REL and BHLHE40 variants are associated with IL-12 and IL-10 responses and tuberculosis risk

Abstract

The major human genes regulating M. tuberculosis (Mtb)-induced immune responses and tuberculosis (TB) susceptibility are poorly understood. Although IL-12 and IL-10 are critical for TB pathogenesis, the genetic factors that regulate their expression in humans are unknown. CNBP, REL, and BHLHE40 are master regulators of IL-12 and IL-10 signaling. We hypothesized that common variants in CNBP, REL, and BHLHE40 were associated with IL-12 and IL-10 production from dendritic cells, and these variants also influence adaptive immune responses to BCG vaccination and TB susceptibility. We characterized the association between common variants in CNBP, REL, and BHLHE40, innate immune responses in dendritic cells and monocyte-derived macrophages, BCG-specific T cell responses, and susceptibility to pediatric and adult TB in human populations. BHLHE40 SNP rs4496464 was associated with increased BHLHE40 expression in MDMs and increased IL-10 from peripheral blood dendritic cells and MDMs after LPS and TB whole cell lysate stimulation. SNP BHLHE40 rs11130215, in linkage disequilibrium with rs4496464, was associated with increased BCG-specific IL2+CD4+ T cell responses and decreased risk for pediatric TB in South Africa. SNPs REL rs842634 and rs842618 were associated with increased IL-12 production from dendritic cells, and SNP REL rs842618 was associated with increased risk for TB meningitis. In summary, we found that genetic variation in REL and BHLHE40 are associated with IL-12 and IL-10 cytokine responses and TB clinical outcomes. Common human genetic regulation of well-defined intermediate cellular traits provides insights into mechanisms of TB pathogenesis.

Introduction

Tuberculosis (TB) is a leading cause of death from infection worldwide. The current BCG vaccine remains the only approved vaccine against TB despite its partial and variable effects across populations (1). Vaccine efforts are hampered by a lack of understanding of the immune correlates of protection (2). Understanding the factors required to induce effective, long-lasting immunity to infections may provide tools to improve TB vaccines.

Twin, Mendelian, linkage, genome-wide association, and candidate gene studies suggest that genetic factors influence susceptibility to TB (3, 4). Multiple clinical TB phenotypes show a high degree of heritability, including host susceptibility to pulmonary TB (5–10), TB meningitis (11, 12), and latent TB infection (13–17). Common genetic variation influences the cellular innate immune response to Mycobacterium tuberculosis (Mtb). Multiple studies demonstrate the impact of genetic variation on innate immune cellular distribution and cytokine responses (18–21). Quantitative trait loci (QTL) of gene expression demonstrate immune cell-specific effects (20, 22). Recent advances permit the evaluation of innate immune cytokine responses from rare cell populations (23, 24). Variants that influence functional responses in immune cells of interest represent attractive secondary traits which can be correlated with TB susceptibility and these correlations may provide insight into genetic mechanisms of disease susceptibility (25).

Dendritic cells (DCs) are essential for host defense against mycobacteria (7, 15, 26), and IL-10 and IL-12, which are produced in large quantities by DC, are particularly important cytokines that shape immune responses to Mtb. Genetic variation in the IL-10 gene has been modestly associated with TB susceptibility (27–29). However, rare inborn mutations in IL-12 signaling genes lead to widely disseminated mycobacterial disease (30, 31).

Recently, several novel mediators of IL-12 and IL-10 signaling were identified. After pathogen receptor recognition, the transcription factor CNBP and its binding partner c-REL translocate to the nucleus and induce IL12B (32, 33). The transcription factors BHLHE40, with CNBP, control IL10 transcription from both myeloid and lymphoid cells (32–34). The role of these genes and their genetic variants in human regulation of dendritic cell and T cell responses is unknown. We hypothesized that variants that influences DC phenotypes would lead to multiple downstream effects, including influence on BCG-specific T cell responses and susceptibility to TB disease. Herein, we investigated the impact of common human genetic variation in the CNBP, REL, and BHLHE40 gene regions on LPS and mycobacteria-induced cytokine responses in DC, BCG-specific T cell responses and TB susceptibility.

Materials and Methods

Study Participants and Ethics Statement

Approval for human study protocols was obtained from the institutional review boards at local sites in Vietnam, South Africa, and the University of Washington School of Medicine. Genomic DNA was purified from blood samples using genomic DNA isolation kits (Qiagen, Inc). DNA concentrations were confirmed using Nanodrop. Seattle cohort study subjects were local volunteers self-described as healthy without history of recurrent of serious infections. 52% of individuals are female, and 48% are male. The ethnic composition of this study group was 69% White, 19% Asian, 2% Black or African American, and 2% Latinx. Average age of study participants was 39, with interquartile range of 29 – 46 at the time of their enrollment. In this study, we tested immune responses on a subset of 35 participants (discovery) and a second set of 48 participants (validation).

For genetic studies in Vietnam, approval for human study protocols was obtained from the human review boards at the University of Washington School of Medicine, the Hospital for Tropical Diseases (Ho Chi Minh City, Vietnam), Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases (Ho Chi Minh City, Vietnam), Health Services of Ho Chi Minh City, Hung Vuong Hospital (Ho Chi Minh City, Vietnam), and the Oxford Tropical Research Ethics Committee. For South African pediatric TB cohorts, the study was conducted according to the U.S. Department of Health and Human Services and Good Clinical Practice guidelines. This study included written informed consent from the parent or legal guardian of the study participant and protocol approval by the University of Cape Town Research Ethics Committee and the University of Washington human subjects review board.

