Associated with Susceptibility to Tuberculosis Signaling and Its Polymorphisms Are Human TOLLIP Regulates TLR2 and TLR4

The Journal of Immunology

Human TOLLIP Regulates TLR2 and TLR4 Signaling and Its Polymorphisms Are Associated with Susceptibility to Tuberculosis

Javeed A. Shah,* Jay C. Vary,† Tran T. H. Chau,‡,x Nguyen D. Bang,{ Nguyen T. B. Yen,{ Jeremy J. Farrar,‡,x Sarah J. Dunstan,‡,x and Thomas R. Hawn*

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 3 10216 and 6.97 3 1027, 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. The Journal of Immunology, 2012, 189: 1737–1746.

uberculosis is one of the leading causes of infectious core structure), lipids, and LAM (5, 6, 12–14). Despite the multiple death worldwide, yet its immune pathogenesis remains lines of evidence demonstrating a critical role for TLR mediation T incompletely understood (1, 2). The innate immune sys- of M. tuberculosis recognition in vitro, the in vivo significance of tem is critical for the initial host defense against mycobacteria (3). individual TLRs has been more difficult to consistently dem- Several lines of evidence suggest that TLR1, TLR2, TLR4, TLR6, onstrate. Although some studies demonstrated increased suscepti- TLR8, and TLR9 are important for host defense against Myco- bility to M. tuberculosis in MyD882/2,andTlr22/2,andTlr2/92/2 bacterium tuberculosis (4-11). For example, in vitro studies in mice, these results have not been uniformly observed under all murine macrophages demonstrate that TLR1, TLR2, TLR4, experimental conditions (10, 18, 21, 22-25). Furthermore, the TLR6, TLR9, and MyD88 regulate M. tuberculosis-induced cy- effects appear to be strongest when multiple TLRs are impaired. tokine secretion (5, 12-21). The mycobacterial cell wall contains a Together, these in vivo studies suggest an important though not number of proinflammatory TLR2 ligands, including lipoproteins, absolute role for the TLR pathway in mediating host protection to mycolylarabinogalactan–peptidoglycan complex (the cell wall in vivo murine M. tuberculosis infection. Through the TLR pathway, proinflammatory (IL-6, TNF, and IL-12) and anti-inflammatory (IL-10) cytokines are secreted from *Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA macrophages stimulated with M. tuberculosis (26, 27). The in- 98195; †Division of Dermatology, University of Washington, Seattle, WA 98195; ‡Centre for Tropical Medicine, Oxford University Clinical Research Unit, Ho Chi terplay between proinflammatory and anti-inflammatory cytokines Minh City, District 5, Vietnam; xNuffield Department of Clinical Medicine, Centre is important in determining the quality and quantity of both the for Tropical Medicine, Oxford University, Oxford OX3 7LJ, United Kingdom; and innate and adaptive immune response to pathogens. The signaling {Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, Ho Chi Minh City, Vietnam mechanisms that specifically modulate IL-10 production after Received for publication December 8, 2011. Accepted for publication June 4, 2012. TLR stimulation are unclear, and understanding how IL-10 secretion is modulated by TLR regulators has important implications This work was supported by National Institute of Allergy and Infectious Diseases at the 2/2 National Institutes of Health Grants 1K24AI089794 (to T.R.H.) and 5T32AI00704432 in M. tuberculosis pathogenesis and vaccine design. IL-10 (to J.A.S.), the Burroughs Wellcome Foundation (to T.R.H.), and the Wellcome Trust mice develop severe colitis in response to gut microbiota (28), of Great Britain (to J.J.F.). making extrapolation of the role of IL-10 in humans difficult. Address correspondence and reprint requests to Dr. Javeed A. Shah, Division of Several investigators reported that IL-102/2 mice have enhanced Allergy and Infectious Diseases, University of Washington, 1959 NE Pacific Street, Box 356423, Seattle, WA 98195. E-mail address: [email protected] M. tuberculosis clearance (29), but others have reported no such The online version of this article contains supplemental material. differences (30, 31). Human studies of the role of IL-10 in tu- Abbreviations used in this article: HTD, Hospital for Tropical Diseases; OR, odds berculosis (TB) have shown that IL-10 levels are increased in lung ratio; PNT, Pham Ngoc Thach; PTB, pulmonary tuberculosis; shRNA, short hairpin tissue of patients with pulmonary TB (32). Furthermore, neutral- RNA; SNP, single nucleotide polymorphism; TB, tuberculosis; TBM, tuberculous ization of IL-10 enhances IFN-g and T cell proliferation in vitro meningitis. (33). Human genetic studies examining whether polymorphisms in Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 the IL-10 gene are associated with susceptibility to TB are in- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103541 1738 TOLLIP VARIANTS AND TB conclusive (34-36). Together, these studies suggest that the role of CD14 Ab associated with magnetic beads (Miltenyi Biotec, Auburn, CA). IL-10 in the pathogenesis of TB remains unclear and further study Monocytes were isolated with .95% purity, based on CD14 positivity in is required. flow cytometric analysis and were maintained in RPMI 1640 supplemented with 10% FCS. Cells were incubated with TLR ligands PAM2, PAM3, and Negative regulators have been discovered at nearly every step of LPS, or with M. tuberculosis whole cell lysates for 24 h. Subsequently, the TLR signaling cascade (37–39) and include Toll-interacting supernatants were evaluated for levels of cytokine (IL-6, IL-10, TNF) via protein (TOLLIP, also called IL-1RAcPIP), A20 (40), Single Ig ELISA Duoset (R&D Systems, Minneapolis, MN). Each sample was IL-1R (SIGIRR, also called TIR8) (41), and IL-1R–associated assayed in duplicate or triplicate, and experiments