Impact of Mycobacterium tuberculosis Glycolipids on the CD4+ T Cell-Macrophage Immunological Synapse.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

Monocyte Transcriptional Responses to Mycobacterium tuberculosis Associate with Resistance to Tuberculin Skin Test and Interferon Gamma Release Assay Conversion.

Heavy exposure to Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB) and among the top infectious killers worldwide, results in infection that is cleared, contained, or progresses to disease. Some heavily exposed tuberculosis contacts show no evidence of infection using the tuberculin skin test (TST) and interferon gamma release assay (IGRA); yet the mechanisms underlying this "resister" (RSTR) phenotype are unclear. To identify transcriptional responses that distinguish RSTR monocytes, we performed transcriptome sequencing (RNA-seq) on monocytes isolated from heavily exposed household contacts in Uganda and gold miners in South Africa after ex vivo M. tuberculosis infection. Gene set enrichment analysis (GSEA) revealed several gene pathways that were consistently enriched in response to M. tuberculosis among RSTR subjects compared to controls with positive TST/IGRA testing (latent TB infection [LTBI]) across Uganda and South Africa. The most significantly enriched gene set in which expression was increased in RSTR relative to LTBI M. tuberculosis-infected monocytes was the tumor necrosis factor alpha (TNF-α) signaling pathway whose core enrichment (leading edge) substantially overlapped across RSTR populations. These leading-edge genes included candidate resistance genes (ABCA1 and DUSP2) with significantly increased expression among Uganda RSTRs (false-discovery rate [FDR], <0.1). The distinct monocyte transcriptional response to M. tuberculosis among RSTR subjects, including increased expression of the TNF signaling pathway, highlights genes and inflammatory pathways that may mediate resistance to TST/IGRA conversion and provides therapeutic targets to enhance host restriction of M. tuberculosis intracellular infection. IMPORTANCE After heavy M. tuberculosis exposure, the events that determine why some individuals resist TST/IGRA conversion are poorly defined. Enrichment of the TNF signaling gene set among RSTR monocytes from multiple distinct cohorts suggests an important role for the monocyte TNF response in determining this alternative immune outcome. These TNF responses to M. tuberculosis among RSTRs may contribute to antimicrobial programs that result in early clearance or the priming of alternative (gamma interferon-independent) cellular responses.

Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane ß-Lactamase Inhibitor To Augment ß-Lactam Action.

Mycobacterium abscessus (Mab) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab, it is advantageous to identify potent ß-lactam and ß-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane ß-lactamase inhibitor to restore in vitro susceptibilities in combination with ß-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 µg/mL and an MIC50/MIC90 of ≤0.06/0.25 µg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the ß-lactamase BlaMab with a relative Michaelis constant (Ki app) of 4 × 10-3 ± 0.8 × 10-3 µM and acylation rate (k2/K) of 1 × 107 M-1 s-1. Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and LdtMab2-4 and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a BlaMab inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with LdtMab2-4 and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and LdtMab2,4 supports new therapeutic approaches using ß-lactams in eradicating Mab. IMPORTANCE Durlobactam (DUR) is a potent inhibitor of BlaMab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with LdtMab2 and LdtMab4. The ability of DUR to protect amoxicillin and imipenem against BlaMab and its intrinsic activity along with the dual ß-lactam target redundancy can explain the rationale behind the potent activity of this combination.

Methylome-wide Analysis Reveals Epigenetic Marks Associated With Resistance to Tuberculosis in Human Immunodeficiency Virus-Infected Individuals From East Africa.

