W. Henry Boom

Toll-like receptor 2-dependent inhibition of macrophage class II MHC expression and antigen processing by 19-kDa lipoprotein of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (MTB) induces vigorous immune responses, yet persists inside macrophages, evading host immunity. MTB bacilli or lysate was found to inhibit macrophage expression of class II MHC (MHC-II) molecules and MHC-II Ag processing. This report characterizes and identifies a specific component of MTB that mediates these inhibitory effects. The inhibitor was extracted from MTB lysate with Triton X-114, isolated by gel electroelution, and identified with Abs to be MTB 19-kDa lipoprotein. Electroelution- or immunoaffinity-purified MTB 19-kDa lipoprotein inhibited MHC-II expression and processing of both soluble Ags and Ag 85B from intact MTB bacilli. Inhibition of MHC-II Ag processing by either MTB bacilli or purified MTB 19-kDa lipoprotein was dependent on Toll-like receptor (TLR) 2 and independent of TLR 4. Synthetic analogs of lipopeptides from Treponema pallidum also inhibited Ag processing. Despite the ability of MTB 19-kDa lipoprotein to activate microbicidal and innate immune functions early in infection, TLR 2-dependent inhibition of MHC-II expression and Ag processing by MTB 19-kDa lipoprotein during later phases of macrophage infection may prevent presentation of MTB Ags and decrease recognition by T cells. This mechanism may allow intracellular MTB to evade immune surveillance and maintain chronic infection.

Regulation of antigen presentation by Mycobacterium tuberculosis : a role for Toll-like receptors

Mycobacterium tuberculosis survives in antigen-presenting cells (APCs) such as macrophages and dendritic cells. APCs present antigens in association with major histocompatibility complex (MHC) class II molecules to stimulate CD4+ T cells, and this process is essential to contain M. tuberculosis infection. Immune evasion allows M. tuberculosis to establish persistent or latent infection in macrophages and results in Toll-like receptor 2 (TLR2)-dependent inhibition of MHC class II transactivator expression, MHC class II molecule expression and antigen presentation. This reduction of antigen presentation might reflect a general mechanism of negative-feedback regulation that prevents excessive T cell-mediated inflammation and that M. tuberculosis has subverted to create a niche for survival in infected macrophages and evasion of recognition by CD4+ T cells.

Inhibition of IFN-γ-Induced Class II Transactivator Expression by a 19-kDa Lipoprotein from Mycobacterium tuberculosis: A Potential Mechanism for Immune Evasion

Mycobacterium tuberculosis (MTB) persists inside macrophages despite vigorous immune responses. MTB and MTB 19-kDa lipoprotein inhibit class II MHC (MHC-II) expression and Ag processing by a Toll-like receptor 2-dependent mechanism that is shown in this study to involve a defect in IFN-γ induction of class II transactivator (CIITA). Exposure of macrophages to MTB or MTB 19-kDa lipoprotein inhibited IFN-γ-induced MHC-II expression, but not IL-4-induced MHC-II expression, by preventing induction of mRNA for CIITA (total, type I, and type IV), IFN regulatory factor-1, and MHC-II. MTB 19-kDa lipoprotein induced mRNA for suppressor of cytokine signaling (SOCS)1 but did not inhibit IFN-γ-induced Stat1 phosphorylation. Furthermore, the lipoprotein inhibited MHC-II Ag processing in SOCS1−/− macrophages. MTB 19-kDa lipoprotein did not inhibit translocation of phosphorylated Stat1 to the nucleus or Stat1 binding to and transactivation of IFN-γ-sensitive promoter constructs. Thus, MTB 19-kDa lipoprotein inhibited IFN-γ signaling independent of SOCS1 and without interfering with the activation of Stat1. Inhibition of IFN-γ-induced CIITA by MTB 19-kDa lipoprotein may allow MTB to evade detection by CD4+ T cells.

Mycobacterium tuberculosis LprG (Rv1411c): A Novel TLR-2 Ligand That Inhibits Human Macrophage Class II MHC Antigen Processing

MHC class II (MHC-II)-restricted CD4+ T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-α production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4+ T cells.