shown were performed at least twice to ensure reproducibility. kinase 3 (IRAK-3, also called IRAK-M) (42). TOLLIP is a 274- aa protein with highly conserved C2 (amino acids 54-186, similar Genotyping and linkage disequilibrium to that found in phosphoinositol-specific phospholipase C-d1I) and Genotyping was performed with Sequenom’s MassARRAY technique as C-terminal ubiquitin-associated domains. It was initially discov- described previously (54). This technique uses allele-specific primer ex- ered as an IL-1R1–interacting protein that linked IRAK to the IL- tension reactions to discriminate genotypes. We also confirmed the SNPs 1R pathway (43), and later was implicated in suppression of the of interest using Taqman genotyping technology (Applied Biosystems, TLR2 (44) and TLR4 pathways (45). However, in vivo murine Carlsbad, CA). We identified haplotype-tagging SNPs from the CHB (Han Chinese in Beijing) and CEU (Utah residents with European ancestry) knockout models demonstrated that TOLLIP induced proin- populations from the International HapMap Project (http://www.hapmap. flammatory pathways, in contrast to in vitro experiments (46). org) and other public databases with the Genome Variation Server (http:// To our knowledge, there are no studies examining the effects of www.ncbi.nlm.nih.gov/SNP/ and www.innateimmunity.net). We searched TOLLIP on TLR signaling pathways in humans. a region on chromosome 11p15.5, 10 kb upstream and downstream of TOLLIP for tagged SNPs using an R2 cutoff of 0.8 for linkage disequi- Several lines of evidence suggest that host genetics regulates librium and a minor allele frequency cut-off of 5%. Stata/Intercooled susceptibility to TB, including twin-based, linkage, candidate gene version 11.0 software program PWLD (StataCorp, College Station, TX) association, and genome-wide association studies. We, along with was used to calculate R2 and D9 as measurements of linkage disequilib- other investigators, have identified associations between common rium between the polymorphisms. polymorphisms in innate immunity genes and susceptibility to TB Molecular biology and clinical phenotypes (22, 47, 48). Much of this work has focused on pattern recognition receptors and their associated adaptor pro- TOLLIP mRNA levels were measured in monocytes from peripheral blood of healthy donors in the Seattle area. RNA was obtained after cell lysis and teins, including TLR 1, 2, 4, 6, and 9. Several candidate gene RNA purification (Qiagen, Valencia, CA). After synthesis of cDNA, real- association studies have shown associations between single time PCR was performed. TOLLIP mRNA levels were normalized with nucleotide polymorphisms (SNPs) in TLR2 and TB, including a GAPDH control using the following primer-probe sets (59-J56-FAM/ 597CC within a Vietnamese population (8, 49). A polymorphism in CTCCGGGAT/ZEN/GGT-39,59-CTCAGGCTGTACCACTTG-39,59- GGCGTGGACTCTTTCTAT-39; Integrated DNA Technologies, Coralville, TLR1 (T1805G, I602S) regulates lipopeptide-induced cytokine IA). Quantitative PCR was performed on a Taqman machine. secretion and TLR1 surface expression, and is associated with susceptibility to TB as well as leprosy (48, 50–52). Genetic studies Gene silencing have also found associations of TLR4 polymorphisms with TB Knockdown studies were performed using plasmids encoding three susceptibility (10, 24, 25). These studies suggest that genetic TOLLIP-specific short hairpin RNA (shRNA; Santa Cruz Biotechnology, control of TLR1/2/4/6 signaling by negative regulators may alter Santa Cruz, CA) that were then packaged into a nonreplicating lentiviral susceptibility to TB. In contrast to TLRs, the functional and clin- particle to improve transfection efficiency using a standard protocol (UCSF Viracore Facility, San Francisco, CA). These lentiviral particles ical significance of genetic variation of TOLLIP and other negative were then incubated with monocytes at a multiplicity of infection of 2 for regulators of TLR signaling is poorly studied and only partially 24 h before cells were stimulated and harvested for cytokine analysis. understood. In this study, we examined the function of TOLLIP in Knockdown was confirmed using quantitative PCR for TOLLIP mRNA human monocytes with knockdown studies and examination of transcript. common TOLLIP variants. We also examined whether these Human subjects polymorphisms were associated with susceptibility to TB in Viet- nam. These studies suggest that TOLLIP is a critical regulator of Approval for human study protocols was obtained from the human subjects review boards at the University of Washington School of Medicine (Seattle, the TLR pathway in humans and that this immune regulation has WA), the Hospital for Tropical Diseases (Ho Chi Minh City, Vietnam), a critical role in the susceptibility to TB. 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 Materials and Methods Ethics Committee (Oxford, U.K.). Materials Genomic DNA was purified from saliva and blood samples. The Seattle study group included 56 healthy volunteers who donated blood and saliva RPMI 1640 medium, L-glutamine, penicillin, and streptomycin were samples. The ethnic composition of these subjects was 70% Caucasian (39/ obtained from Life Technologies (Carlsbad, CA). Ultrapure LPS (TLR4 ligand) isolated from Salmonella minnesota R595 was obtained from 56), 27% Asian (15/56), and 4% African American (2/56). Study subjects List Biological Labs (Campbell, CA). The synthetic lipopeptides PAM2 with tuberculous meningitis (TBM) were recruited from two centers in Ho (PAM2CSKKKK, S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl) Chi Minh City, Vietnam: Pham Ngoc Thach (PNT) Hospital for Tuberculosis 3-lysine, TLR2/6 ligand) and PAM3 (Pam3CSKKKK, N-palmitoyl-S- and the