BACKGROUND: Tuberculosis (TB) is the most deadly infectious disease globally and is highly prevalent in the developing world. For individuals infected with both Mycobacterium tuberculosis (Mtb) and human immunodeficiency virus (HIV), the risk of active TB is 10% or more annually. Previously, we identified in a genome-wide association study (GWAS) a region on chromosome 5 associated with resistance to TB, which included epigenetic marks that could influence gene regulation. We hypothesized that HIV-infected individuals exposed to Mtb who remain disease free carry epigenetic changes that strongly protect them from active TB. METHODS: We conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania and integrated data from our GWAS. RESULTS: We identified 3 regions of interest that included markers that were differentially methylated between TB cases and controls with latent TB infection: chromosome 1 (RNF220, P = 4 × 10-5), chromosome 2 (between COPS8 and COL6A3, P = 2.7 × 10-5), and chromosome 5 (CEP72, P = 1.3 × 10-5). These methylation results co-localized with associated single-nucleotide polymorphisms (SNPs), methylation QTLs, and methylation × SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung. CONCLUSIONS: Epigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

Toll-Like Receptor 2-Tpl2-Dependent ERK Signaling Drives Inverse Interleukin 12 Regulation in Dendritic Cells and Macrophages.

This study investigated responses to Toll-like receptor 2 (TLR2)-driven extracellular signal-related kinase (ERK) signaling in dendritic cells (DCs) versus macrophages. TLR2 signaling was induced with Pam3Cys-Ser-Lys4, and the role of ERK signaling was interrogated pharmacologically with MEK1/2 inhibitor U0126 or genetically with bone marrow-derived macrophages or DCs from Tpl2-/- mice. We assessed cytokine production via enzyme-linked immunosorbent assay (ELISA) or V-Plex, and mRNA levels were assessed via reverse transcriptase quantitative PCR (qRT-PCR). In macrophages, blockade of ERK signaling by pharmacologic or genetic approaches inhibited interleukin 10 (IL-10) expression and increased expression of the p40 subunit shared by IL-12 and IL-23 (IL-12/23p40). In DCs, blockade of ERK signaling similarly inhibited IL-10 expression but decreased IL-12/23p40 expression, which is opposite to the effect of ERK signaling blockade on IL-12/23p40 in macrophages. This difference in IL-12/23p40 regulation correlated with the differential expression of transcription factors cFos and IRF1, which are known to regulate IL-12 family members, including IL-12 and IL-23. Thus, the impact of ERK signaling in response to TLR2 stimulation differs between macrophages and DCs, potentially regulating their distinctive functions in the immune system. ERK-mediated suppression of IL-12/23p40 in macrophages may prevent excessive inflammation and associated tissue damage following TLR2-stimulation, while ERK-mediated induction of IL-12/23p40 in DCs may promote priming of T helper 1 (Th1) responses. A greater understanding of the role that ERK signaling plays in different immune cell types may inform the development of host-directed therapy and optimal adjuvanticity for a number of infectious pathogens.

Mycobacterium tuberculosis Lipoprotein and Lipoglycan Binding to Toll-Like Receptor 2 Correlates with Agonist Activity and Functional Outcomes.

Mycobacterium tuberculosis causes persistent infection due to its ability to evade host immune responses. M. tuberculosis induces Toll-like receptor 2 (TLR2) signaling, which influences immune responses to M. tuberculosis TLR2 agonists expressed by M. tuberculosis include lipoproteins (e.g., LprG), the glycolipid phosphatidylinositol mannoside 6 (PIM6), and the lipoglycan lipomannan (LM). Another M. tuberculosis lipoglycan, mannose-capped lipoarabinomannan (ManLAM), lacks TLR2 agonist activity. In contrast, PILAM, from Mycobacterum smegmatis, does have TLR2 agonist activity. Our understanding of how M. tuberculosis lipoproteins and lipoglycans interact with TLR2 is limited, and binding of these molecules to TLR2 has not been measured directly. Here, we directly measured M. tuberculosis lipoprotein and lipoglycan binding to TLR2 and its partner receptor, TLR1. LprG, LAM, and LM were all found to bind to TLR2 in the absence of TLR1, but not to TLR1 in the absence of TLR2. Trimolecular interactions were revealed by binding of TLR2-LprG or TLR2-PIM6 complexes to TLR1, whereas binding of TLR2 to TLR1 was not detected in the absence of the lipoprotein or glycolipid. ManLAM exhibited low affinity for TLR2 in comparison to PILAM, LM, and LprG, which correlated with reduced ability of ManLAM to induce TLR2-mediated extracellular-signal-regulated kinase (ERK) activation and tumor necrosis factor alpha (TNF-α) secretion in macrophages. We provide the first direct affinity measurement and kinetic analysis of M. tuberculosis lipoprotein and lipoglycan binding to TLR2. Our results demonstrate that binding affinity correlates with the functional ability of agonists to induce TLR2 signaling.

