Antimycobacterial Immune Response Research Articles

Gamma-Delta T-Cell Responses During Subcutaneous Mycobacterium avium Subspecies paratuberculosis Challenge in Sensitized or Naïve Calves Using Matrix Biopolymers

We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

Suppression of the NF-κB Pathway by Diesel Exhaust Particles Impairs Human Antimycobacterial Immunity

Epidemiological studies suggest that chronic exposure to air pollution increases susceptibility to respiratory infections, including tuberculosis in humans. A possible link between particulate air pollutant exposure and antimycobacterial immunity has not been explored in human primary immune cells. We hypothesized that exposure to diesel exhaust particles (DEP), a major component of urban fine particulate matter, suppresses antimycobacterial human immune effector cell functions by modulating TLR-signaling pathways and NF-κB activation. We show that DEP and H37Ra, an avirulent laboratory strain of Mycobacterium tuberculosis, were both taken up by the same peripheral human blood monocytes. To examine the effects of DEP on M. tuberculosis-induced production of cytokines, PBMC were stimulated with DEP and M. tuberculosis or purified protein derivative. The production of M. tuberculosis and purified protein derivative-induced IFN-γ, TNF-α, IL-1β, and IL-6 was reduced in a DEP dose-dependent manner. In contrast, the production of anti-inflammatory IL-10 remained unchanged. Furthermore, DEP stimulation prior to M. tuberculosis infection altered the expression of TLR3, -4, -7, and -10 mRNAs and of a subset of M. tuberculosis-induced host genes including inhibition of expression of many NF-κB (e.g., CSF3, IFNG, IFNA, IFNB, IL1A, IL6, and NFKBIA) and IFN regulatory factor (e.g., IFNG, IFNA1, IFNB1, and CXCL10) pathway target genes. We propose that DEP downregulate M. tuberculosis-induced host gene expression via MyD88-dependent (IL6, IL1A, and PTGS2) as well as MyD88-independent (IFNA, IFNB) pathways. Prestimulation of PBMC with DEP suppressed the expression of proinflammatory mediators upon M. tuberculosis infection, inducing a hyporesponsive cellular state. Therefore, DEP alters crucial components of antimycobacterial host immune responses, providing a possible mechanism by which air pollutants alter antimicrobial immunity.

Myc Against Mycobacteria

Cytoplasmic Myc promotes antimycobacterial immune responses in macrophages.

Patterns of in vitro cell-death, metaloproteinase-9 and pro-inflammatory cytokines in human monocytes induced by the BCG vaccine, Moreau strain

Mononuclear cells have been implicated in the primary inflammatory response against mycobacteria. Yet, little is known about the interaction of Mycobacterium bovis bacillus Calmette-Guerin (BCG) with human monocytes. Here, we investigated the potential of BCG Moreau strain to induce in vitro specific cell-death utilizing a flow cytometry approach that revealed an increase in apoptosis events in BCG-stimulated monocytes from healthy adults. We also detected a concomitant release of interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), but not metalloproteinase (MMP)-9. In addition, annexin V-propidium iodide double staining demonstrated an enhancement of monocytes necrosis, but not apoptosis, following BCG Moreau strain stimulation of umbilical vein cells from naïve, neonate. This pattern was paralleled by different pro-inflammatory cytokine levels, as well as MMP-9 induction when compared to the adults. Our findings support the hypothesis that BCG induces distinct cell-death patterns during the maturation of the immune system and that this pattern might set the stage for a subsequent antimycobacterial immune response that might have profound effects during vaccination.

Recognition of mycobacterial antigens by phagocytes

Recognition of mycobacterial antigens by receptors of phagocytes is not only a key element of the first line of defense, but also an important link to the specific phase of the immune response. The immune response is based on the existence of a number of pattern recognition receptors (PRR) that recognize conservative microbial structures called pathogen-associated molecular patterns (PAMP). These receptors are involved in the processes of opsonization and phagocytosis of pathogens, activation of the complement system, induction of apoptosis and signal transduction cell systems. The initiated signal cascade is supposed to lead to the mobilization of immune forces against the penetrating pathogen and is aimed at its fast elimination from the body. Understanding the role of these receptors in the antimycobacterial immune response appears to be fully justified in view of their potential application in distinguishing persons particularly sensitive to tuberculosis as well as in the development of new generation vaccines for prophylaxis and therapy and new biomarkers for improvement of the difficult and time-consuming diagnosis of mycobacterial infections.