Hospital for Tropical Diseases (HTD). These 500-bed hospitals serve [2,3-bis-(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine, TLR2/1 li- the local community and act as tertiary referral centers for TB (PNT) and gand) were obtained from EMC Microcollections (Tuebingen, Germany). infectious diseases (HTD) for southern Vietnam. Individuals at least 15 y old, Whole cell lysate from M. tuberculosis strain H37Rv was obtained as part admitted to these centers with clinical meningitis (defined as nuchal rigidity of National Institutes of Health, National Institute of Allergy and Infec- and abnormal cerebrospinal fluid parameters), a negative HIV test result, and M. tuberculosis tious Diseases Contract No. HHSN266200400091C, entitled Tuberculosis a positive Ziehl–Neelsen stain for acid-fast bacilli or cul- Vaccine Testing and Research Materials (Colorado State University, Fort ture, or both, from cerebrospinal fluid (“definite TBM”) were recruited for Collins, CO). genetics studies during 2001 to 2008. In addition to definite TBM, the cohort included subjects with “probable TBM,” defined as clinical menin- Cell culture and ELISA gitis plus at least one of the following: chest radiograph consistent with active TB, acid-fast bacilli found in any specimen other than cerebrospinal Primary monocytes were isolated from human subjects as described pre- fluid, and clinical evidence of other extrapulmonary TB (55). viously (53). Human monocytes were isolated from peripheral blood with From 2003 through 2004, subjects with pulmonary TB were recruited Ficoll gradient separation followed by positive selection using human anti- from a network of district TB control units within Ho Chi Minh City that The Journal of Immunology 1739 provide directly observed therapy to TB patients. These subjects were Nine TOLLIP polymorphisms were genotyped in the Vietnam and outpatients who were at least 15 y old, had no previous history of treatment Seattle cohorts. Two (rs4744015 and rs5744016) were monogenic and one for TB, no evidence of miliary or extrapulmonary TB, chest radiograph (rs5743890) was not in Hardy-Weinberg equilibrium and thus not analyzed results consistent with nonmiliary pulmonary TB, negative HIV test results, further. Six are shown in all subsequent analyses. If we correct for multiple and sputum smear positive for acid-fast bacilli or M. tuberculosis cultured comparisons with a Bonferroni test (p value/6), only p values less than from sputum. In addition, pulmonary TB subjects enrolled were recruited 0.008 remain significant. However, as we had two single nucleotide from PNT hospital from 2006 through 2008. polymorphisms of interest based upon prior functional data, we show Control subjects were enrolled at Hung Vuong Hospital in Ho Chi Minh uncorrected p values in these tables. City from blood collected from the umbilical cord of babies after birth. All The Pearson-x2 test and Student t test were used to assess categorical case and control participants were unrelated, and greater than 95% were of and continuous clinical variables, respectively. For analysis of associations the Vietnamese Kinh ethnicity. Written, informed consent was obtained between TOLLIP polymorphisms and mRNA levels or cytokines, we used from patients or their relatives if the patient could not provide consent an ANOVA analysis with Mann–Whitney tests for significance. (i.e., was unconscious). Parents provided consent for cord-blood controls. Out of the study subjects that self-identified their ancestry as Vietnamese Kinh, we formally tested for levels of background genetic differences Results between cases and controls by genotyping the cohort for a panel of 24 TOLLIP regulates the TLR signaling pathway in humans 2 independent SNP markers (56). The mean x statistic for the 24 SNPs In order to determine the functional role of TOLLIP in the human comparing allelic frequencies between cases and control was 1.6 (p = 0.20), suggesting that no significant population stratification was present in our innate immune system, we performed shRNA knockdown ex- study population (57). periments on peripheral blood monocytes from healthy volunteers (Fig. 1). Using a lentiviral delivery system in primary monocytes, Statistical methods we knocked down TOLLIP mRNA expression by more than 50% All analyzed SNPs were tested for Hardy-Weinberg equilibrium in control compared with monocytes alone or those treated with control subjects using a x2 goodness-of-fit test. In our primary analysis, we ex- lentiviral particles (Fig. 1A; p , 0.001, Student t test). We also amined whether polymorphism genotype frequencies were associated with tested and confirmed that TLR2 and TLR4 mRNA expression was any type of TB in a case-population study design using Stata MP 11 and not altered by TOLLIP shRNA (Fig. 1A; p = 0.78 for TLR2, p = the user written package “genassoc” (58). For secondary analyses, SNPs were investigated for associations under additional genetic models (dom- 0.14 for TLR4; Student t test comparing control to TOLLIP inant, recessive, and additive) and for association with the clinical subtypes shRNA viral infection). After stimulation with PAM2 (TLR2/6 of TB (pulmonary or meningeal). In the recessive model, carriers of allele ligand) and PAM3 (TLR2/1 ligand), TOLLIP-deficient mono- 1 (00 and 01 genotypes) were compared with homozygous subjects for cytes produced elevated levels of IL-6 compared with either 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 untransfected monocytes or control-lentiviral particle-transfected genotype). Measures of linkage disequilibrium were assessed in controls monocytes (p , 0.01; Fig 1B–F). Transfection with control using “pwld” in Stata. shRNA led to a mild increase in IL-6 that we attributed to the