A blood RNA signature for tuberculosis disease risk: a prospective cohort study

BACKGROUND Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease might lead to interventions that combat the tuberculosis epidemic. We aimed to assess whether global gene expression measured in whole blood of healthy people allowed identification of prospective signatures of risk of active tuberculosis disease. METHODS In this prospective cohort study, we followed up healthy, South African adolescents aged 12-18 years from the adolescent cohort study (ACS) who were infected with M tuberculosis for 2 years. We collected blood samples from study participants every 6 months and monitored the adolescents for progression to tuberculosis disease. A prospective signature of risk was derived from whole blood RNA sequencing data by comparing participants who developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex quantitative real-time PCR (qRT-PCR), the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. Participants of the independent cohorts were household contacts of adults with active pulmonary tuberculosis disease. FINDINGS Between July 6, 2005, and April 23, 2007, we enrolled 6363 participants from the ACS study and 4466 from independent South African and Gambian cohorts. 46 progressors and 107 matched controls were identified in the ACS cohort. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% CI 63·2-68·9) and a specificity of 80·6% (79·2-82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA sequencing and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6-64·3) and a specificity of 82·8% (76·7-86) in the 12 months preceding tuberculosis. INTERPRETATION The whole blood tuberculosis risk signature prospectively identified people at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. FUNDING Bill & Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union, and the South African Medical Research Council.Background Identification of blood biomarkers that prospectively predict progression of Mycobacterium tuberculosis infection to tuberculosis disease may lead to interventions that impact the epidemic. Methods Healthy, M. tuberculosis infected South African adolescents were followed for 2 years; blood was collected every 6 months. A prospective signature of risk was derived from whole blood RNA-Sequencing data by comparing participants who ultimately developed active tuberculosis disease (progressors) with those who remained healthy (matched controls). After adaptation to multiplex qRT-PCR, the signature was used to predict tuberculosis disease in untouched adolescent samples and in samples from independent cohorts of South African and Gambian adult progressors and controls. The latter participants were household contacts of adults with active pulmonary tuberculosis disease. Findings Of 6,363 adolescents screened, 46 progressors and 107 matched controls were identified. A 16 gene signature of risk was identified. The signature predicted tuberculosis progression with a sensitivity of 66·1% (95% confidence interval, 63·2–68·9) and a specificity of 80·6% (79·2–82·0) in the 12 months preceding tuberculosis diagnosis. The risk signature was validated in an untouched group of adolescents (p=0·018 for RNA-Seq and p=0·0095 for qRT-PCR) and in the independent South African and Gambian cohorts (p values <0·0001 by qRT-PCR) with a sensitivity of 53·7% (42·6–64·3) and a specificity of 82·8% (76·7–86) in 12 months preceding tuberculosis. Interpretation The whole blood tuberculosis risk signature prospectively identified persons at risk of developing active tuberculosis, opening the possibility for targeted intervention to prevent the disease. Funding Bill and Melinda Gates Foundation, the National Institutes of Health, Aeras, the European Union and the South African Medical Research Council (detail at end of text).

Mycobacterium tuberculosis LprA is a lipoprotein agonist of TLR2 that regulates innate immunity and APC function

TLR2 recognizes components of Mycobacterium tuberculosis (Mtb) and initiates responses by APCs that influence both innate and adaptive immunity. Mtb lipoproteins are an important class of TLR2 ligand, but only two, LpqH and LprG, have been characterized to date. In this study, we characterize a third Mtb lipoprotein, LprA, and determine its effects on host macrophages and dendritic cells. LprA is a cell wall-associated lipoprotein with no homologs outside the slow-growing mycobacteria. Using Mycobacterium smegmatis as an expression host, we purified 6× His-tagged LprA both with and without its acyl modifications. Acylated LprA had agonist activity for both human and murine TLR2 and induced expression of TNF-α, IL-10, and IL-12. LprA also induced dendritic cell maturation as shown by increased expression of CD40, CD80, and class II MHC (MHC-II). In macrophages, prolonged (24 h) incubation with LprA decreased IFN-γ-induced MHC-II Ag processing and presentation, consistent with an observed decrease in MHC-II expression (macrophage viability was not affected and apoptosis was not induced by LprA). Reduced MHC-II Ag presentation may represent a negative feedback mechanism for control of inflammation that may be subverted by Mtb for immune evasion. Thus, Mtb LprA is a TLR2 agonist that induces cytokine responses and regulates APC function.

Mycobacterium tuberculosis 19-kDa Lipoprotein Inhibits IFN-γ-Induced Chromatin Remodeling of MHC2TA by TLR2 and MAPK Signaling

During infection of macrophages, prolonged signaling by Mycobacterium tuberculosis (Mtb) or its 19-kDa lipoprotein (LpqH; Rv3763) inhibits IFN-γ-induced expression of several immune function genes, including class II transactivator (CIITA), which regulates class II MHC. Mtb does not inhibit early IFN-γ signaling events, e.g., Stat1α activation. This study analyzed downstream mechanisms that regulate the transcription of MHC2TA, the gene encoding CIITA. Chromatin immunoprecipitation showed that IFN-γ induced acetylation of histones H3 and H4 at the CIITA promoter IV (pIV). In contrast, IFN-γ-dependent histone acetylation at CIITA pIV was inhibited by Mtb or 19-kDa lipoprotein. Mtb 19-kDa lipoprotein also inhibited IFN-γ-dependent recruitment of Brahma-related gene 1, a chromatin remodeling protein, to CIITA pIV. Mtb 19-kDa lipoprotein did not inhibit histone acetylation in TLR2−/− macrophages. Furthermore, 19-kDa lipoprotein did not inhibit CIITA expression or IFN-γ-dependent histone acetylation of CIITA pIV in macrophages treated with inhibitors of MAPKs p38 or ERK. Thus, CIITA expression was inhibited by TLR2-induced MAPK signaling that caused histone hypoacetylation at CIITA pIV and suppression of CIITA transcription. Chromatin remodeling at MHC2TA is a novel target of inhibition by Mtb. These mechanisms may diminish class II MHC expression by infected macrophages, contributing to immune evasion by Mtb.

