TNF influences chemokine expression of macrophages in vitro and that of CD11b+ cells in vivo during Mycobacterium tuberculosis infection

Granulomas, focal accumulations of immune cells, form in the lung during Mycobacterium tuberculosis infection. Chemokines, chemotactic cytokines, are logical candidates for inducing migration of T lymphocytes and monocytes to and within the lung. TNF influences chemokine expression in some models. TNF-deficient mice infected with M. tuberculosis are highly susceptible to disease, and granuloma formation is inhibited. Through in vitro assays, we demonstrate that neutralization of TNF in M. tuberculosis-infected macrophages led to a reduction in many inflammatory chemokines, such as C-C chemokine ligand 5, CXC ligand 9 (CXCL9), and CXCL10. In TNF-deficient mice, immune cells migrated to the lungs early after infection, but did not organize to form granulomas within the lung. Although chemokine expression, as measured in whole lung tissue, was not different, the expression of chemokines in the CD11b(+) subset of cells isolated ex vivo from the lungs of TNF-deficient mice had reduced expression of C-C chemokine ligand 5, CXCL9, and CXCL10 at early time points after TNF neutralization. Local expression of CXCR3-binding chemokines within the lungs, as determined by in situ hybridization, was also affected by TNF. Therefore, TNF affects the expression of chemokines by macrophages in vitro and CD11b(+) cells in vivo, which probably influences the local chemokine gradients and granuloma formation.     

Transmembrane TNF is sufficient to initiate cell migration and granuloma formation and provide acute, but not long-term, control of Mycobacterium tuberculosis infection

TNF is critical for immunity against Mycobacterium tuberculosis infection; however, the relative contributions of the soluble and transmembrane forms of TNF in this immunity are unknown. Using memTNF mice, which express only the transmembrane form of TNF, we have addressed this question. Wild-type (WT), TNF-/-, and transmembrane TNF (memTNF) mice were infected with M. tuberculosis by aerosol. TNF-/- mice developed overwhelming infection with extensive pulmonary necrosis and died after only 33 days. memTNF mice, like WT mice, contained bacterial growth for over 16 wk, developed an Ag-specific T cell response, and initially displayed compact granulomas, comprised of both lymphocytes and macrophages. Expression of mRNA for the chemokines CXCL10, CCL3, CCL5, and CCL7 was comparable in both WT and memTNF mice. As the infection progressed, however, the pulmonary lesions in memTNF mice became larger and more diffuse, with increased neutrophil accumulation and necrosis. This was accompanied by increased influx of activated memory T cells into the lungs of memTNF mice. Eventually, these mice succumbed to infection with a mean time to death of 170 days. The expression of memTNF on T cells is functionally important because the transfer of T cells from memTNF, but not TNF-/- mice, into either RAG-/- or TNF-/- mice conferred the same survival advantage on the M. tuberculosis-infected recipient mice, as the transfer of WT T cells. Therefore, memTNF, in the absence of soluble TNF, is sufficient to control acute, but not chronic, M. tuberculosis infection, in part through its expression on T cells.     

Reactivation of latent tuberculosis infection in TNF-deficient mice

TNF-deficient mice are highly susceptible to Mycobacterium tuberculosis H37Rv infection. Here we asked whether TNF is required for postinfectious immunity in aerosol-infected mice. Chemotherapy for 4 wk commencing 2 wk postinfection reduced CFU to undetectable levels. While wild-type mice had a slight rise in CFU, but controlled infection upon cessation of chemotherapy, TNF-deficient mice developed reactivation of infection with high bacterial loads in lungs, spleen, and liver, which was fatal within 13-18 wk. The increased susceptibility of TNF-deficient mice was accompanied by diminished recruitment and activation of T cells and macrophages into the lung, with defective granuloma formation and reduced inducible NO synthase expression. Reduced chemokine production in the lung might explain suboptimal recruitment and activation of T cells and uncontrolled infection. Therefore, despite a massive reduction of the mycobacterial load by chemotherapy, TNF-deficient mice were unable to compensate and mount a protective immune response. In conclusion, endogenous TNF is critical to maintain latent tuberculosis infection, and in its absence no specific immunity is generated.     

Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guérin infection in the absence of secreted TNF and lymphotoxin-alpha

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-alpha (LT-alpha) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-alpha(-/-), TNF(-/-), and LT-alpha(-/-) mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-alpha(-/-) mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-alpha and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.     

Structural deficiencies in granuloma formation in TNF gene-targeted mice underlie the heightened susceptibility to aerosol Mycobacterium tuberculosis infection, which is not compensated for by lymphotoxin

TNF and lymphotoxin-alpha (LT alpha) may act at various stages of the host response to Mycobacterium tuberculosis. To dissect the effects of TNF independent of LT alpha, we have used C57BL/6 mice with a disruption of the TNF gene alone (TNF-/-). Twenty-one days following aerosol M. tuberculosis infection there was a marked increase in the number of organisms in the lungs of TNF-/- mice, and by 28-35 days all animals had succumbed, with widespread dissemination of M. tuberculosis. In comparison with the localized granulomas containing activated macrophages and T cells in lungs and livers of C57BL/6 wild-type (wt) mice, cellular infiltrates in TNF-/- mice were poorly formed, with extensive regions of necrosis and neutrophilic infiltration of the alveoli. Phenotypic analysis of lung homogenates demonstrated similar numbers of CD4+ and CD8+ T cells in TNF-/- and wt mice, but in TNF-deficient mice the lymphocytes were restricted to perivascular and peribronchial areas rather than colocated with macrophages in granulomas. T cells from TNF-/- mice retained proliferative and cytokine responses to purified protein derivative, and delayed-type hypersensitivity to purified protein derivative was demonstrable. Macrophages within the lungs of TNF-/- and wt mice showed similar levels of MHC class II and inducible nitric oxide synthase expression, and levels of serum nitrite were comparable. Thus, the enhanced susceptibility of TNF-/- is not compensated for by the presence of LT alpha, and the critical role of TNF is not in the activation of T cells and macrophages but in the local organization of granulomas.     

CCR5-deficient mice control Mycobacterium tuberculosis infection despite increased pulmonary lymphocytic infiltration

The control of Mycobacterium tuberculosis infection is dependent on the development of an adaptive immune response, which is mediated by granulomas. The granuloma is a dynamic structure that forms in the lung and consists primarily of macrophages and lymphocytes. For this structure to be effective in containment of the bacillus, it must develop in an organized and timely manner. The formation of the granuloma is dependent on recruitment of activated cells through adhesion molecules and chemokines. M. tuberculosis infection causes an increase in the expression of beta-chemokines CCL3, CCL4, and CCL5, and their receptor CCR5, in the lungs. In this study, we demonstrate that CCR5-transgenic knockout mice were capable of recruiting immune cells to the lung to form granulomas. CCR5(-/-) mice successfully induced a Th1 response and controlled infection. Unexpectedly, M. tuberculosis infection in these mice resulted in greater numbers of lymphocytes migrating to the lung and higher levels of many inflammatory cytokines, compared with wild-type mice, without apparent long-term detrimental effects. In the absence of CCR5, there were more dendritic cells in the lung-draining lymph nodes and more primed T lymphocytes in these mice. Bacterial numbers in the lymph nodes were also higher in CCR5(-/-) mice. Therefore, CCR5 may play a role in the migration of dendritic cells to and from the lymph nodes during M. tuberculosis infection.     