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 three areas within the TOLLIP gene for 24 h. These cells were then incubated with TLR ligands for 24 h, 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 of two. IL-6 (B–F), TNF (G–K), and IL-10 (L–P) cytokine responses after media, PAM2, PAM3, or LPS stimulation in primary monocytes incubated with nothing, control shRNA, or TOLLIP shRNA- encoding lentiviral particles. M. tuberculosis stimulation is from an independent experiment. *p , 0.01, ANOVA with Mann–Whitney U test. 1740 TOLLIP VARIANTS AND TB innate inflammatory response to lentivirus infection and cytosolic recessive model. (p , 0.01; AA = 49, AG = 31, GG = 4; Fig. 2A). DNA. In contrast, LPS (TLR4 ligand)-induced IL-6 levels were The other 4 TOLLIP SNPs were not significantly associated with similar under all conditions (Fig. 1E). TOLLIP knockdown led to mRNA expression (Fig. 2B, 2C, 2E, 2F). Three SNPs (rs3793964, increased TNF production after stimulation with PAM2, PAM3, CC = 36, CT = 24, TT = 16; rs3829223, CC = 19, CC = 32, TT = LPS, or M. tuberculosis whole cell lysate (p , 0.01; Fig. 1G–K). 27; and rs3168046, CC = 15, CT = 22, TT = 14) showed a trend In cells stimulated with PAM2, PAM3, or M. tuberculosis whole toward an association with mRNA expression (Fig. 2C, 2E, 2F; cell lysate, there were no significant differences in IL-10 produc- p = 0.26, 0.22, and 0.14, respectively). These SNPs were in tion between cells stimulated with nonspecific lentiviral particles linkage disequilibrium with SNP rs3750920 (Supplemental Fig. or TOLLIP-specific viral particles (Fig 1M, 1N). Intriguingly, 1A; R2 = 0.43, 0.55, 0.73 for pairwise comparisons of three SNPs TOLLIP knockdown in LPS-stimulated cells led to significant with rs3750920). These results suggest that at least one TOLLIP decreases in secreted IL-10 from monocytes (p , 0.01; Fig 1O). polymorphism is associated with mRNA expression and is a ge- In a separate experiment with stimulation with M. tuberculosis netic marker of human TOLLIP deficiency. H37Rv whole cell lysate, we found that TOLLIP knockdown led Association of TOLLIP SNP rs5743899 with cytokine to increased IL-6 and TNF secretion, but not IL-10 compared with responses to TLR ligands controls (p , 0.01; Fig. 1F, 1K, 1P). These data suggest that TOLLIP regulates the innate immune We next examined whether polymorphisms rs5743899 and response in humans via two mechanisms—by suppressing proin- rs3750920 were associated with cytokine responses in monocytes. flammatory cytokines via TLR2 and TLR4 and by inducing IL-10 We stimulated PBMCs with TLR ligands (PAM2, PAM3, and LPS) through a TLR4-specific mechanism. as well as M. tuberculosis whole cell lysate, and measured secreted IL-6 and IL-10 in the culture supernatants (Fig. 3A, 3B). Minor Association of TOLLIP SNPs with mRNA Expression homozygotes (GG) of polymorphism rs5743899 were associated Next, we examined whether common variants of TOLLIP regulated with significantly higher levels of IL-6 compared with hetero- its function. We genotyped nine haplotype-tagging SNPs in 84 zygotes or major homozygotes (Fig 3C, 3D, 3E; AA individuals = healthy volunteers and examined their association with TOLLIP 35, AG = 21, GG = 7; p , 0.01, ANOVA with Mann–Whitney U mRNA expression in monocytes. (Fig. 2, polymorphisms listed in test) after stimulation with PAM2 (TLR2/TLR6) or PAM3 (TLR2/ genomic order) For SNP rs3750920, the minor homozygote was TLR1), but not LPS (TLR4). Furthermore, IL-6 levels were sig- significantly associated with increased mRNA expression com- nificantly increased after stimulation with M. tuberculosis whole pared with either heterozygotes or major homozygotes (p , 0.01 cell lysate (Fig. 3F). In addition, IL-10 levels were significantly by genotypic model; CC = 26, CT = 32, TT = 16; Fig. 2D). This decreased in a recessive pattern for GG individuals after LPS association was also significant in a recessive model comparing TT stimulation (p = 0.03; Fig 3G). homozygotes with CT/CC genotypes (p , 0.01). SNP rs5743899 There were not any significant changes in IL-10 production after genotype GG was associated with decreased mRNA transcript in a PAM2 or PAM3 stimulation. By way of contrast, minor homo-