South African study participants were enrolled at the South African Tuberculosis Vaccine Initiative field site in Worcester, South Africa, near Cape Town as part of a larger study on BCG vaccination with 11,680 infants (35, 36). This area has one of the highest rates of TB incidence in the world with an incidence of 3% among children under 3 years of age in the study population (35, 36). A nested genetics case-control study was performed with identification of cases and controls during a 2-year prospective observation period after vaccination at birth. Household controls were children without TB disease over the 2-year follow period living with an individual with active TB disease. Community controls had no history of TB disease in the first two years of life. Community-wide passive surveillance systems identified patients with TB disease and children with symptoms suggestive of TB disease. The criteria for detection of TB cases have been described previously (37). All infants who had symptoms compatible with TB disease or who had contact with an adult with TB disease were admitted to a dedicated research ward for clinical examination, chest radiography, tuberculin skin testing, two early-morning gastric aspirations, and two sputum inductions for Mtb smear and culture. Participants were defined as having “definite TB” if they had a positive Mtb culture, a positive smear, or a positive Mtb PCR from one of their samples. Participants with a chest radiograph compatible with or suggestive of TB combined with one or more additional laboratory or clinical features consistent with TB (smear negative, cough > 2 weeks, PPD skin test ≥ 15 mm, failure to thrive, and recent weight loss) were defined as having “probable TB.” Individuals without radiography consistent with TB who were diagnosed with TB by the treating physician and had 2 or more clinical features suggestive of TB were defined as “possible TB.” All others were described as “not TB.” All infants admitted to the research ward were also tested for HIV infection and positive tests resulted in exclusion from the study. The following were additional exclusion criteria at 10 weeks of age: mother known to be infected with HIV; BCG not received by infant within 24 hours of birth; significant perinatal complications in the infant; any acute or chronic disease in the infant at the time of enrollment; clinically apparent anemia in the infant; household contact with any person with TB disease or any person who was coughing.

Whole blood from South African infants was collected as previously described (37, 38). Infants were vaccinated with BCG on the day of birth, and at 10 weeks of age, heparinized blood was collected from BCG-vaccinated infants. None of the infants in this study had active tuberculosis at the time of their 10-week blood draw or during 2 years of follow-up observation. Flow cytometry was performed on a total of 181 infants, divided into discovery (n = 89) and validation (n = 92) cohorts.

Study subjects from the Vietnam cohort were described previously and are briefly summarized here (12). Subjects with tuberculous meningitis (TBM) were recruited from two centers in Ho Chi Minh City, Vietnam: Pham Ngoc Thach (PNT) Hospital for Tuberculosis and the Hospital for Tropical Diseases (HTD). Subjects with pulmonary TB were recruited from a network of district TB control units within Ho Chi Minh City that provide directly observed therapy to TB patients. In addition, pulmonary TB subjects were recruited from PNT hospital from 2006 through 2008. Vietnamese population controls are otherwise healthy adults with primary angle closure glaucoma which have been previously described (39). All case and control participants were unrelated, and greater than 95% were of the Vietnamese Kinh ethnicity. Previous genetic studies of this population indicate minimal population substructure (12). Written, informed consent was obtained from patients or their relatives if the patient could not provide consent (i.e., was unconscious). Individuals in the TBM group were defined as follows. Individuals at least 15 years old, admitted to these centers with clinical meningitis (defined as nuchal rigidity and abnormal cerebrospinal fluid parameters), a negative HIV test result, and a positive Ziehl–Neelsen stain for acid-fast bacilli or Mtb culture, or both, from cerebrospinal fluid (“definite TBM”) were recruited for genetics studies from 2001 to 2008. In addition to definite TBM, the cohort included subjects with “probable TBM,” defined as clinical meningitis plus at least one of the following: chest radiograph consistent with active TB, acid-fast bacilli found in any specimen other than cerebrospinal fluid, and clinical evidence of other extrapulmonary TB. The pulmonary TB group was defined as follows: participants were outpatients who were at least 18 years old, had no previous history of treatment for TB, no evidence of miliary or extrapulmonary TB, chest radiograph results consistent with non-miliary pulmonary TB, negative HIV test results, and sputum smear positive for acid-fast bacilli or M. tuberculosis cultured from sputum.

Reagents and Antibodies

RPMI 1640 medium, L-glutamate were obtained from Life Technologies. Ultrapure LPS (TLR4 ligand) isolated from Salmonella Minnesota R595 was obtained from List Biological Labs. Whole cell lysate from M. tuberculosis strain H37Rv was obtained as part of National Institutes of Health, National Institute of Allergy and Infectious Diseases Contract No. HHSN266200400091C, entitled Tuberculosis Vaccine Testing and Research Materials (Colorado State University, Fort Collins, CO). Lyophilized live Bacille Calmette-Guerin (BCG, 20 x 10⁶ CFU/mL) was obtained from Statens Serum Institute (Copenhagen, Denmark).

Bacterial Source and Stock

For whole blood stimulation assays, BCG Russia strain (gift of David Sherman) was grown to optical density value 1.0 at 600nm (OD600) in 7H9 broth medium supplemented with 0.2% glycerol and 10% OADC enrichment (complete medium for bacterial culture) and harvested by centrifugation at 4000rpm for 25min. The bacterial pellet was resuspended and adjusted to OD600 value of 1.0 in RPMI with 10% BSA supplementation. These aliquots were frozen at −80°C until use.