Immunological mechanisms of human resistance to persistent Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis is a leading cause of mortality worldwide and establishes a long-lived latent infection in a substantial proportion of the human population. Multiple lines of evidence suggest that some individuals are resistant to latent M. tuberculosis infection despite long-term and intense exposure, and we term these individuals 'resisters'. In this Review, we discuss the epidemiological and genetic data that support the existence of resisters and propose criteria to optimally define and characterize the resister phenotype. We review recent insights into the immune mechanisms of M. tuberculosis clearance, including responses mediated by macrophages, T cells and B cells. Understanding the cellular mechanisms that underlie resistance to M. tuberculosis infection may reveal immune correlates of protection that could be utilized for improved diagnostics, vaccine development and novel host-directed therapeutic strategies.

Resistance and Susceptibility to Mycobacterium tuberculosis Infection and Disease in Tuberculosis Households in Kampala, Uganda.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major public health problem. Household contact studies identify children and adults along the spectrum from Mtb exposure to disease. In the Kawempe Community Health Study (conducted in Kampala, Uganda), 872 culture-confirmed pulmonary TB cases and their 2,585 contacts were enrolled during 2002-2012 and followed for up to 2 years each. Risk factors identified by time-to-event analysis for secondary TB differed among children, women, and men. Younger age (P = 0.0061), human immunodeficiency virus (HIV) (P = 0.0002), thinness (P = 0.01), absent bacille Calmette-Guérin vaccination (P = 0.002), and epidemiologic risk score (P < 0.0001) were risks for children. For women, risks were HIV (P < 0.0001), thinness (World Health Organization criteria; P < 0.0001), and epidemiologic risk score (P = 0.003). For men, HIV (P = 0.0007) and low body mass index (P = 0.008) resulted in faster progression to TB. Tuberculin skin testing (TST) identified contacts with Mtb infection and those with persistently negative TST. Risks for faster time to Mtb infection were identified, and included age (P = 0.0007), baseline TST induration (P < 0.0001), and epidemiologic risk score (P < 0.0001) only in children. Those with persistently negative TST comprised 10% of contacts but had no unique epidemiologic characteristics among adults. The burden of Mtb infection and disease is high in TB households, and risk factors for progression from exposure to infection and disease differ among children, women, and men.

Effectiveness of WHO's pragmatic screening algorithm for child contacts of tuberculosis cases in resource-constrained settings: a prospective cohort study in Uganda.