Lipocalin 2 Regulates Inflammation during Pulmonary Mycobacterial Infections

Pulmonary tuberculosis (TB), caused by the intracellular bacteria Mycobacterium tuberculosis, is a worldwide disease that continues to kill more than 1.5 million people every year worldwide. The accumulation of lymphocytes mediates the formation of the tubercle granuloma in the lung and is crucial for host protection against M.tuberculosis infection. However, paradoxically the tubercle granuloma is also the basis for the immunopathology associated with the disease and very little is known about the regulatory mechanisms that constrain the inflammation associated with the granulomas. Lipocalin 2 (Lcn2) is a member of the lipocalin family of proteins and binds to bacterial siderophores thereby sequestering iron required for bacterial growth. Thus far, it is not known whether Lcn2 plays a role in the inflammatory response to mycobacterial pulmonary infections. In the present study, using models of acute and chronic mycobacterial pulmonary infections, we reveal a novel role for Lcn2 in constraining T cell lymphocytic accumulation and inflammation by inhibiting inflammatory chemokines, such as CXCL9. In contrast, Lcn2 promotes neutrophil recruitment during mycobacterial pulmonary infection, by inducing G-CSF and KC in alveolar macrophages. Importantly, despite a common role for Lcn2 in regulating chemokines during mycobacterial pulmonary infections, Lcn2 deficient mice are more susceptible to acute M.bovis BCG, but not low dose M.tuberculosis pulmonary infection.     

Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo

Differentiation of macrophages into foamy (lipid-laden) macrophages is a common pathological observation in tuberculous granulomas both in experimental settings as well as in clinical conditions; however, the mechanisms that regulate intracellular lipid accumulation in the course of mycobacterial infection and their significance to pathophysiology of tuberculosis are not well understood. In this study, we investigated the mechanisms of formation and function of lipid-laden macrophages in a murine model of tuberculosis. Mycobacterium bovis bacillus Calmette-Guérin (BCG), but not Mycobacterium smegmatis, induced a dose- and time-dependent increase in lipid body-inducible nonmembrane-bound cytoplasmic lipid domain size and numbers. Lipid body formation was drastically inhibited in TLR2-, but not in TLR4-deficient mice, indicating a role for TLR2 in BCG recognition and signaling to form lipid bodies. Increase in lipid bodies during infection correlated with increased generation of PGE2 and localization of cyclooxygenase-2 within lipid bodies. Moreover, we demonstrated by intracellular immunofluorescent localization of newly formed eicosanoid that lipid bodies were the predominant sites of PGE2 synthesis in activated macrophages. Our findings demonstrated that BCG-induced lipid body formation is TLR2 mediated and these structures function as signaling platforms in inflammatory mediator production, because compartmentalization of substrate and key enzymes within lipid bodies has impact on the capacity of activated leukocytes to generate increased amounts of eicosanoids during experimental infection by BCG.     

An in vitro dual model of mycobacterial granulomas to investigate the molecular interactions between mycobacteria and human host cells

In the majority of individuals infected with Mycobacterium tuberculosis, the bacilli cause a long-term asymptomatic infection called latent tuberculosis, a state during which the bacilli reside within granulomas. Latently infected individuals have around 10% risk of progression to clinical disease at a later stage. Determining the state of the mycobacteria and the host cells during this latent phase, i.e. within the granulomas, would greatly improve our understanding of the physiopathology of tuberculosis, and thus enable the development of new therapeutic means to treat the one-third of the world's population who are latently infected. We have developed an in vitro model of human mycobacterial granulomas, enabling the cellular and molecular analysis of the very first steps in the host granulomatous response to either mycobacterial compounds or live mycobacterial species. In vitro mycobacterial granulomas mimic natural granulomas very well, with the progressive recruitment of macrophages around live bacilli or mycobacterial antigen-coated beads, their differentiation into multinucleated giant cells and epithelioid cells, and the final recruitment of a ring of activated lymphocytes. Besides morphological similarities, in vitro granulomas also functionally resemble natural ones, with the development of intense cellular co-operation and intracellular mycobactericidal activities.     

Induction of TNF in human alveolar macrophages as a potential evasion mechanism of virulent Mycobacterium tuberculosis.