Stimulation of whole blood samples, cell culture, and flow cytometry

LPS (10 ng/ml), BCG (10⁶ CFU/ml), and TB whole cell lysate (TBWCL; 50 μg/ml) were added to 500 μl of whole blood. Media-stimulated cells were included as a negative control. Brefeldin A (BFA, Sigma) was added at a concentration of 500 μg/mL (50 times the desired final concentration of 10 ng/mL). Additionally, Protein Transport Inhibitor containing monensin (monensin, BD) was added according to manufacturer’s protocol to all wells 4 hours prior to the completion of the experiment. Primary monocytes were isolated from human subjects as described previously (40). Briefly, peripheral blood was obtained and PBMCs were collected via Ficoll gradient separation and cryopreserved. Subsequently, CD14+ cells were purified using human monocyte negative selection kit (Miltenyi Biotec, Inc.). Monocytes were >95% pure for CD14+ using this method.

Antibodies

The following antibodies (clones and source) were used in these experiments: PE-Texas Red anti-CD3 (UCHT1, Beckman Coulter), APC anti-CD11c (S-HCL-3, BD), V500 anti-CD14 (M5E2, BD), BV650 anti-CD16 (3G8, Biolegend), Biotin anti CD66a/c/e (ASL-3, Biolegend), PE-Cy7 anti-CD123 (6H6, Biolegend), SB600 anti-HLA-DR (LN3, eBioscience), FITC anti-IL-10 (BT-10, eBioscience), EF450 anti-IL-12 (C8.6, eBioscience), BV786 streptavidin (BD), near-IR Avid fixable live-dead stain (Invitrogen). The concentrations of all antibodies were titrated prior to use.

Infant T cell assays were collected as described previously (37, 38). Infants were vaccinated with BCG on the day of birth, and at 10 weeks of age, heparinized blood was collected from BCG- vaccinated infants and 1 mL was incubated ex vivo with 1.26x10⁶ CFU of BCG (Danish strain 1331). None of the infants in this study had active tuberculosis at the time of their blood draw at 10 weeks of age or during 2 years of follow-up observation. Whole blood was incubated for 12 hours with either media control, BCG, or SEB, then fixed and frozen in liquid nitrogen. Flow cytometry was performed on a total of 181 infants, divided into discovery (n = 89) and validation (n = 92) cohorts.

Frozen samples were thawed in a 37°C water bath and spun, then pellets were resuspended in 200 μl PermWash Solution (BD) and incubated at room temperature for 10 min. After one wash in PermWash, cells were stained for 30 min at room temperature. After two further washes with PermWash, cells were immediately analyzed on an LSRII Flow Cytometer (BD). Positivity thresholds were determined by gating on fluorescence minus one control samples followed by selective testing of thresholds control samples.

Genotyping and linkage disequilibrium

Genotyping was performed with Illumina MegaEx Chip for the Seattle cohorts and Illumina OmniExpress in Vietnam. Imputation in Vietnam was performed as described previously (41). Selected genotyping was also performed using a Fluidigm Genotyping 96 x 96 array. We selected a region 10 kb upstream and downstream of genes of interest using a minor allele frequency cut-off of 5%. SNPs were excluded if they demonstrated Hardy-Weinberg equilibrium (HWE) p<0.001. In the Seattle cohort, genotypes were determined by Illumina MEGAex array annotated to the human genome (hg19) with annotatr basic genes (v1.14.0) in R (v4.0.2) (42). Linkage disequilibrium (LD) Pearson coefficient of correlation (R²) was calculated between SNPs with two or more genotypes and within 10 kb of a gene using the genetics package (v1.3.8.1.2). Haplotype tags were selected by the SNPinfo Web Server with a linkage disequilibrium threshold = 0.8, maximum distance of 250kB between SNPs, and minimum of 2 SNPs tagged using each SNP, using a European population for the Seattle cohort and a East Asian population for the Vietnam cohort (43).

Genotyping in the South Africa cohort was performed on haplotype-tagging SNPs in the REL and BHLHE40 gene region using the Fluidigm Genotyping 96 x 96 array using the SNP selection approach detailed above. Haplotype tags were selected by the SNPinfo Web Server using the African population (43).

SNPs of interest were identified using a haplotype-tagging approach from whole genome data in the Seattle cohort. The association between SNPs and development of TBM was compared by Chi-squared analysis between cases and controls. Statistical significance of cytokine responses were initially determined using a linear model. In case-control studies, an allelic model was used as a screening approach. For SNPs of interest, a dominant genotypic model (major allele homozygotes are compared to a composite of heterozygotes and minor allele homozygotes) and recessive genotypic model (minor allele homozygotes are compared to a composite of heterozygotes and major allele homozygotes) were also evaluated to further define the genetic model. Haplotype blocks and tagging SNPs were identified in CNBP, REL, and BHLHE40 using the SNP info webserver (https://manticore.niehs.nih.gov/cgi-bin/snpinfo/snptag.cgi) selecting for genotype data “dbSNP,” and the European population (44). We used 10kB flanking regions, including any upstream genomic regions that may participate in gene regulation. We used an LD threshold of 0.8, minor allele frequency cutoff of 0.05, and 2 SNPs minimum tagged by each SNP. Measures of correlation (R²) and allelic linkage (D^’) between SNPs of interest were calculated using the R package genetics for the Seattle population. The values of R² and D’ range from 0 to 1, with lower values indicating allelic independence and higher values suggesting that alleles are in complete LD (e.g., that the alleles are fully co-inherited).