Lysophosphatidic acid enhances antimycobacterial response during in vivo primary Mycobacterium tuberculosis infection

Lysophospholipids may play an important protective role during primary infection of Mycobacterium tuberculosis (MTB) by enhancing innate antimycobacterial immune response of both macrophages and alveolar epithelial cells. Here, we show that treatment with lysophosphatidic acid (LPA) of mice aerogenically infected with MTB immediately after infection results in a significant early reduction of pulmonary CFUs and of histopathological damage in comparison with control mice. In contrast, treatment of acute disease does not result in any improvement of both microbiological and histopathological parameters. Altogether, these results show that LPA treatment can exert protective effect if administrated during primary infection, only.

S1 HIV-1 infection of macrophages modulates host responses to Mycobacterium tuberculosis to contribute to immunopathogenesis and viral propagation

Introduction and ObjectivesHuman immunodeficiency virus (HIV)-1 infection increases the risk of active tuberculosis (TB) by 50-fold. The concurrent HIV and TB pandemics therefore represent a major threat to global health....

Cysteine-Cysteinyl Chemokine Receptor 6 Mediates Invariant Natural Killer T Cell Airway Recruitment and Innate Stage Resistance during Mycobacterial Infection

This study examined the contribution of cysteine-cysteinyl chemokine receptor 6 (CCR6) to the innate pulmonary antimycobacterial immune response. Using a mouse model of Mycobacterium bovis BCG airway infection, we detected maximal induction of the CCR6 agonist CCL20 in lungs at 1 week after infection. Infected CCR6 knockout (CCR6–/–) mice displayed an early impairment of bacterial clearance, but ultimately eliminated the attenuated organisms with the onset of adaptive immunity. Flow-cytometric analyses of bronchoalveolar lavages and dispersed lungs revealed a 60% reduction in TCR-α/β+ T cells in airways but no compromise of TCR-γ/δ+ T cells. The subset of CD1d-restricted, CD8-TCR-α/β+ natural killer cells, which mediate innate mycobacterial resistance, was profoundly reduced (90%). Analysis of the adaptive response using ovalbumin-specific transgenic TCR T cell (OT-II) transfer combined with infection with recombinant M. bovis BCG producing ovalbumin peptide indicated no impairment of adaptive T cell activation in CCR6–/– mice. There was also no impairment of the induction of cytokine-producing cells in draining lymphoid tissue of CCR6–/– mice. Taken together, our findings indicate that CCR6 is not required for induction of the adaptive antimycobacterial response, but is likely critical to airway compartment mobilization of TCR-α/β+CCR6+ innate and adaptive effector T cells.

Assessment of immune response to repeat stimulation with BCG vaccine using in vitro PBMC model

BackgroundTuberculosis (TB) is one of the most prevalent cause of death due to a single pathogen. Bacillus Calmette Guérin (BCG) is the only vaccine available for clinical use that protects against miliary TB; however, this vaccine has shown variable levels of efficacy against pulmonary TB. In India, a single dose of BCG vaccine is given and there are few countries where repeated doses of BCG are given. The incidence of TB in India is very high inspite of primary vaccination in neonatal period and therefore requires consideration for repeated immunization.MethodsTo improve BCG immunogenicity, we have evaluated specific antimycobacterial immune responses (anti-BCG IgG and IFN-γ), T cell activity-ADA, CD4 and CD8 T cell count, and CD4/CD8 ratio in a peripheral blood mononuclear cells (PBMC) model using boost immunization protocols with the BCG vaccine. PBMC were induced with a repeat dose of BCG at 24 and 72 hrs of cell culture.ResultsAt the end of the experimental time, supernatant was collected to estimate anti-BCG IgG titer, interferon γ, ADA activity, CD 4 and CD8 T cell count, and CD4/CD8 ratio. We demonstrated that PBMC induced with a repeat dose of BCG showed an increased specific anti-mycobacterial immune responses, T cell activity, and ADA activity as compared to PBMC induced with BCG alone or without BCG induction.ConclusionThe repeat BCG stimulation of PBMC obtained from BCG vaccinated individuals shows enhanced immune activation with respect to increased anti-BCG titre, IFN-γ and ADA activity without concomitant increase in CD4 and CD8 cells. This study provides some basic data in future experiments in animal models with respect to repeat BCG vaccination.