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. (A) rs5743899: AA = 49, AG = 31, GG = 4. (B) rs5743942: CC = 17, CT = 31, TT = 20. (C) rs3793964: CC = 36, CT = 24, TT = 16. (D) rs3750920: CC = 26, CT = 32, TT = 16. (E) rs3829223: CC = 19, CC = 32, TT = 27. (F) rs3168046: CC = 15, CT = 22, TT = 14. *p , 0.01; #p = 0.15, ANOVA analysis with Mann–Whitney U test. The Journal of Immunology 1741

FIGURE 3. TOLLIP SNP rs5743899 and cytokine responses after TLR and M. tuberculosis stimulation of PBMCs. PBMCs 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, M. tuberculosis whole cell lysate at 1 mg/ml) for 24 h. Secreted IL-6 (A) and IL-10 (B) 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 U test in a recessive model. zygotes from rs3750920 were not associated with IL-6 levels when berculosis whole cell lysate stimulation, as well as decreased IL- compared with heterozygotes or major homozygotes (Fig. 4A–D; 10 after LPS stimulation. CC = 18 individuals, CT = 24, TT = 12). Furthermore, no associations were noted in IL-10 production (Fig 4E–H). These data Association of TOLLIP SNPs with susceptibility to TB demonstrate that the minor homozygote of rs5743899 is associ- To assess the role of TOLLIP deficiency in human disease, we ated with increased levels of IL-6 after PAM2, PAM3, or M. tu- performed a candidate gene case-population association study to