BACKGROUND: Tuberculosis is a leading cause of global childhood mortality; however, interventions to detect undiagnosed tuberculosis in children are underused. Child contact tracing has been widely recommended but poorly implemented in resource-constrained settings. WHO has proposed a pragmatic screening approach for managing child contacts. We assessed the effectiveness of this screening approach and alternative symptom-based algorithms in identifying secondary tuberculosis in a prospectively followed cohort of Ugandan child contacts. METHODS: We identified index patients aged at least 18 years with microbiologically confirmed pulmonary tuberculosis at Old Mulago Hospital (Kampala, Uganda) between Oct 1, 1995, and Dec 31, 2008. Households of index patients were visited by fieldworkers within 2 weeks of diagnosis. Coprevalent and incident tuberculosis were assessed in household contacts through clinical, radiographical, and microbiological examinations for 2 years. Disease rates were compared among children younger than 16 years with and without symptoms included in the WHO pragmatic guideline (presence of haemoptysis, fever, chronic cough, weight loss, night sweats, and poor appetite). Symptoms could be of any duration, except cough (>21 days) and fever (>14 days). A modified WHO decision-tree designed to detect high-risk asymptomatic child contacts was also assessed, in which all asymptomatic contacts were classified as high risk (children younger than 3 years or immunocompromised [HIV-infected]) or low risk (aged 3 years or older and immunocompetent [HIV-negative]). We also assessed a more restrictive algorithm (ie, assessing only children with presence of chronic cough and one other tuberculosis-related symptom). FINDINGS: Of 1718 household child contacts, 126 (7%) had coprevalent tuberculosis and 24 (1%) developed incident tuberculosis, diagnosed over the 2-year study period. Of these 150 cases of tuberculosis, 95 (63%) were microbiologically confirmed with a positive sputum culture. Using the WHO approach, 364 (21%) of 1718 child contacts had at least one tuberculosis-related symptom and 85 (23%) were identified as having coprevalent tuberculosis, 67% of all coprevalent cases detected (diagnostic odds ratio 9·8, 95% CI 6·8-14·5; p<0·0001). 1354 (79%) of 1718 child contacts had no symptoms, of whom 41 (3%) had coprevalent tuberculosis. The WHO approach was effective in contacts younger than 5 years: 70 (33%) of 211 symptomatic contacts had coprevalent disease compared with 23 (6%) of 367 asymptomatic contacts (p<0·0001). This approach was also effective in contacts aged 5 years and older: 15 (10%) of 153 symptomatic contacts had coprevalent disease compared with 18 (2%) of 987 asymptomatic contacts (p<0·0001). More coprevalent disease was detected in child contacts recommended for screening when the study population was restricted by HIV-serostatus (11 [48%] of 23 symptomatic HIV-seropositive child contacts vs two [7%] of 31 asymptomatic HIV-seropositive child contacts) or to only culture-confirmed cases (47 [13%] culture confirmed cases of 364 symptomatic child contacts vs 29 [2%] culture confirmed cases of 1354 asymptomatic child contacts). In the modified algorithm, high-risk asymptomatic child contacts were at increased risk for coprevalent disease versus low-risk asymptomatic contacts (14 [6%] of 224 vs 27 [2%] of 1130; p=0·0021). The presence of tuberculosis infection did not predict incident disease in either symptomatic or asymptomatic child contacts: in symptomatic contacts, eight (5%) of 169 infected contacts and six (5%) of 111 uninfected contacts developed incident tuberculosis (p=0·80). Among asymptomatic contacts, incident tuberculosis occurred in six (<1%) of 795 contacts infected at baseline versus four (<1%) of 518 contacts uninfected at baseline, respectively (p=1·00). INTERPRETATION: WHO's pragmatic, symptom-based algorithm was an effective case-finding tool, especially in children younger than 5 years. A modified decision-tree identified 6% of asymptomatic child contacts at high risk for subclinical disease. Increasing the feasibility of child-contact tracing using these approaches should be encouraged to decrease tuberculosis-related paediatric mortality in high-burden settings, but this should be partnered with increasing access to microbiological point-of-care testing. FUNDING: National Institutes of Health, Tuberculosis Research Unit, AIDS International Training and Research Program of the Fogarty International Center, and the Center for AIDS Research.

Proteomics and Network Analyses Reveal Inhibition of Akt-mTOR Signaling in CD4+ T Cells by Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan.

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose-capped lipoarabinomannan (ManLAM) inhibits CD4+ T-cell activation by inhibiting proximal T-cell receptor (TCR) signaling when activated by anti-CD3. To understand the impact of ManLAM on CD4+ T-cell function when both the TCR-CD3 complex and major costimulator CD28 are engaged, we performed label-free quantitative MS and network analysis. Mixed-effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti-CD3- and anti-CD28-activated CD4+ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF-κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x-Akt-mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T-cell proteome by affecting Akt-mTOR signaling, resulting in broad functional impairment of CD4+ T-cell activation beyond inhibition of proximal TCR-CD3 signaling.