518 A novel support tool for Breast Multidisciplinary Meetings: an advanced evidence based computer decision support technology

Following Mycobacterium avium subspecies paratuberculosis (Map) infection, some calves are apparently able to successfully clear the pathogen whereas others become persistently infected; however the reasons for this remain unknown. The importance of innate immunity, and in particular the role of γδ T lymphocytes, during early anti-mycobacterial immune response is recognized but specific mechanisms remain incompletely characterized. The objective of this study was to investigate how bovine WC1+ γδ T lymphocytes mediate macrophage function during early Map infection. To achieve this objective, Map-infected monocyte-derived macrophages (MDMs) were co-cultured either in direct contact with, or separated by a semi-permeable membrane from, autologous WC1+ γδ T lymphocytes. Nitrites, IL-17A, IFN-γ, IL-4 and IL-10 from cell culture supernatants were measured. Expression of CD25 on WC1+ γδ T lymphocytes, expression of MHC-I and MHC-II on MDMs and the viability of Map recovered from MDM cultures 72 h after Map infection were also assessed. Map viability was significantly reduced when WC1+ γδ T lymphocytes were co-cultured in direct contact with Map-infected MDMs. Both MDMs and WC1+ γδ T lymphocytes generated increased concentrations of IFN-γ and IL-4 in our system, and MDM/WC1+ γδ T lymphocyte synergism was identified for IFN-γ production. MDMs but not WC1+ γδ T lymphocytes were a significant source of IL-17A. The presence of WC1+ γδ T lymphocytes was associated with higher expression of MHC-I on MDMs and increased concentration of nitrites in supernatants 72 h after Map infection. In conclusion, this study showed that WC1+ γδ lymphocytes had differential effects on Map-infected macrophages in vitro.

Decreased Expression of T-Cell Costimulatory Molecule CD28 on CD4 and CD8 T Cells of Mexican Patients with Pulmonary Tuberculosis

Patients with tuberculosis frequently develop anergy, a state of T-cell hyporesponsiveness in which defective T-cell costimulation could be a factor. To know if the expression of T-cell costimulatory molecules was altered in tuberculosis, we analyzed the peripheral blood T-cell phenotype of 23 Mexican patients with pulmonary tuberculosis. There was severe CD4 (P < .001) and CD8 (P < .01) lymphopenia and upregulation of costimulatory molecule CD30 on CD4 and CD8 T cells (P < .05); this increase was higher in relapsing tuberculosis. The main finding was severe downregulation of the major costimulatory molecule CD28 on both CD8 and CD4 T cells (P < .001). Depletion of the CD4/CD28 subset, a hitherto undescribed finding, is relevant because CD4 T cells constitute the main arm of the cell-mediated antimycobacterial immune response.

Gamma–delta T cell subsets are differentially associated with granuloma development and organization in a bovine model of mycobacterial disease

The characteristic lesion in bovine tuberculosis is well-organized respiratory granulomas. This is typically associated with a strong T-helper 1 biased cell-mediated immune response and eventual containment of the infection. In bovine paratuberculosis, the classic lesion is unorganized granulomatous intestinal inflammation. Clinical paratuberculosis is associated with a T-helper 2 biased humoral immune response and eventual death because of inability of the host to contain the infection. Recent reports have suggested that gamma-delta (gammadelta) T cells play a significant role in granuloma development and/or maintenance during initial stages of infection and may influence the subsequent adaptive immune response. The objective of this study was to use an in vivo bovine model to evaluate gammadelta T cells during the early host immune response to mycobacterial infection. We used immunofluorescent staining, hyperspectral microscopy, and computerized assisted morphometry to evaluate staining and distribution of gammadelta T cells during development of organized and unorganized granulomas. Our data suggest that bovine gammadelta T cell subsets are differentially recruited to early infection sites, and may be instrumental during the initial antimycobacterial host immune response as well as for granuloma organization.