Statistical methods

In our primary analysis, we examined whether polymorphism genotype frequencies were associated with cytokine production using a simple generalized linear model. For genetic analysis, background-corrected values were used to estimate cytokine induction by inflammatory stimuli. We modeled effect size for each of these observations with R*2. For each result with p < 0.05 in this model, we added a second p-value that adjusts for ethnicity using a linear regression with ethnic origin as a covariate. For secondary analyses, SNPs were investigated for associations under additional genetic models (dominant, recessive, and additive). In the recessive model, carriers of allele 1 (00 and 01 genotypes) were compared with homozygous subjects for allele 2 (11 genotype). In the dominant model, carriers of allele 2 (01 and 11 genotypes) were compared with homozygous subjects for allele 1 (00 genotype). For genetic studies of continuous variables, simple linear models were used, modelling the additive contribution of genotypes, and effect sizes were estimated using the slope of the linear regression model (model R²; glm command, STATA 14.1). Analyses adjusted for ethnicity are linear regressions evaluating the relationship between genotype and cytokine, with ethnicity as a covariate (regress command, STATA 14.1).

Within the Vietnam GWAS, no correction for sex or ethnicity was provided. This is due to prior data demonstrating that sex is not a confounding factor in genetic studies (45). For Vietnam GWAS studies, all participants were unrelated, and all were of the Kinh Vietnamese ethnicity. In addition, we previously genotyped a panel of ancestry informative markers and found no evidence of population admixture between case and control participants (46). Furthermore, principal components analysis confirmed the genetic homogeneity of this population (47). Therefore, we did not perform regression with ethnicity as a covariate.

Graphs were created with Prism version 8.0 (GraphPad, Inc.). Results are reported without correction for multiple comparisons due to the heterogeneous sources of data (mixture of cellular and clinical), including varied availability of validation datasets for some datasets.

Results

Flow cytometry analysis of cytokine production in peripheral blood DCs

To evaluate genetic regulation of IL-10 and IL-12 production from healthy human donors, we used flow cytometry to measure the proportion of peripheral blood MHC-II+CD11c+ DCs producing IL-10 and IL-12 after stimulation of whole blood with LPS or TB whole cell lysate (TBWCL; Figure 1A). Initially, we chose LPS as a stimulation because it is a well-characterized ligand which potently induces IL-12 and IL-10 from innate immune cells both in vitro and in vivo. We also used TBWCL due to its breadth of Mycobacterial ligands and relevance for studying TB pathogenesis as well as its induction of IL10 and IL12. We demonstrate the kinetics of IL-12 and IL-10 responses in Figure S1A. LPS (10 ng/ml) and TBWCL (50 μg/ml) both strongly induced IL-12 (p < 0.0001 for both tests, Figure 1B) and IL-10 (p < 0.0001 for both tests, Figure 1C) from DCs 24 hours after stimulation. We also measured cytokine responses to LPS (10 ng/ml) and live BCG (10⁶ CFU/ml) six hours after stimulation (Figure 1D). We chose BCG (10⁶ cfu/ml) because it was a live bacillus relevant to Mtb pathogenesis and due to its capacity to induce T cell responses in humans, nonhuman primates, and mice that are relevant to Mtb control. We found that LPS and BCG induced IL-12 six hours after stimulation in CD11c+ DCs (p < 0.001 for LPS, p = 0.01 for BCG, Figure 1D). However, we did not detect IL-10 above background levels from DCs at this time point (data not shown).

Figure 1. IL-10 and IL-12 responses from peripheral blood DCs in whole blood stimulation assay.

Peripheral whole blood was collected from healthy volunteers and incubated with either negative control or immune stimuli followed by BFA and monensin 2 hours afterward.

A) Gating strategy. From left to right, singlets were selected, then leukocytes. CD66+ cells were excluded, and HLA-DR+ cells were selected. CD14− and CD16− cells, followed by CD11c+ cells were selected, and the proportion of cytokine positive cells were measured as compared to total number of HLA-DR+CD14-CD16-CD11c+ DCs.

B) Proportion of IL-12+CD11c+ DCs after media control, LPS (10 ng/ml), or Mtb whole cell lysate (TBWCL; 50 μg/ml) stimulation for 24 hours.

C) Proportion of IL-10+CD11C+ DCs after media, LPS, or TBWCL for 24 hours.

D) Proportion of IL-12+CD11c+ DCs after media, LPS, or live BCG (10⁶ CFU) stimulation for 6 hours. Bars demonstrate median values. Data provided are not corrected for background cytokine positivity. Dots represent individual values. N = 46.

Discovery analysis of genetic associations with IL-12 responses to LPS and TBWCL.

We next examined whether candidate gene variants were associated with LPS or TB whole cell lysate- (TBWCL) induced IL-12 in DCs. We interrogated 5 haplotype-tagging SNPs from CNBP, 6 from REL, and 20 from BHLHE40 in a local cohort of healthy volunteers (complete SNP list in Figure S1B–D). REL SNP rs842634 was associated with increased IL-12 after TBWCL and LPS stimulation (Figure 2A, p = 0.037, generalized linear model (GLM), ethnicity-adjusted p = 0.017, R² = 0.10; Figure 2B, p = 0.044, adjusted p = 0.042, R² = 0.09). A second haplotype-tagging SNP, REL SNP rs842618, was associated with increased IL-12 after TBWCL and LPS stimulation (Figure 2C, p = 0.013, adjusted p = 0.03, R² = 0.13; Figure 2D, p = 0.04, adjusted p = 0.04, R² = 0.09). CNBP SNP rs11709852 was associated with increased IL-12 production after TBWCL stimulation, but not LPS stimulation (Figure 2E, p = 0.003, adjusted p = 0.24; Figure 2F, p = 0.48). However, after adjustment for ethnicity, this association did not meet criteria for statistical significance. No SNPs from BHLHE40 were associated with IL-12 (Data Set S1).