The ability of macrophages to release cytokines is crucial to the host response to intracellular infection. In particular, macrophage-derived TNF plays an important role in the host response to infection with the intracellular pathogen Mycobacterium tuberculosis. In mice, TNF is indispensable for the formation of tuberculous granulomas, which serve to demarcate the virulent bacterium. TNF is also implicated in many of the immunopathological features of tuberculosis. To investigate the role of TNF in the local immune response, we infected human alveolar macrophages with virulent and attenuated mycobacteria. Infection with virulent strains induced the secretion of significantly higher levels of bioactive TNF than attenuated strains correlating with their ability to multiply intracellularly. Treatment of infected macrophages with neutralizing anti-TNF Abs reduced the growth rate of intracellular bacteria, whereas bacterial replication was augmented by addition of exogenous TNF. Infected and uninfected macrophages contributed to cytokine production as determined by double-staining of M. tuberculosis and intracellular TNF. The induction of TNF by human alveolar macrophages at the site of infection permits the multiplication of intracellular bacteria and may therefore present an evasion mechanism of human pathogens.     

A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4(+) memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4(+) cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.     

The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection

The recruitment of leukocytes to infectious foci depends strongly on the local release of chemoattractant mediators. The human CXC chemokine receptor 3 (CXCR3) is an important node in the chemokine signaling network and is expressed by multiple leukocyte lineages, including T cells and macrophages. The ligands of this receptor originate from an ancestral CXCL11 gene in early vertebrates. Here, we used the optically accessible zebrafish embryo model to explore the function of the CXCR3-CXCL11 axis in macrophage recruitment and show that disruption of this axis increases the resistance to mycobacterial infection. In a mutant of the zebrafish ortholog of CXCR3 (cxcr3.2), macrophage chemotaxis to bacterial infections was attenuated, although migration to infection-independent stimuli was unaffected. Additionally, attenuation of macrophage recruitment to infection could be mimicked by treatment with NBI74330, a high-affinity antagonist of CXCR3. We identified two infection-inducible CXCL11-like chemokines as the functional ligands of Cxcr3.2, showing that the recombinant proteins exerted a Cxcr3.2-dependent chemoattraction when locally administrated in vivo. During infection of zebrafish embryos with Mycobacterium marinum, a well-established model for tuberculosis, we found that Cxcr3.2 deficiency limited the macrophage-mediated dissemination of mycobacteria. Furthermore, the loss of Cxcr3.2 function attenuated the formation of granulomatous lesions, the typical histopathological features of tuberculosis, and led to a reduction in the total bacterial burden. Prevention of mycobacterial dissemination by targeting the CXCR3 pathway, therefore, might represent a host-directed therapeutic strategy for treatment of tuberculosis. The demonstration of a conserved CXCR3-CXCL11 signaling axis in zebrafish extends the translational applicability of this model for studying diseases involving the innate immune system.KEY WORDS: Macrophage biology, Tuberculosis, Chemokine, CXCR3, CXCL11, Mycobacterium, Zebrafish, Immunology     

Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV-/-, FucT-VII-/-, FucT-IV-/-/FucT-VII-/-, or wild-type control mice. In lymph nodes, infected FucT-IV-/-/FucT-VII-/- and, to a lesser extent, FucT-VII-/- mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV-/-/FucT-VII-/- mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.     

Mycobacterium smegmatis laminin-binding glycoprotein shares epitopes with Mycobacterium tuberculosis heparin-binding haemagglutinin

Mycobacterium tuberculosis, the causative agent of tuberculosis, produces a heparin-binding haemagglutinin adhesin (HBHA), which is involved in its epithelial adherence. To ascertain whether HBHA is also present in fast-growing mycobacteria, Mycobacterium smegmatis was studied using anti-HBHA monoclonal antibodies (mAbs). A cross-reactive protein was detected by immunoblotting of M. smegmatis whole-cell lysates. However, the M. tuberculosis HBHA-encoding gene failed to hybridize with M. smegmatis chromosomal DNA in Southern blot analyses. The M. smegmatis protein recognized by the anti-HBHA mAbs was purified by heparin-Sepharose chromatography, and its amino-terminal sequence was found to be identical to that of the previously described histone-like protein, indicating that M. smegmatis does not produce HBHA. Biochemical analysis of the M. smegmatis histone-like protein shows that it is glycosylated like HBHA. Immunoelectron microscopy demonstrated that the M. smegmatis protein is present on the mycobacterial surface, a cellular localization inconsistent with a histone-like function, but compatible with an adhesin activity. In vitro protein interaction assays showed that this glycoprotein binds to laminin, a major component of basement membranes. Therefore, the protein was called M. smegmatis laminin-binding protein (MS-LBP). MS-LBP does not appear to be involved in adherence in the absence of laminin but is responsible for the laminin-mediated mycobacterial adherence to human pneumocytes and macrophages. Homologous laminin-binding adhesins are also produced by virulent mycobacteria such as M. tuberculosis and Mycobacterium leprae, suggesting that this adherence mechanism may contribute to the pathogenesis of mycobacterial diseases.     