FIGURE 4. TOLLIP polymorphism rs3750920 and cytokine responses after TLR and MTb stimulation of PBMCs. Identical experimental details as Figure 3, except that data were stratified by genotype rs3750920; CC = 18 individuals, CT = 24, TT = 12. IL-6 (A–D) responses and IL-10 (E–H) responses after stimulation with media or TLR ligands (LPS at 10 ng/ml, PAM2 at 250 ng/ml, PAM3 at 250 ng/ml, M. tuberculosis whole cell lysate at 1 mg/ml) for 24 h. 1742 TOLLIP VARIANTS AND TB examine associations between TOLLIP polymorphisms and sus- (Table II). Finally, we evaluated whether these two SNPs were ceptibility to TB. We examined 671 cases (394 pulmonary TB associated with death or disability from TB, as well as with CSF [PTB] and 277 TBM) and 760 cord blood controls in our cohort. We and serum cytokine responses at the time of presentation (for first analyzed polymorphisms rs3750920 and rs5743899 because subjects with TBM). We did not find any significant associations of their association with functional phenotypes. TOLLIP poly- with these outcomes (data not shown). These data suggest that two morphisms had similar linkage disequilibrium patterns in Seattle TOLLIP polymorphisms are associated with susceptibility to TB and Vietnam, as well as the Northern European (CEU) and Han as well as TOLLIP expression levels or regulation of TLR- Chinese (CHB) HapMap populations (Fig. 5, Supplemental Figs. 1 mediated cytokine secretion. and 2). Polymorphisms rs3750920 and rs5743899 were associated with susceptibility to TB using a genotypic model (Table I; Discussion 2 2 rs3750920, p = 7.03 3 10 16; and rs5743899, p = 6.97 3 10 7). Our study shows that TOLLIP regulates human TLR signaling Three other SNPs (rs3793964, rs3829223, and rs3168046) were pathways in monocytes by suppressing proinflammatory cytokines associated with TB, and these SNPs were in a high degree of (IL-6 and TNF) and inducing anti-inflammatory cytokine (IL-10) linkage disequilibrium with SNP rs3750920 in the Vietnamese secretion in peripheral blood monocytes. In addition, we found two population (Supplemental Table I). All these SNPs were associ- common genetic variants of TOLLIP that are associated with ated with TB after a conservative Bonferroni correction for mul- TOLLIP mRNA expression or TLR-mediated cytokine release, or tiple comparisons. One SNP (rs5743942) was associated with TB both. Finally, these genetic markers of TOLLIP deficiency were in an unadjusted analysis, but not after a Bonferroni correction. associated with an increased risk for developing TB in a case- population study in Vietnam. Secondary analysis of rs3750920 and rs5743899 Our data from knockdown of TOLLIP in peripheral blood For the two significant polymorphisms, we examined these asso- monocytes indicates that TOLLIP is a negative regulator of TNF ciations further under different genetic models and clinical phe- and IL-6 after PAM2, PAM3, and LPS stimulation. Although these notypes. Testing the association with TB under different genetic data demonstrate that TOLLIP regulates human TLR2 and TLR4 models for rs3750920, we found that the strongest association was function, the findings appear to be in contrast to murine models, consistent with a dominant model (Table II; odds ratio [OR], where Tollip2/2 bone marrow-derived macrophages and dendritic 0.453; p = 6.28 3 10212). In contrast, SNP rs5743899 was most cells had decreased LPS-induced IL-6 and TNF in comparison strongly statistically associated with all forms of TB in a recessive with wild type cells (46). The reasons for this discrepancy are model (OR, 1.641; p = 0.0004). We also examined the associations uncertain. It is possible that the unique gene splicing profile of with different clinical forms of TB (e.g., PTB) and TBM. Both murine TOLLIP led to the proinflammatory profile in the knock- SNPs were associated with PTB and TBM to a similar degree out (59) or that cell type or assay conditions were different be-

FIGURE 5. Chromosomal map and linkage disequilibrium plots of TOLLIP polymorphisms in a Vietnamese cohort. (A) Chromosomal map shows genomic location of polymorphisms. Boxes show exons within gene on chromosome 11. rs3168046 was located on the 39 UTR for the TOLLIP gene. (B, C) Linkage disequilibrium plots with R2 and D9 values for controls in Vietnam. White boxes on top of plot show frequency of polymorphisms. Degree of shading is proportionate to D9 or R2 value. The Journal of Immunology 1743