Toll like Receptor 2 engagement on CD4+ T cells promotes TH9 differentiation and function.

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4+ T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4+ T-cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4+ T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4+ T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4+ T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4+ T cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells.

A chromosome 5q31.1 locus associates with tuberculin skin test reactivity in HIV-positive individuals from tuberculosis hyper-endemic regions in east Africa.

One in three people has been infected with Mycobacterium tuberculosis (MTB), and the risk for MTB infection in HIV-infected individuals is even higher. We hypothesized that HIV-positive individuals living in tuberculosis-endemic regions who do not get infected by Mycobacterium tuberculosis are genetically resistant. Using an "experiment of nature" design that proved successful in our previous work, we performed a genome-wide association study of tuberculin skin test positivity using 469 HIV-positive patients from prospective study cohorts of tuberculosis from Tanzania and Uganda to identify genetic loci associated with MTB infection in the context of HIV-infection. Among these individuals, 244 tested were tuberculin skin test (TST) positive either at enrollment or during the >8 year follow up, while 225 were not. We identified a genome-wide significant association between a dominant model of rs877356 and binary TST status in the combined cohort (Odds ratio = 0.2671, p = 1.22x10-8). Association was replicated with similar significance when examining TST induration as a continuous trait. The variant lies in the 5q31.1 region, 57kb downstream from IL9. Two-locus analyses of association of variants near rs877356 showed a haplotype comprised of rs877356 and an IL9 missense variant, rs2069885, had the most significant association (p = 1.59x10-12). We also replicated previously linked loci on chromosomes 2, 5, and 11. IL9 is a cytokine produced by mast cells and TH2 cells during inflammatory responses, providing a possible link between airway inflammation and protection from MTB infection. Our results indicate that studying uninfected, HIV-positive participants with extensive exposure increases the power to detect associations in complex infectious disease.

Mycobacterium tuberculosis Membrane Vesicles Inhibit T Cell Activation.

Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhibition of effector CD4+ T cell responses by M. tuberculosis may contribute to immune evasion. TCR signaling is inhibited by M. tuberculosis cell envelope lipoglycans, such as lipoarabinomannan and lipomannan, but a mechanism for lipoglycans to traffic from M. tuberculosis within infected macrophages to reach T cells is unknown. In these studies, we found that membrane vesicles produced by M. tuberculosis and released from infected macrophages inhibited the activation of CD4+ T cells, as indicated by reduced production of IL-2 and reduced T cell proliferation. Flow cytometry and Western blot demonstrated that lipoglycans from M. tuberculosis-derived bacterial vesicles (BVs) are transferred to T cells, where they inhibit T cell responses. Stimulation of CD4+ T cells in the presence of BVs induced expression of GRAIL, a marker of T cell anergy; upon restimulation, these T cells showed reduced ability to proliferate, confirming a state of T cell anergy. Furthermore, lipoarabinomannan was associated with T cells after their incubation with infected macrophages in vitro and when T cells were isolated from lungs of M. tuberculosis-infected mice, confirming the occurrence of lipoarabinomannan trafficking to T cells in vivo. These studies demonstrate a novel mechanism for the direct regulation of CD4+ T cells by M. tuberculosis lipoglycans conveyed by BVs that are produced by M. tuberculosis and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.

ERK Signaling Is Essential for Macrophage Development.

