL-2 was detected in this cohort. (D) Frequency and cytokine expression profile of BCG-specific CD4⁺ T cells in validation cohort (n: C/C = 40, G/C = 35, G/G = 5). (E) Frequency and cytokine expression profile of frequency of all BCG-specific IL-2⁺ CD4⁺ T cells measured in a combined cohort (n: C/C = 85, G/C = 69, G/G = 15). Statistical significance determined by Mann-Whitney test of recessive genetic model (C/C + G/C vs. G/G genotype). (F) CD4⁺ T-cell proliferation after BCG restimulation. Peripheral blood mononuclear cells from 65 infants were stained with Oregon Green overnight and incubated with media or BCG for 6 days. Cells were assessed for Oregon Green and stratified by rs5743854. Tukey plot with the Stimulation Index (percentage of BCG-restimulated, Oregon Green_low cells/percentage of media-restimulated, Oregon Green_low cells). Statistical significance determined using linear model. *P < 0.05, **P < 0.01. FSC = forward scatter; SSC = side scatter; TNF = tumor necrosis factor.

Figure 3.

Toll-interacting protein (TOLLIP) deficiency is associated with increased proinflammatory cytokine secretion after Toll-like receptor (TLR) stimulation. Peripheral blood mononuclear cells were collected from 64 healthy volunteers in Seattle and stimulated with LPS (A, 10 ng/ml) or Mycobacterium tuberculosis whole-cell lysate (B, 1 μg/ml) for 24 hours. Supernatants were collected, IL-6 responses were measured by ELISA, and the results were stratified by rs5743854 genotype. C/C individuals = 35, C/G = 21, G/G = 7; Mann-Whitney test of recessive model (G/G vs. G/C + C/C) was used to determine statistical significance. (C) Empty vector (control) THP-1 cells and TOLLIP-deficient (TOLLIP-KO) THP-1 cells were generated with CRISPR/Cas9, and TOLLIP protein expression was assessed by Western blot with β-actin comparison as a loading control. (D and E) Concentrations of tumor necrosis factor (D) or IL-6 (E) from supernatants of control and TOLLIP-KO cells were compared after 10 ng/ml LPS stimulation (TLR4 agonist) and 250 ng/ml PAM2 stimulation (TLR2/6 agonist) overnight. Student’s t test was used to determine statistical significance. **P < 0.01; ***P < 0.0001. M. Tb = Mycobacterium tuberculosis; TNF = tumor necrosis factor.

Figure 4.

Toll-interacting protein (TOLLIP) regulates immune response and Mycobacterium tuberculosis (MTb) replication. Empty vector (control) and TOLLIP-deficient (TOLLIP-KO) THP-1 cells were generated and stimulated in the following manner. (A) Concentration of tumor necrosis factor from supernatants of control and TOLLIP-KO cells was compared after 10 ng/ml LPS stimulation or MTb H37Rv infection (multiplicity of infection [MOI] 5) overnight. (B) Reactive nitrogen species production was compared in control with TOLLIP-KO cells infected with MTb H37Rv (MOI 25) or media for 24 hours. Nitrite concentrations were determined in supernatants using the Griess reagent system. Limit of detection for reagent system was 1 µM. Cytokine and nitrite results are the mean ± SEM of three replicates per condition and are representative of three independent experiments. Statistical significance was measured by Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.0001. (C) MTb replication in control and TOLLIP-KO cells over 7 days. Cells were cultured and then infected with MTb Erdman strain expressing the lux gene (MOI 5). Luminescence was measured for each group over the next 7 days. Mycobacterial replication experiments are shown as the mean ± SEM. These experiments were performed independently at least three times to ensure reproducibility. Statistical significance was measured by Student’s t test for each time point by comparing TOLLIP-KO and control cells. *P < 0.05, **P < 0.01, ***P < 0.0001. RLU = relative luciferase units; TNF = tumor necrosis factor.