Positive role of mitogen-activated protein kinase phosphatase-1 in regulating the anti-mycobacterial immune responses

Positive role of mitogen-activated protein kinase phosphatase-1 in regulating the anti-mycobacterial immune responses

Antimycobacterial immune responses in patients with pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of Mycobacterium tuberculosis has frequently been discussed in the aetiology of sarcoidosis; however, studies still remain contradictory. We addressed the question of mycobacterial involvement in the pathogenesis of sarcoidosis by analysing cellular immune responses to mycobacterial antigens. We examined the interferon (IFN)-gamma production by enzyme-linked immunospot in response to purified protein derivate (PPD) mycobacterial-specific antigen early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10 by peripheral blood mononuclear cells (PBMCs) and bronchoalveolar-lavage mononuclear cells (BALMCs) of patients with pulmonary sarcoidosis, smear-negative tuberculosis and controls. Release of IFN-gamma in response to ex vivo contact with PPD, ESAT-6 or CFP-10 by BALMC and PBMC were comparable among patients with sarcoidosis and controls (PBMC P = 0.2326; BALMC P = 0.1767) and were less frequently observed in both groups compared to patients with tuberculosis (BALMC P < 0.05; PBMC P < 0.0001). Within PBMC, the immunophenotype of sarcoidosis patients differed from that of patients with tuberculosis, as well as from that of controls, while within BALMC it resembled that of patients with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local and systemic immune responses is not elevated in patients with sarcoidosis when compared to controls. The immunophenotype represents the local resemblance of the granulomatous reaction underlying tuberculosis and sarcoidosis while showing systemical difference. These observations do not support a role of an infection with M. tuberculosis in the pathogenesis of sarcoidosis.

Interaction of Mycobacterium tuberculosis with the host: consequences for vaccine development

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a major worldwide health problem that causes more than 2 million deaths annually. In addition, an estimated 2 billion people are latently infected with M. tuberculosis. The bacterium is one of the oldest human pathogens and has evolved complex strategies for survival. Therefore, to be successful in the high endemic regions, any future TB vaccine strategy will have to be tailored in accordance with the resulting complexity of the TB infection and anti-mycobacterial immune response. In this review, we will discuss what is presently known about the interaction of M. tuberculosis with the immune system, and how this knowledge is used in new and more advanced vaccine strategies.

Antimycobacterial immuneresponses in patients with pulmonary sarcoidosis

Sarcoidosis is a multisystem granulomatous disease of unknown origin. Pathogenetic involvement of Mycobacterium tuberculosis has frequently been discussed, however, studies still remain contradictory. We examined the interferon (IFN)-γ production by enzyme-linked-immunospot in response to purified protein derivate (PPD) and mycobacterial specific antigens early-secretory-target (ESAT)-6 and culture-filtrate-protein (CFP)-10 by peripheral blood mononuclear cells (PBMC) and bronchoalveolar-lavage mononuclear cells (BALMC) of patients with pulmonary sarcoidosis, smear-negative tuberculosis and controls. Release of IFN-γ in response to ex vivo contact with PPD, ESAT-6 or CFP-10 by BALMC and PBMC were comparable among patients with sarcoidosis and controls (PBMC p=0.1630; BALMC p=0.2199) and were less freuqently observed in both groups compared to patients with tuberculosis (BALMC p&lt;0,0131; PBMC p&lt;0.0001). Within PBMC the immunephenotype of sarcoidosis-patients differed from patients with tuberculosis, as well as from controls, while within BALMC it resembled the one of patients with tuberculosis. In contrast to patients with tuberculosis, the frequency of mycobacteria-specific local and systemic immunresponses is not elevated in patients with sarcoidosis when compared to controls. The immunephenotype represents the local resemblance of the granulomatous reaction underlying tuberculosis and sarcoidosis, while showing systemical difference. These observations do not support a role of a mycobacterial infection in the pathogenesis of sarcoidosis.

Lymphocyte proliferation to mycobacterial antigens is detectable across a spectrum of HIV-associated tuberculosis