Table I. TOLLIP gene region single nucleotide polymorphisms and genotype frequencies in the control and tuberculosis groups

Genotype Genotype Analysis

SNPa Group 00 01 11 x2 pH-W rs3750920 Control 308 (0.415) 355 (0.479) 78 (0.105) — — — TB 328 (0.610) 134 (0.253) 75 (0.129) 69.8 7.03 3 10216 0.101 rs5743899 Control 252 (0.334) 391 (0.520) 110 (0.145) — — — TB 261 (0.397) 252 (0.383) 144 (0.220) 28.4 6.97 3 1027 0.163 aSNPs are arranged in the order that they are located on the chromosome. SNPs are listed by reference SNP ID. 0, Common allele; 1, allele with minor frequency. Hardy-Weinberg (H-W) equilibrium was calculated from control subjects only. tween the human and murine experiments. Possibly, another set of mimic the phenotype elicited by shRNA knockdown. The mech- regulatory molecules may exist apart from TOLLIP and distin- anism that leads to its loss of function is unclear. We found that guish TLR2 and TLR4 signaling pathways in mice and humans. minor homozygotes of rs5743899 were associated with a trend of These species-specific findings underscore the well-recognized less TOLLIP mRNA in comparison with heterozygotes and major and important concept that murine data cannot always be ex- homozygotes, suggesting that differences in TOLLIP expression trapolated and assumed to be similar to humans. levels regulate cytokine responses. Our analysis of the case- In contrast to the proinflammatory cytokines, TLR2- and TLR4- population data supports this theory, because there is an associa- mediated IL-10 secretion was decreased after TOLLIP knockdown tion between rs5743899 and risk for developing TB in a recessive in peripheral blood monocytes. To our knowledge, TOLLIP is the model. The causative polymorphisms are not known, and this only signal transduction molecule to downregulate IL-6 and TNF complicates interpretation of the data. In addition to regulation of while upregulating IL-10. LPS upregulates IL-10, TNF, and IL-6 in transcription, the possible mechanisms include linkage disequi- monocytes, presumably by activation of similar transcription librium of the associated SNPs with a nonsynonymous coding factors such as NF-kB (60). However, there are several examples region functional SNP that changes protein localization or con- of the IL-10 induction pathway diverting from the IL-6 or TNF formation and leads to abnormal TOLLIP function. The C2 domain induction pathway. For example, IL-10 is induced through the of TOLLIP contains a phosphorylation domain that is hypothesized TLR2/TLR4 pathway (61) and modified by CARD9 as well as to be important for IRAK-4 binding (66). This may affect TLR IFN-b. (62) In addition, Card92/2 mice are unable to control M. signaling transduction and lead to the cytokine differences noted tuberculosis infection and do not produce significant IL-10, de- in this study. A mutation in this domain is a potential location for spite IL-6 and TNF production. (63) Furthermore, differential a loss-of-function mutation, and in vitro cell culture models have regulation of proinflammatory and anti-inflammatory signaling shown that abolishing the phosphorylation domain reduces the has been observed in HIV-infected macrophages that upregulate regulatory capacity of TOLLIP (45). Further study is needed to IL-10 and downregulate IL-12. (64) Several possible mechanisms determine the molecular mechanism of action that leads to this could explain how TOLLIP could differentially regulate proin- phenotype. flammatory and anti-inflammatory cytokine production. For ex- In contrast to rs5743899, polymorphism rs3750920 was asso- ample, the C-terminal domain of TOLLIP contains a Tom-1 ciated with protection from TB and increased levels of TOLLIP binding domain and likely plays a role in cellular trafficking and mRNA. The functional change that leads to the association of ubiquitination of proteins that could affect TLR signal transduc- rs3750920 with TB is a subject of ongoing study. There are several tion. In addition, TOLLIP can traffic from the cytosol to the cell possible mechanisms that could lead to rs3750920 affecting risk nucleus via sumoylation, suggesting a role for TOLLIP regulation for developing TB, but not IL-6 or TNF cytokine responses in of transcription factors (65). Although the mechanism by which monocytes. First, polymorphisms at rs3750920 might affect TOLLIP differentially regulates cytokines is not clear, this finding cytokines that were not examined during this study, such as IL-1b may help to elucidate the signaling pathway of IL-10. or IL-18, which could affect inflammasome activity. Second, To our knowledge, this report is the first description of TOLLIP rs3750920 may affect cytokines that are not expressed in mono- polymorphisms that are associated with a loss of function as they cytes, such as IL-12p70 or IL-23, but that are expressed in much