Mice deficient in LRG-47 display increased susceptibility to mycobacterial infection associated with the induction of lymphopenia

Although IFN-gamma is essential for host control of mycobacterial infection, the mechanisms by which the cytokine restricts pathogen growth are only partially understood. LRG-47 is an IFN-inducible GTP-binding protein previously shown to be required for IFN-gamma-dependent host resistance to acute Listeria monocytogenes and Toxoplasma gondii infections. To examine the role of LRG-47 in control of mycobacterial infection, LRG-47(-/-) and wild-type mice were infected with Mycobacterium avium, and host responses were analyzed. LRG-47 protein was strongly induced in livers of infected wild-type animals in an IFN-gamma-dependent manner. LRG-47(-/-) mice were unable to control bacterial replication, but survived the acute phase, succumbing 11-16 wk postinfection. IFN-gamma-primed, bone marrow-derived macrophages from LRG-47(-/-) and wild-type animals produced equivalent levels of TNF and NO upon M. avium infection in vitro and developed similar intracellular bacterial loads. In addition, priming for IFN-gamma production was observed in T cells isolated from infected LRG-47(-/-) mice. Importantly, however, mycobacterial granulomas in LRG-47(-/-) mice showed a marked lymphocyte deficiency. Further examination of these animals revealed a profound systemic lymphopenia and anemia triggered by infection. As LRG47(-/-) T lymphocytes were found to both survive and confer resistance to M. avium in recipient recombinase-activating gene-2(-/-) mice, the defect in cellular response and bacterial control in LRG-47(-/-) mice may also depend on a factor(s) expressed in a nonlymphocyte compartment. These findings establish a role for LRG-47 in host control of mycobacteria and demonstrate that in the context of the IFN-gamma response to persistent infection, LRG-47 can have downstream regulatory effects on lymphocyte survival.     

A TNF-regulated recombinatorial macrophage immune receptor implicated in granuloma formation in tuberculosis

Macrophages play a central role in host defense against mycobacterial infection and anti- TNF therapy is associated with granuloma disorganization and reactivation of tuberculosis in humans. Here, we provide evidence for the presence of a T cell receptor (TCR) αβ based recombinatorial immune receptor in subpopulations of human and mouse monocytes and macrophages. In vitro, we find that the macrophage-TCRαβ induces the release of CCL2 and modulates phagocytosis. TNF blockade suppresses macrophage-TCRαβ expression. Infection of macrophages from healthy individuals with mycobacteria triggers formation of clusters that express restricted TCR Vβ repertoires. In vivo, TCRαβ bearing macrophages abundantly accumulate at the inner host-pathogen contact zone of caseous granulomas from patients with lung tuberculosis. In chimeric mouse models, deletion of the variable macrophage-TCRαβ or TNF is associated with structurally compromised granulomas of pulmonary tuberculosis even in the presence of intact T cells. These results uncover a TNF-regulated recombinatorial immune receptor in monocytes/macrophages and demonstrate its implication in granuloma formation in tuberculosis.     