Figure 2. REL SNPs rs842634 and rs842618 are associated with IL-12 production after TBWCL stimulation of peripheral blood DCs for 24 hours.

A-B) Proportion of CD11c+ DCs producing IL-12 after A) Mtb whole cell lysate (TBWCL; 50 μg/ml) stimulation or B) LPS (10 ng/ml) stimulation for 24 hours. Data are stratified by rs842634 genotype; N = 19 T/T, 21 T/C, and 7 C/C.

C-D) Proportion of CD11c+ DCs producing IL-12 after A) Mtb whole cell lysate (TBWCL; 50 μg/ml) stimulation or B) LPS (10 ng/ml) stimulation for 24 hours. Data are stratified by rs842618 genotype; N = 17 T/T, 24 T/C, and 6 C/C.

E-F) Proportion of CD11c+ DCs producing IL-12 after C) TBWCL or D) LPS stimulation for 24 hours. Data are stratified by rs11798052 genotype; N = 34 G/G, 5 G/A, and 2 A/A. All data presented in this figure represent background-corrected values (proportion of cytokine-producing cells after ligand stimulation – proportion of cytokine-producing cells after media control stimulation).

* p < 0.05; statistical significance determined by generalized linear model.

REL variants rs842634 and rs842618 are associated with LPS and BCG-induced IL-12 secretion after 6-hour stimulation in an independent dataset.

We evaluated the association of our candidate SNPs with an earlier timepoint with different healthy donors. We examined whole blood incubated with BCG (10⁶ CFU/ml) or LPS (10 ng/ml) for 6 hours, followed by measurement of cytokine responses, as described above. REL SNP rs842634 was associated with increased IL-12 after BCG infection (Figure 3A; p = 0.046; ethnicity-adjusted p = 0.040, R²=0.11) and LPS stimulation (Figure 3B; p = 0.024; ethnicity-adjusted p = 0.014, R² = 0.15), and REL SNP rs842618 was associated with increased IL-12 after BCG (Figure 3C, p = 0.042; ethnicity-adjusted p = 0.014, R² = 0.10) and LPS (Figure 3D, p = 0.002; ethnicity-adjusted p = 0.004, R² = 0.26).

Figure 3. REL SNPs rs842634 and rs842618 are associated with IL-12 production in peripheral blood DCs after 6 hours of BCG or LPS stimulation.

A-B) Proportion of CD11c+ DCs producing IL-12 after A) live BCG stimulation (10⁶ CFU) or B) LPS (10 ng/ml) stimulation for 6 hours. Data are stratified by rs842634 genotype; N = 15 T/T, 16 T/C, and 4 C/C.

C-D) Proportion of CD11c+ DCs producing IL-12 after A) live BCG stimulation (10⁶ CFU) or B) LPS (10 ng/ml) stimulation for 6 hours. Data are stratified by rs842618 genotype; N = 16 T/T, 15 T/C, and 4 C/C.

* p < 0.05; ** p < 0.01, *** p < 0.001; statistical significance determined by generalized linear model. All data presented in this figure represent background-corrected values (proportion of cytokine-producing cells after ligand stimulation – proportion of cytokine-producing cells after media control stimulation).

BHLHE40 SNP rs4496464 is associated with IL-10 secretion from DCs

Next, we evaluated for associations between genetic variants in CNBP, REL, and BHLHE40 with IL-10 production from DCs. BHLHE40 SNP rs4496464 was associated with increased IL-10 production after TBWCL stimulation (Figure 4A; p = 0.005; ethnicity-adjusted p = 0.005, R² = 0.15). In contrast, rs4496464 was not associated with IL-10 after LPS stimulation (Figure 4B, p = 0.18, R² = 0.04). Although rs79248174 was associated with IL10 after TBWCL stimulation, this was due to a single data point, and therefore, this SNP was not included for further analysis (Data Set S1). BHLHE40 SNP rs4496464 was not associated with IL-12 expression after stimulation with either TBWCL or LPS (Figure 4C and Figure 4D). No REL SNPs were associated with IL-10 expression after LPS stimulation (data not shown).

Figure 4. BHLHE40 SNP rs4496464 is associated with IL-10 production from peripheral blood DCs after Mtb whole cell lysate stimulation.

A-B) Proportion of CD11c+ DCs producing IL-10 after A) Mtb whole cell lysate (TBWCL; 50 μg/ml) or B) LPS (10 ng/ml) stimulation for 24 hours. Data are stratified by rs4496494 genotype; N = 40 A/A, 7 G/A and 2 G/G.

C-D) Proportion of CD11c+ DCs producing IL-12 after C) TBWCL or D) LPS stimulation for 24 hours. Data are stratified by rs4496494 genotype. N = 38 A/A, 7 G/A, 2 G/G.

All data presented in this figure represent background-corrected values (proportion of cytokine-producing cells after ligand stimulation – proportion of cytokine-producing cells after media control stimulation).