Table II. Associations of rs3750920 and rs5743899 with TB meningitis and pulmonary tuberculosis

Genotype Dominant Analysisa Recessive Analysis

SNP b Groupc 00 01 11 ORd CI (95%) p Value OR CI (95%) p Value Rs3750920 Control 308 (0.415) 355 (0.479) 78 (0.105) — — — — — — TBM 144 (0.415) 60 (0.255) 35 (0.128) 0.441 0.34–0.64 5.26E207 1.240 0.92–2.3 0.387 PTB 184 (0.617) 74 (0.248) 40 (0.134) 0.445 0.33–0.59 6.34E209 1.324 0.85–2.0 0.176 TB all 328 (0.610) 134 (0.253) 75 (0.129) 0.453 0.36–0.57 6.28E212 1.380 0.96–2.0 0.061 rs5743899 Control 252 (0.334) 391 (0.520) 110 (0.145) — — — — — — TBM 119 (0.422) 107 (0.379) 56 (0.199) 0.689 0.52–0.920 0.010 1.448 1.0–2.09 0.040 PTB 118 (0.372) 123 (0.388) 76 (0.240) 0.825 0.63–1.07 0.145 1.792 1.3–2.5 0.0003 TB all 261 (0.397) 252 (0.383) 144 (0.220) 0.763 0.61–0.95 0.015 1.641 1.23–2.2 0.0004 aA dominant and recessive allelic analysis was performed. bSNPs are listed by reference SNP ID. c“All TB” includes both PTB and TBM. dFor calculations of ORs, each group was compared with the control group. 0, common allele; 1, allele with minor frequency. CI, Confidence interval. 1744 TOLLIP VARIANTS AND TB higher quantities in macrophages (67) and dendritic cells. Third, inflammatory pathways. Although further study is required, mod- SNP rs3750920 may regulate protein trafficking and functions ulating TOLLIP activity and function may lead to important such as endocytosis and phagocytosis. TOLLIP is known to bind breakthroughs in TB vaccine design and immune drug development. to ubiquitinated proteins and localize with the protein Tom1 to the early endosome, and it may be important in bacterial entry (68– Acknowledgments 70). Finally, TOLLIP polymorphisms may regulate effector We thank the clinical staff from the Hospital of Tropical Diseases and Pham mechanisms that lead to bacterial killing (such as NO production). Ngoc Thach Hospital who initially diagnosed and studied the patients with Studies are ongoing to examine these possibilities and to deter- TBM and PTB; Dr. Nguyen Thi Hieu from Hung Vuong Obstetric Hospital mine the causative SNP in linkage disequilibrium with rs3750920. Vietnam; Dr. Tran Tinh Hien from the Hospital for Tropical Diseases Viet- The genetic and mRNA data from these two SNPs likely represent nam; Dr. Guy Thwaites from Imperial College London; all Vietnamese indi- two independent effects with distinct mechanisms. viduals who participated in this study; Dr. Alan Aderem and Dr. Marta Janer Interestingly, our genetic data suggests that a hypofunctional from the Institute for Systems Biology for advice and Sarah Li for technical assistance; and Glenna Peterson, Rick Wells, Dr. William Berrington, Dr. TOLLIP genotype (rs5743899 GG) is associated with an increased Ann Misch, Dr. Dave Horne, and Dr. Chetan Seshadri from the University of risk of TB, as well as increased levels of proinflammatory cyto- Washington for assistance. kines. There is a long history of observations in the TB field of the potentially deleterious effects of too much inflammation. Steroid treatment of patients with TBM and pericarditis is used to dampen Disclosures the immunopathologic consequences of inflammation (55, 71). In The authors have no financial conflicts of interest. this regard, TNF has been shown to have protective and deleterious effects in different models of TB. For example, although References knockout of TNF in murine models is deleterious with increased 1. World Health Organization. 2011. Global tuberculosis control - surveillance, M. tuberculosis replication, evidence also suggests that a hyper- planning, financing. World Health Organization, Geneva. 2. Dye, C., S. Scheele, P. Dolin, V. Pathania, and M. C. Raviglione. 1999. Con- inflammatory state can lead to increased TB replication and sensus statement. Global burden of tuberculosis: estimated incidence, preva- worsened disease (72–74). Furthermore, studies of leukotriene A4 lence, and mortality by country. 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