Exosomes isolated from mycobacteria-infected mice or cultured macrophages can recruit and activate immune cells in vitro and in vivo

More than 2 billion people are infected with Mycobacterium. tuberculosis; however, only 5-10% of those infected will develop active disease. Recent data suggest that containment is controlled locally at the level of the granuloma and that granuloma architecture may differ even within a single infected individual. Formation of a granuloma likely requires exposure to mycobacterial components released from infected macrophages, but the mechanism of their release is still unclear. We hypothesize that exosomes, which are small membrane vesicles containing mycobacterial components released from infected macrophages, could promote cellular recruitment during granuloma formation. In support of this hypothesis, we found that C57BL/6 mouse-derived bone marrow macrophages treated with exosomes released from M. tuberculosis-infected RAW264.7 cells secrete significant levels of chemokines and can induce migration of CFSE-labeled macrophages and splenocytes. Exosomes isolated from the serum of M. bovis bacillus Calmette-Guérin-infected mice could also stimulate macrophage production of chemokines and cytokines ex vivo, but the level and type differed during the course of a 60-d infection. Of interest, the exosome concentration in serum correlated strongly with mouse bacterial load, suggesting some role in immune regulation. Finally, hollow fiber-based experiments indicated that macrophages treated with exosomes released from M. tuberculosis-infected cells could promote macrophage recruitment in vivo. Exosomes injected intranasally could also recruit CD11b(+) cells into the lung. Overall, our study suggests that exosomes may play an important role in recruiting and regulating host cells during an M. tuberculosis infection.     

Mycobacterial lipopeptides elicit CD4+ CTLs in Mycobacterium tuberculosis-infected humans

In searching for immunogenic molecules with the potential to induce protective immune responses against tuberculosis, we developed an ex vivo model to study frequency, phenotype, and effector functions of human T lymphocytes recognizing hydrophobic Ags of Mycobacterium tuberculosis (M.Tb). To obtain unbiased results, we characterized T lymphocytes responding to a crude cell wall extract (chloroform methanol extract of M.Tb (M.Tb-CME)) containing a broad spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion of T lymphocytes recognized M.Tb-CME (290 IFN-gamma+ T cells/10(5) PBMCs) and developed to effector memory cells as determined by the expression of CD45RO and the chemokine receptors CXCR3 and CCR5. Expanded lymphocytes fulfilled all criteria required for an efficient immune response against tuberculosis: 1) release of macrophage-activating Th1 cytokines and chemokines required for the spatial organization of local immune responses, 2) cytolytic activity against Ag-pulsed macrophages, and 3) recognition of infected macrophages and killing of the intracellular bacteria. Phenotypically, M.Tb-CME-expanded cells were CD4+ and MHC class II restricted, challenging current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8+ T cell subset. Pretreatment of M.Tb-CME with protease or chemical delipidation abrogated the biological activity, suggesting that responses were directed toward mycobacterial lipopeptides. These findings suggest that lipidated peptides are presented by M.Tb-infected macrophages and elicit CD4+ cytolytic and antimicrobial T lymphocytes. Our data support an emerging concept to include hydrophobic microbial Ags in vaccines against tuberculosis.     

Macrophage response to Mycobacterium tuberculosis during HIV infection: relationships between macrophage activation and apoptosis

Human macrophages represent the first line of defense for the containment of Mycobacterium tuberculosis infection. After phagocytosis, macrophages express activation surface markers and produce proinflammatory cytokines and chemokines whose main role is to control pathogen spreading by recruiting peripheral lymphocytes and monocytes at the site of inflammation. However, in the case of a concomitant human immunodeficiency virus (HIV) infection, these signals strongly enhance the susceptibility to viral infection both at the viral entry and replication levels. Under these conditions, viral expansion extends beyond tissue macrophages to T cells and vice-versa, according to the emerging viral phenotype. In absence of an efficient immune response, Mycobacterium tuberculosis can replicate in macrophages in an uncontrolled fashion culminating in macrophage death by apoptosis. As a consequence, a more severe form of immunedepression, involving both innate and specific immune responses, could be responsible for both ematogenous mycobacterial dissemination and extrapulmonary form of tuberculosis in HIV-infected patients.     

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.