* p < 0.05; ** p < 0.01, *** p < 0.001; generalized linear model.

Rs4496464 is associated with BHLHE40 mRNA expression in monocyte-derived macrophages

We evaluated whether rs4496464 genotypes were associated with BHLHE40 mRNA expression in peripheral blood monocyte-derived macrophages (MDM) from healthy donors. The uncommon G allele of rs4496464 was associated with increased BHLHE40 in unstimulated monocytes using a dominant model of inheritance (Figure 5A; p = 0.026, A/A vs (G/A + G/G), Mann-Whitney U-test). No other BHLHE40 SNPs were associated with expression. The T allele of REL variant rs842618 was associated with increased mRNA expression in monocytes (Figure 5B; p = 0.0015, R² = 0.20). However, REL SNP rs842634 was not associated with mRNA expression in monocytes (Figure 5C).

Figure 5. BHLHE40 SNP rs4496464 is associated with BHLHE40 mRNA and SNP rs842618 is associated with REL mRNA in monocyte-derived macrophages.

A) BHLHE40 mRNA expression, normalized to GAPDH expression, was measured from RNA extracted from MDMs isolated from healthy volunteers and stratified by rs4496464; n = 26 A/A, 7 G/A, and 1 G/G.

B-C) REL mRNA expression, normalized to GAPDH, was measured from MDMs isolated from healthy volunteers and stratified by B) SNP rs842618 (N =18 C/C, 12 T/C, and 2 T/T) or C) SNP rs842634 (N =14 T/T, 12 T/C, and 6 C/C). * p < 0.05, ** p <0.01; linear model.

BHLHE40 rs4496464 is associated with IL-10 production in LPS and TBWCL stimulated monocyte-derived macrophages.

To validate our association between rs496464 and IL-10 expression in DCs, we measured IL-10 secreted from monocyte-derived macrophages (MDMs) stimulated with either LPS (50 ng/ml) or TBWCL (25 μg/ml) overnight (Figure 6A, n = 26). The rs4496464 G allele was associated with increased IL-10 after LPS stimulation (Figure 6B, p = 0.01, generalized linear model). SNP rs4496464 was also associated with increased IL-10 after TBWCL (Figure 6C, p = 0.005, generalized linear model). SNP rs4496464 was not associated with TNF secretion after either LPS (Figure 6D) or TBWCL stimulation (Figure 6E), which suggests that variation in BHLHE40 is associated with IL-10 production specifically, over proinflammatory cytokine responses.

Figure 6. BHLHE40 SNP rs4496464 is associated with IL-10 production from monocyte-derived macrophages.

Peripheral blood monocytes were differentiated into macrophages by M-CSF for 5 days, then incubated with either LPS (50 ng/ml) or Mtb whole cell lysate (TBWCL; 25 μg/ml).

A) Overall IL-10 cytokine concentrations from cellular supernatants MDMs after 24 hours of stimulation.

B-C) Concentration of IL-10 in cellular supernatants after B) LPS stimulation or C) TBWCL stimulation for 24 hours, stratified by rs4496494 genotype. N = 20 A/A, 6 G/A, 2 G/G.

D-E) Concentration of TNF in cellular supernatants after D) LPS stimulation or E) TBWCL stimulation for 24 hours and stratified by rs4496464.

* P < 0.05, ** P < 0.01, *** P < 0.001; generalized linear model. All data presented in this figure represent background-corrected values (proportion of cytokine-producing cells after ligand stimulation – proportion of cytokine-producing cells after media control stimulation).

REL rs842618 is associated with TB meningitis.

Our data suggests that rs842634 and rs842618 are associated with increased IL-12 in DCs and rs4496464 is associated with increased IL-10 production from peripheral blood monocytes and DCs in our local population. We hypothesized that these polymorphisms were associated with susceptibility to TB due to their influence on these critical immune phenotypes. Within a large genome wide association study comparing Vietnamese individuals with adult pulmonary TB (PTB; n =1598) or TB meningitis (TBM; N = 407) with control subjects (N = 1139), we evaluated if SNPs in REL and BHLHE40 were associated with adult PTB or TBM and in LD with our SNPs of interest (Figure S2). REL rs842634 was not associated with TBM, but the minor T allele of REL SNP rs842618 was associated with increased TBM susceptibility (p = 0.03; OR 1.27, allelic model, Table 1 and Data Set S2). These data best fit a dominant model (Table 1, p = 0.035, OR 1.32, 95% CI 1.02 – 1.73) No BHLHE40 SNPs were associated with TBM. We did not identify any associations between SNPs in REL, or BHLHE40 SNPs with PTB (Data Set S2). We performed a haplotype analysis comparing the contribution of rs842634 and rs842618 with the TB meningitis phenotype. SNPs rs842634 and rs842618 are interrelated and the presence of both minor alleles strengthens the association between these genotypes and TBM susceptibility (p = 0.003 describing an interaction between these two SNPs). Together, these data suggest that a causal REL SNP linked to rs842618 is associated with both increased IL-12 production, increased REL mRNA and increased adult TBM susceptibility, but not morbidity or mortality.

Table 1. Association of REL SNPs with adult TB meningitis in Vietnam.

Number of individuals with major homozygous (AA), heterozygous (Aa), and minor homozygous (aa) genotypes described.