Macrophages depend on colony stimulating factor 1 (also known as M-CSF) for their growth and differentiation, but the requirements for intracellular signals that lead to macrophage differentiation and function remain unclear. M-CSF is known to activate ERK1 and ERK2, but the importance of this signaling pathway in macrophage development is unknown. In these studies, we characterized a novel model of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice in which the ERK2 isoform is deleted from macrophages in the background of global ERK1 deficiency. Cultures of M-CSF-stimulated bone marrow precursors from these mice yielded reduced numbers of macrophages. Whereas macrophages developing from M-CSF-stimulated bone marrow of Erk2(flox/flox) Lyz2(Cre/Cre) mice showed essentially complete loss of ERK2 expression, the reduced number of macrophages that develop from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow show retention of ERK2 expression, indicating selective outgrowth of a small proportion of precursors in which Cre-mediated deletion failed to occur. The bone marrow of Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) mice was enriched for CD11b+ myeloid cells, CD11b(hi) Gr-1(hi) neutrophils, Lin- c-Kit+ Sca-1+ hematopoietic stem cells, and Lin- c-Kit+ CD34+ CD16/32+ granulocyte-macrophage progenitors. Culture of bone marrow Lin- cells under myeloid-stimulating conditions yielded reduced numbers of monocytes. Collectively, these data indicate that the defect in production of macrophages is not due to a reduced number of progenitors, but rather due to reduced ability of progenitors to proliferate and produce macrophages in response to M-CSF-triggered ERK signaling. Macrophages from Erk1(-/-) Erk2(flox/flox) Lyz2(Cre/Cre) bone marrow showed reduced induction of M-CSF-regulated genes that depend on the ERK pathway for their expression. These data demonstrate that ERK1/ERK2 play a critical role in driving M-CSF-dependent proliferation of bone marrow progenitors for production of macrophages.

Toll-like receptor 2-dependent extracellular signal-regulated kinase signaling in Mycobacterium tuberculosis-infected macrophages drives anti-inflammatory responses and inhibits Th1 polarization of responding T cells.

Mycobacterium tuberculosis survives within macrophages and employs immune evasion mechanisms to persist in the host. Protective T helper type 1 (Th1) responses are induced, and the immune response in most individuals is sufficient to restrict M. tuberculosis to latent infection, but most infections are not completely resolved. As T cells and macrophages respond, a balance is established between protective Th1-associated and other proinflammatory cytokines, such as interleukin-12 (IL-12), interferon gamma (IFN-γ), and tumor necrosis factor alpha, and anti-inflammatory cytokines, such as IL-10. The mechanisms by which M. tuberculosis modulates host responses to promote its survival remain unclear. In these studies, we demonstrate that M. tuberculosis induction of IL-10, suppression of IL-12, and inhibition of class II major histocompatibility complex (MHC-II) molecules in infected macrophages are all driven by Toll-like receptor 2 (TLR2)-dependent activation of the extracellular signal-regulated kinases (ERK). Elimination of ERK signaling downstream of TLR2 by pharmacologic inhibition with U0126 or genetic deletion of Tpl2 blocks IL-10 secretion and enhances IL-12 p70 secretion. We demonstrate that M. tuberculosis regulation of these pathways in macrophages affects T cell responses to infected macrophages. Thus, genetic blockade of the ERK pathway in Tpl2(-/-) macrophages enhances Th1 polarization and IFN-γ production by antigen-specific CD4(+) T cells responding to M. tuberculosis infection. These data indicate that M. tuberculosis and its potent TLR2 ligands activate ERK signaling in macrophages to promote anti-inflammatory macrophage responses and blunt Th1 responses against the pathogen.

Mycobacterium tuberculosis lipoprotein LprG binds lipoarabinomannan and determines its cell envelope localization to control phagolysosomal fusion.

Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.

Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Competition for iron influences host-pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

Wasting among Uganda men with pulmonary tuberculosis is associated with linear regain in lean tissue mass during and after treatment in contrast to women with wasting who regain fat tissue mass: prospective cohort study.