BackgroundIdentifying novel TB diagnostics is a major public health priority. We explored the diagnostic characteristics of antimycobacterial lymphocyte proliferation assays (LPA) in HIV-infected subjects with latent or active TB.MethodsHIV-infected subjects with bacille Calmette Guérin (BCG) scars and CD4 counts ≥ 200 cells/mm3 entering a TB booster vaccine trial in Tanzania had baseline in vivo and in vitro immune tests performed: tuberculin skin tests (TST), LPA and five day assays of interferon gamma (IFN-γ) release. Assay antigens were early secreted antigenic target 6 (ESAT-6), antigen 85 (Ag85), and Mycobacterium tuberculosis whole cell lysate (WCL). Subjects were screened for active TB at enrollment by history, exam, sputum smear and culture. We compared antimycobacterial immune responses between subjects with and without latent or active TB at enrollment.ResultsAmong 1885 subjects screened, 635 had latent TB and 13 had active TB. Subjects with latent TB were more likely than subjects without TB to have LPA responses to ESAT-6 (13.2% vs. 5.5%, P < 0.0001), Ag85 (18.7% vs. 3.1%, P < 0.0001), and WCL (45.7% vs. 17.1%, P < 0.0001). Subjects with active TB also were more likely than those without active TB to have detectable LPA responses to ESAT-6 (38.5% vs. 8.1%, P = 0.0001), Ag85 (46.2% vs. 8.5%, P < 0.0001), and WCL (61.5% vs. 27.0%, P = 0.0053). In subjects with a positive TST, LPA responses to ESAT-6, Ag85 and WCL were more common during active TB (p < 0.0001 for all tests). In diagnosing active TB, in vivo and in vitro tests of mycobacterial immune responses had sensitivity and specificity as follows: TST 84.6% and 65.5%, ESAT-6 LPA 38.5% and 92.0%, Ag85 LPA 46.2% and 91.5%, and WCL LPA 61.5% and 73.0%. Detectable LPA responses were more common in patients with higher CD4 counts, and higher HIV viral loads.ConclusionLymphoproliferative responses to mycobacteria are detectable during HIV-associated active TB, and are less sensitive but more specific than TST.Trial registrationClinicalTrials.gov Identifier NCT00052195.

Exploiting Immunotherapy in Mycobacterium Tuberculosis-Infected MICE: Sphingosine 1-Phosphate Treatment Results in a Protective or Detrimental Effect Depending on the Stage of Infection

Sphingosine 1-phosphate (S1P) is a natural lysophospholipid able to enhance antimycobacterial innate immune response. In the present study, we address the possible therapeutic role of S1P administered during primary or acute infection in mice aerogenically infected with Mycobacterium tuberculosis (MTB). Results show that the administration of S1P during primary infection significantly reduces the presence of MTB-infected cells within pulmonary granulomas and mycobacterial burden in the lung and in the spleen. However, if S1P treatment was started during acute infection, a detrimental effect was observed in terms of pulmonary histopathology and mycobacterial burden in the lung and in the spleen. Taken together, these results show that S1P can exert a therapeutic effect as a treatment of primary infection only.

CpG oligodeoxynucleotides promote phospholipase D dependent phagolysosome maturation and intracellular mycobacterial killing in M. tuberculosis infected type II alveolar epithelial cells

CpG oligodeoxynucleotides have been previously shown to enhance antimycobacterial response in human monocytes/macrophages. The present study reports evidences showing the capability of CpG oligodeoxynucleotides to induce (i) host phospholipase D (PLD) activation, (ii) PLD dependent reactive oxygen intermediate production, (iii) PLD dependent phagolysosome maturation and (iv) PLD dependent intracellular mycobacterial killing in type II alveolar epithelial cells. These are the first evidences showing that alveolar epithelial cells may represent efficient effecter cells during primary innate antimycobacterial immune response.

Lipocalin 2-Dependent Inhibition of Mycobacterial Growth in Alveolar Epithelium

Mycobacterium tuberculosis invades alveolar epithelial cells as well as macrophages. However, the role of alveolar epithelial cells in the host defense against M. tuberculosis remains unknown. In this study, we report that lipocalin 2 (Lcn2)-dependent inhibition of mycobacterial growth within epithelial cells is required for anti-mycobacterial innate immune responses. Lcn2 is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits the in vitro growth of mycobacteria through sequestration of iron uptake. Lcn2-deficient mice are highly susceptible to intratracheal infection with M. tuberculosis. Histological analyses at the early phase of mycobacterial infection in Lcn2-deficient mice reveal increased numbers of mycobacteria in epithelial cell layers, but not in macrophages, in the lungs. Increased intracellular mycobacterial growth is observed in alveolar epithelial cells, but not in alveolar macrophages, from Lcn2-deficient mice. The inhibitory action of Lcn2 is blocked by the addition of endocytosis inhibitors, suggesting that internalization of Lcn2 into the epithelial cells is a prerequisite for the inhibition of intracellular mycobacterial growth. Taken together, these findings highlight a pivotal role for alveolar epithelial cells during mycobacterial infection, in which Lcn2 mediates anti-mycobacterial innate immune responses within the epithelial cells.