		Control				Case
locus	Gene	AA	Aa	aa	Total	AA	Aa	aa	Total	Allelic p	Dom p	OR (95% CI)
rs842618	REL	883	231	13	1075	289	99	7	395	0.032	0.035	1.33 (1.02 – 1.73)
rs842634	REL	901	218	11	1130	299	92	6	397	0.052	0.064	1.21 (0.72-2.0)

Total: total N in group after genotyping. Allelic p: p value in an allelic genetic model. Dom p: p value in a dominant genetic model of inheritance. OR: odds ratio in an allelic genetic model. CI: confidence interval.

BHLHE40 variants are associated with pediatric TB in South Africa.

We next evaluated whether variants in REL or BHLHE40 were associated with pediatric TB in South Africa (Figure S2) (48). SNP rs4496464 was not associated with pediatric TB. However, in this gene, BHLHE40 SNP rs11130215 was associated with decreased risk for pediatric TB in an allelic model (Table 2 and Data Set 2; p = 0.001) which best fit a dominant model of inheritance p = 3.3x10⁻⁴, OR 0.5 (0.33 – 0.75). Rs11130215 was in low LD with rs4496464 in the South African cohort (R² 0.10, D’ 0.30). To adjust for ethnic heterogeneity, we genotyped a panel of 95 ancestry informational markers (AIMs) and performed principal components analysis, as described previously (37). The association between rs11130215 and pediatric TB remained statistically significant after adjustment for gender and the top five principal components of the tested AIMs (Table 2, p = 0.01, OR 0.24 - 0.83). No REL SNPs were associated with pediatric TB. Together, these data suggest that a BHLHE40 polymorphism (rs11130215) with low linkage to rs4496464 is associated with decreased risk for pediatric TB.

Table 2. Association of SNPs with pediatric TB in South Africa.

Number of individuals with major homozygous (AA), heterozygous (Aa), and minor homozygous (aa) genotypes described.

		Control				Case
locus	Gene	AA	Aa	aa	Total	AA	Aa	aa	Total	Allelic p	Dom p	OR (95% CI)
rs11130215	BHLHE40	99	169	65	333	78	67	25	170	0.001	3.3x10−4	0.5 (0.33 – 0.75
											0.012*	0.56 (0.28-0.87)*
rs4496464	BHLHE40	158	141	35	334	86	66	17	169	0.51	0.48	1.21 (0.72-2.0)
											0.39*	1.30 (0.71-2.4)*

Allelic p: p value in an allelic genetic model. Dom p: p value in a dominant genetic model by logistic regression with adjustment for ancestry and sex. OR: odds ratio; CI: confidence interval.

^*Adjusted for ethnicity and sex by logistic regression.

BHLHE40 SNP rs11130215 is associated with BCG-induced T cell responses in South African infants.

We next examined whether rs842618, rs842634, rs496464, or rs11130215 were associated with adaptive immune responses, as a possible mechanism of TB susceptibility from DC regulation of T cell responses. We tested this hypothesis in a cohort of South African infants that were vaccinated with BCG at birth and whose BCG-specific CD4+ IL-2, TNF, and IFNγ+T cell responses were measured at 10 weeks of age by flow cytometry (36, 37) (Figure 7A). Overall media (Figure 7B), BCG-induced (Figure 7C), and SEB-induced (Figure 7D) responses are shown. We evaluated the association between genetic variation in our SNPs of interest: rs842634, rs842618, rs4496464, and rs11130215, with the frequency of BCG-induced IL-2, TNF, and IFNγ in CD4+ T-cells. SNPs rs842618 and rs842634 were monoallelic in the South African cohort and not analyzed further. SNP rs4496464 was associated with a trend toward increased IL2+CD4+ T cell frequency, but this did not achieve statistical significance (Figure 7E, p = 0.15, generalized linear model). This SNP was not associated with TNF or IFNγ frequency in CD4+ T cells (Figure 7F–G). The G allele of BHLHE40 rs11130215 was associated with increased frequency of BCG-specific IL2+CD4+ cells (Figure 7H, p = 0.015, generalized linear model), but not TNF or IFNγ (Figure 7I–J). In a second cohort of South African infants, rs11130215 was associated with a trend toward increased IL-2 expression that did not achieve statistical significance (Figure 7J, p = 0.06, generalized linear model). However, when these data were combined, we found that this SNP was associated with increased IL-2 from CD4+ T cells (Figure 7L, p = 0.006, generalized linear model). Taken together, these data suggest that a BHLHE40 SNP rs11130215 G allele is associated with increased IL-2+CD4+ T cell frequency and decreased risk for pediatric TB in a genetic cohort of South African infants.

Figure 7. BHLHE40 SNP rs11130215 is associated with BCG-induced IL-2+CD4+ T-cell responses in South African infants.

BCG-specific CD4+ T cell responses were measured in 181 South African infants at 10 weeks of age, divided into discovery (n = 89) and validation (n = 92) cohorts, using flow cytometry and stratified by genotype of interest. Background correction was performed by subtracting the proportion of cytokine-producing cells after BCG or SEB stimulation from media control stimulation.

A) Gating strategy for T cell responses measured in South African infants. Briefly, singlets were selected, followed by CD14− cells. After selecting lymphocyte populations, CD3+ cells, then CD4+ cells were selected. Gates for cytokines are shown and representative images of samples stimulated with media, BCG, and SEB is shown.

B-D) B) Media control, C) BCG-induced, and D) staphylococcus enterotoxin B (SEB)-induced IL-2, TNF, and IFNγ+ CD4+ T cell responses. N = 88.