BACKGROUND: Nutritional changes during and after tuberculosis treatment have not been well described. We therefore determined the effect of wasting on rate of mean change in lean tissue and fat mass as measured by bioelectrical impedance analysis (BIA), and mean change in body mass index (BMI) during and after tuberculosis treatment. METHODS: In a prospective cohort study of 717 adult patients, BMI and height-normalized indices of lean tissue (LMI) and fat mass (FMI) as measured by BIA were assessed at baseline, 3, 12, and 24 months. RESULTS: Men with wasting at baseline regained LMI at a greater rate than FMI (4.55 kg/m2 (95% confidence interval (CI): 1.26, 7.83 versus 3.16 (95% CI: 0.80, 5.52)) per month, respectively during initial tuberculosis therapy. In contrast, women with wasting regained FMI at greater rate than LMI (3.55 kg/m2 (95% CI: 0.40, 6.70) versus 2.07 (95% CI: -0.74, 4.88)), respectively. Men with wasting regained BMI at a rate of 6.45 kg/m2 (95% CI: 3.02, 9.87) in the first three months whereas women, had a rate of 3.30 kg/m2 (95% CI: -0.11, 6.72). There were minimal changes in body composition after month 3 and during months 12 to 24. CONCLUSION: Wasted tuberculosis patients regain weight with treatment but the type of gain differs by gender and patients may remain underweight after the initial phase of treatment.

Longitudinal changes in CD4(+) T-cell memory responses induced by BCG vaccination of newborns.

BACKGROUND: Improved vaccination strategies against tuberculosis are needed, such as approaches to boost immunity induced by the current vaccine, BCG. Design of these strategies has been hampered by a lack of knowledge of the kinetics of the human host response induced by neonatal BCG vaccination. Furthermore, the functional and phenotypic attributes of BCG-induced long-lived memory T-cell responses remain unclear. METHODS: We assessed the longitudinal CD4 T-cell response following BCG vaccination of human newborns. The kinetics, function, and phenotype of these cells were measured using flow cytometric whole-blood assays. RESULTS: We showed that the BCG-specific CD4 T-cell response peaked 6-10 weeks after vaccination and gradually waned over the first year of life. Highly activated T-helper 1 cells, predominantly expressing interferon γ, tumor necrosis factor α, and/or interleukin 2, were present at the peak response. Following contraction, BCG-specific CD4 T cells expressed high levels of Bcl-2 and displayed a predominant CD45RACCR7 central memory phenotype. However, cytokine and cytotoxic marker expression by these cells was more characteristic of effector memory cells. CONCLUSIONS: Our findings suggest that boosting of BCG-primed CD4 T cells with heterologous tuberculosis vaccines may be best after 14 weeks of age, once an established memory response has developed.

Lean tissue mass wasting is associated with increased risk of mortality among women with pulmonary tuberculosis in urban Uganda.

OBJECTIVES: We assessed the impact of wasting on survival in patients with tuberculosis by using a precise height-normalized lean tissue mass index (LMI) estimated by bioelectrical impedance analysis and body mass index (BMI). METHODS: In a retrospective cohort study, 747 adult pulmonary patients with tuberculosis who were screened for HIV and nutritional status were followed for survival. RESULTS: Of 747 patients, 310 had baseline wasting by BMI (kg/m(2)) and 103 by LMI (kg/m(2)). Total deaths were 105. Among men with reduced BMI, risk of death was 70% greater (hazard ratio [HR] 1.7, 95% confidence interval [95% CI] 1.03-2.81) than in men with normal BMI. Survival did not differ by LMI among men (HR 1.1; 95% CI 0.5-2.9). In women, both the BMI and LMI were associated with survival. Among women with reduced BMI, risk of death was 80% greater (HR 1.8; 95% CI 0.9-3.5) than in women with normal BMI; risk of death was 5-fold greater (HR 5.0; 95% CI 1.6-15.9) for women with low LMI compared with women with normal LMI. CONCLUSIONS: Wasting assessed by reduced BMI is associated with an increased risk for death among both men and women whereas reduced LMI is among women with tuberculosis.