The Mycobacterium tuberculosis 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Regulated HLA-DR and FcγR1 on Human Macrophages through Toll-Like Receptor 2

ABSTRACT Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4+ T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-γ) has a central role in host defenses against M. tuberculosis by activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing. M. tuberculosis interferes with IFN-γ receptor (IFN-γR) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein from M. tuberculosis inhibits IFN-γ-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa lipoprotein also inhibited IFN-γ-induced expression of FcγRI. Thus, M. tuberculosis, through 19-kDa lipoprotein activation of TLR-2, inhibits IFN-γR signaling in human macrophages, resulting in decreased MHC-II Ag processing and recognition by MHC-II-restricted CD4 T cells. These findings provide a mechanism for M. tuberculosis persistence in macrophages.

Prolonged Toll-Like Receptor Signaling by Mycobacterium tuberculosis and Its 19-Kilodalton Lipoprotein Inhibits Gamma Interferon-Induced Regulation of Selected Genes in Macrophages

ABSTRACT Infection of macrophages with Mycobacterium tuberculosis or exposure to M. tuberculosis 19-kDa lipoprotein for >16 h inhibits gamma interferon (IFN-γ)-induced major histocompatibility complex class II (MHC-II) expression by a mechanism involving Toll-like receptors (TLRs). M. tuberculosis was found to inhibit murine macrophage MHC-II antigen (Ag) processing activity induced by IFN-γ but not by interleukin-4 (IL-4), suggesting inhibition of IFN-γ-induced gene regulation. We designed an approach to test the ability of M. tuberculosis-infected cells to respond to IFN-γ. To model chronic infection with M. tuberculosis with accompanying prolonged TLR signaling, macrophages were infected with M. tuberculosis or incubated with M. tuberculosis 19-kDa lipoprotein for 24 h prior to the addition of IFN-γ. Microarray gene expression studies were then used to determine whether prolonged TLR signaling by M. tuberculosis broadly inhibits IFN-γ regulation of macrophage gene expression. Of 347 IFN-γ-induced genes, M. tuberculosis and 19-kDa lipoprotein inhibited induction of 42 and 36%, respectively. Key genes or gene products were also examined by quantitative reverse transcription-PCR and flow cytometry, confirming and extending the results obtained by microarray studies. M. tuberculosis inhibited IFN-γ induction of genes involved in MHC-II Ag processing, Ag presentation, and recruitment of T cells. These effects were largely dependent on myeloid differentiation factor 88, implying a role for TLRs. Thus, prolonged TLR signaling by M. tuberculosis inhibits certain macrophage responses to IFN-γ, particularly those related to MHC-II Ag presentation. This inhibition may promote M. tuberculosis evasion of T-cell responses and persistence of infection in tuberculosis.

Immunogenicity of Novel DosR Regulon-Encoded Candidate Antigens of Mycobacterium tuberculosis in Three High-Burden Populations in Africa

ABSTRACT Increasing knowledge about DosR regulon-encoded proteins has led us to produce novel Mycobacterium tuberculosis antigens for immunogenicity testing in human populations in three countries in Africa to which tuberculosis (TB) is endemic. A total of 131 tuberculin skin test-positive and/or ESAT-6/CFP10-positive, human immunodeficiency virus-negative adult household contacts of active pulmonary TB cases from South Africa (n = 56), The Gambia (n = 26), and Uganda (n = 49) were tested for gamma interferon responses to 7 classical and 51 DosR regulon-encoded M. tuberculosis recombinant protein antigens. ESAT-6/CFP10 fusion protein evoked responses in >75% of study participants in all three countries. Of the DosR regulon-encoded antigens tested, Rv1733c was the most commonly recognized by participants from both South Africa and Uganda and the third most commonly recognized antigen in The Gambia. The four most frequently recognized DosR regulon-encoded antigens in Uganda (Rv1733c, Rv0081, Rv1735c, and Rv1737c) included the three most immunogenic antigens in South Africa. In contrast, Rv3131 induced the highest percentage of responders in Gambian contacts (38%), compared to only 3.4% of Ugandan contacts and no South African contacts. Appreciable percentages of TB contacts with a high likelihood of latent M. tuberculosis infection responded to several novel DosR regulon-encoded M. tuberculosis proteins. In addition to significant similarities in antigen recognition profiles between the three African population groups, there were also disparities, which may stem from genetic differences between both pathogen and host populations. Our findings have implications for the selection of potential TB vaccine candidates and for determining biosignatures of latent M. tuberculosis infection, active TB disease, and protective immunity.