E-G) We measured the frequency of BCG-specific D) IL-2+, E) TNF+, and F) IFNγ+ CD4+ T cells after 12 hours of re-stimulation and stratified by rs4496464. A/A N = 29, G/A N = 44, G/G N = 11.

H-J) We measured the frequency of BCG-specific G) IL-2+, H) TNF+, and I) IFNγ+ CD4+ T cells after 12 hours of re-stimulation and stratified by rs11130215 in a discovery cohort. A/A N = 24, G/A N = 31, G/G N = 19.

K) Proportion of BCG-specific IL-2+CD4+ T cells, stratified by rs11130215, in an independent validation set. A/A N = 26, G/A N = 47, G/G N = 20.

L) Combined datasets from E) and J).

All data visualized as Tukey plots, with middle bar representing median, thick bars with interquartile range, and whiskers drawn to 10-90^th percentile. Outliers are represented with dots. All data presented in this figure represent background-corrected values (proportion of cytokine-producing cells after BCG – proportion of cytokine-producing cells after media control stimulation). * p < 0.05, ** p < 0.01, generalized linear model.

Discussion

IL-12 and IL-10 are both essential for an effective host response to tuberculosis, and overexpression of either cytokine can similarly lead to adverse outcomes. In this paper, we found that variation in REL SNP rs842618 and BHLHE40 SNPs rs4496464 are associated with secretion of IL-12 and IL-10, respectively, from peripheral blood DCs using a flow cytometry-based assay. To our knowledge, this assay has not been used previously to evaluate the genetics of DC immune responses (20, 49). REL SNP rs842618 was associated with increased expression of IL-12, increased REL mRNA expression, and increased susceptibility to TBM. SNPs in BHLHE40 associated with increased IL-10 and pediatric TB. These data represent the most comprehensive evaluation of the human genetic loci associated with IL-10 and IL-12 production in TB pathogenesis.

REL SNP rs842618 was associated with IL-12 expression in peripheral blood DC and monocytes, REL mRNA expression, and TBM risk in a Vietnamese cohort. These studies provide evidence that common variation in an upstream regulator of IL-12 and innate immune activation are associated with TB outcomes. Study of the literature and publicly available datasets demonstrates strong ancillary evidence for its functional significance. Whole genome studies demonstrate that variation in rs842618 is associated with REL mRNA expression in lymphoblastoid cells immortalized rom European donors (50) and in monocytes overall(51). As a possible mechanism of action, rs842618 alters two transcription factor motifs, RREB-1 and Foxp1(52). These data provide evidence from the literature supporting our translational observations that this variant is associated with TBM in Vietnam. Identification of genetic factors that modulate dendritic cell proinflammatory cytokines provides insight into the optimal balance of cytokines to control Mtb in adults.

Variants in the BHLEH40 gene region were associated with immune outcomes and TB susceptibility in a South African and European population. To our knowledge, these data represent the first association of variants within this gene region with innate immune activation. Prior genetic studies have provided evidence of association with developmental and metabolic phenotypes, including cleft palate (53), diabetes mellitus(54), and late-onset Alzheimer’s disease(55, 56). The relationship between these phenotypes and TB remains unexplored. BHLHE40 SNP rs4496464 was associated with IL-10 production from DC and macrophages after TBWCL stimulation, but not with IL-10 from LPS-stimulated macrophages only. These data suggest that IL-10 production might be preserved via an alternate mechanism in the setting of TLR4 stimulation. Rs11103215 was also associated with increased BCG-specific IL-2+CD4+ T cells with stable frequencies of TNF+ and IFNγ+ CD4+ T cells in South African infants and decreased risk for developing pediatric TB. Our observations are consistent with a model whereby modest increases in BHLHE40 are associated with increased IL-10 in macrophages, expanded IL-2+CD4+ T cell responses, and protection from TB. Notably, these data support findings from the mouse model, where BHLHE40 deficiency was associated with early Mtb death due to excessive neutrophil-dominant inflammatory response (34). Study of the factors that influence IL-10 expression may provide insight into a suite of macrophage or T cell changes that may provide insight into TB susceptibility and control.

This study has several limitations. We do not yet have evidence of functional SNPs that directly regulate gene function. Future fine-mapping studies with in vitro mechanistic assays will be required to determine the specific alleles that regulate cellular function and clinical outcomes together. Some of these observations do not achieve statistical significance after adjustments for multiple comparisons with associations with clinical outcomes. Although this limitation is true for the clinical findings, the evidence supporting a genetic regulatory role of human cellular IL12/IL10 responses was robust and provided support for the possible clinical associations. Given this, we used a threshold of p < 0.05 as a measure of statistical significance, without the conservative Bonferroni correction. Further studies will be needed in additional cohorts, particularly after discovery of the causal SNP that regulates cytokine production. Third, case-control studies of TB outcomes may have misclassification of controls, as we examined population controls in studies in our Vietnamese cohort. However, classification errors that arise from such control populations likely lead to reduction in the statistical power of these studies.

To our knowledge, this study represents the most comprehensive analysis to date of genetic regulation of dendritic cell IL-12 and IL-10 production by common polymorphisms and their association with TB outcomes. Although further studies are required, overlapping genetic studies of immune outcomes and TB clinical susceptibility may lead to important breakthroughs in TB vaccine design and immune drug development.

Key points:

• SNPs in REL and BHLHE40 are associated with IL-12 and IL-10 and TB susceptibility.

• Genetic associations with secondary traits may identify TB susceptibility factors.