IFN-gamma enhances bovine macrophage responsiveness to Mycobacterium bovis: Impact on bacterial replication, cytokine release and macrophage apoptosis

We sought to determine the impact of bovine IFN-gamma on the interaction between Mycobacterium bovis and bovine macrophages. Bovine macrophages released small amounts of nitric oxide (NO), TNF-alpha, IL-1beta and IL-12 upon infection with bacille Calmette-Guérin (BCG). Prior pulsing of cells with IFN-gamma significantly enhanced the release of NO and IL-12. Infection of bovine macrophages with virulent M. bovis led to the release of higher levels of pro-inflammatory mediators, compared to levels released upon BCG infection. IFN-gamma treatment of macrophages enhanced the release of pro-inflammatory mediators, but did not modify bacterial replication in M. bovis-infected macrophages. Treatment of macrophages with a combination of IFN-gamma and LPS led to a reduction in bacterial replication. Infected cells treated with IFN-gamma/LPS progressed mostly through an apoptotic pathway, whereas untreated infected cells eventually died by necrosis. Agents that prevented the acquisition of bacteriostatic activity by activated macrophages also prevented the induction of apoptosis in infected macrophages (IL-10 and neutralizing anti-TNF-alpha). We conclude that virulent M. bovis is a major determinant of release of pro-inflammatory cytokines by macrophages. IFN-gamma amplifies the macrophage cytokine release in response to M. bovis. Induction of apoptosis is closely linked to the emergence of macrophage resistance to M. bovis replication, which is dependent on endogenous TNF-alpha release.     

Bovine dendritic cells are more permissive for Mycobacterium bovis replication than macrophages, but release more IL-12 and induce better immune T-cell proliferation

Bovine dendritic cells (DCs) were obtained by incubating blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The ability of DCs to phagocytose and allow the replication of virulent Mycobacterium bovis in vitro was studied, and compared with bovine blood monocyte-derived macrophages. In addition, the release of cytokines by M. bovis-infected DCs was assessed. DCs were shown to phagocytose M. bovis efficiently, and allowed a more substantial replication of M. bovis when compared to macrophages, as assessed by the metabolic activity of intracellular bacteria. During the course of M. bovis infection, it was found that macrophages released substantial amounts of pro-inflammatory factors such as tumour necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and interleukin-1 beta (IL-1 beta). M. bovis-infected DCs released much smaller quantities of NO, IL-1 beta and TNF-alpha (5- to 10-fold lower amounts), when compared to macrophages. Treating cells with interferon-gamma (IFN-gamma) before and during the in vitro infection process was shown to increase the release of NO, TNF-alpha and IL-1 beta by M. bovis-infected macrophages, but not by M. bovis-infected DCs. M. bovis-infected macrophages released more interleukin-10 (IL-10) than infected DCs. Treating cells with IFN-gamma/LPS was shown to reduce M. bovis metabolic activity in infected macrophages, but had no such impact on M. bovis metabolic activity in infected DCs. A variety of T-cell-derived cytokines (IFN-gamma, GM-CSF, IL-4) had no impact on the replication of M. bovis in infected DCs. On the other hand, DCs infected with M. bovis sustained a more efficient replication of autologous sensitized T lymphocytes compared to M. bovis-infected macrophages. M. bovis-infected DCs released more substantial amounts of interleukin-12 (IL-12) than similarly infected macrophages. These data suggest a complementary role for DCs and macrophages with regard to bacteriostatic activity and induction of an efficient immune response against M. bovis.     

Acylation determines the toll-like receptor (TLR)-dependent positive versus TLR2-, mannose receptor-, and SIGNR1-independent negative regulation of pro-inflammatory cytokines by mycobacterial lipomannan

Mycobacterium tuberculosis lipomannans (LMs) modulate the host innate immune response. The total fraction of Mycobacterium bovis BCG LM was shown both to induce macrophage activation and pro-inflammatory cytokines through Toll-like receptor 2 (TLR2) and to inhibit pro-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages through a TLR2-independent pathway. The pro-inflammatory activity was attributed to tri- and tetra-acylated forms of BCG LM but not the mono- and di-acylated ones. Here, we further characterize the negative activities of M. bovis BCG LM on primary murine macrophage activation. We show that di-acylated LMs exhibit a potent inhibitory effect on cytokine and NO secretion by LPS-activated macrophages. The inhibitory activity of mycobacterial mannose-capped lipoarabino-mannans on human phagocytes was previously attributed to their binding to the C-type lectins mannose receptor or specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN). However, we found that di-acylated LM inhibition of LPS-induced tumor necrosis factor secretion by murine macrophages was independent of TLR2, mannose receptor, or the murine ortholog SIGNR1. We further determined that tri-acyl-LM, an agonist of TLR2/TLR1, promoted interleukin-12 p40 and NO secretion through the adaptor proteins MyD88 and TIRAP, whereas the fraction containing tetra-acylated LM activated macrophages in a MyD88-dependent fashion, mostly through TLR4. TLR4-dependent pro-inflammatory activity was also seen with M. tuberculosis LM, composed mostly of tri-acylated LM, suggesting that acylation degree per se might not be sufficient to determine TLR2 versus TLR4 usage. Therefore, LM acylation pattern determines the anti-inflammatory versus pro-inflammatory effects of LM through different pattern recognition receptors or signaling pathways and may represent an additional mean of regulating the host innate immunity by mycobacteria.     

Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism

The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3+ cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon.     

Gene expression profiling of lipoarabinomannan-treated mouse macrophage cultures infected with Mycobacterium bovis BCG

The mannosylated lipoarabinomanan (ManLAM) from mycobacterial species possesses strong anti-apoptotic action. Here we examined the ability of ManLAM isolated from Mycobacterium tuberculosis H37Rv to alter expression profiles of apoptosis-related genes in mouse macrophages infected with Mycobacterium bovis BCG Danish strain. ManLAM suppressed BCG-induced apoptosis and activities of caspase-1, -3, -8 and 9. Mouse Apoptosis Gene Array showed that ManLAM significantly down-regulated pro-apoptotic and proinflammatory genes: caspase-1, -3, -7, -8 and -9, TNF-alpha/TNFSF2, Fas/TNFRSF6, Bax-alpha, as well as IL-12 p35 and iNOS simultaneously up-regulating anti-apoptotic genes such as Bcl-2 and Mcl-1. The effect of ManLAM was contrary to BCG-induced up-regulation of proapoptotic and pro-inflammatory genes and consistent with the functional data.     

Role of apoptosis-regulating signal kinase 1 in innate immune responses by Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.     

CD43 controls the intracellular growth of Mycobacterium tuberculosis through the induction of TNF-alpha-mediated apoptosis

Establishment of Tuberculosis infection begins with the successful entry and survival of the pathogen within macrophages. We previously showed that macrophage CD43 is required for optimal uptake and growth inhibition of Mycobacterium tuberculosis both in vitro and in vivo. Here, we explore the mechanisms by which CD43 restricts mycobacterial growth in murine macrophages. We found that although M. tuberculosis grows more readily in resting CD43-/- macrophages, priming of cells with IFN-gamma returns the bacterial growth rate to that seen in CD43+/+ cells. To discern the mechanisms by which M. tuberculosis exhibits enhanced growth within resting CD43-/- macrophages, we assessed the induction of inflammatory mediators in response to infection. We found that absence of CD43 resulted in reduced production of TNF-alpha, IL-12 and IL-6 by M. tuberculosis-infected macrophages. We also found that infected resting, but not activated CD43-/- macrophages, showed decreased apoptosis and increased necrosis. Exogenous addition of the pro-inflammatory cytokine TNF-alpha restored control of M. tuberculosis growth and induction of apoptosis to CD43+/+ levels. We propose that CD43 is involved in the inflammatory response to M. tuberculosis and, through the induction of pro-inflammatory mediators, can regulate apoptosis to control intracellular growth of the bacterium.     

Matrix metalloproteinase proteolysis of the mycobacterial HSP65 protein as a potential source of immunogenic peptides in human tuberculosis

Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB). Mycobacterial secretory protein ESAT-6 induces matrix metalloproteinase (MMP)-9 in epithelial cells neighboring infected macrophages. MMP-9 then enhances recruitment of uninfected macrophages, which contribute to nascent granuloma maturation and bacterial growth. Disruption of MMP-9 function attenuates granuloma formation and bacterial growth. The abundant mycobacterial 65 kDa heat shock protein (HSP65) chaperone is the major target for the immune response and a critical component in M. tuberculosis adhesion to macrophages. We hypothesized that HSP65 is susceptible to MMP-9 proteolysis and that the resulting HSP65 immunogenic peptides affect host adaptive immunity. To identify MMPs that cleave HSP65, we used MMP-2 and MMP-9 gelatinases, the simple hemopexin domain MMP-8, membrane-associated MMP-14, MMP-15, MMP-16 and MMP-24, and glycosylphosphatidylinositol-linked MMP-17 and MMP-25. We determined both the relative cleavage efficiency of MMPs against the HSP65 substrate and the peptide sequence of the cleavage sites. Cleavage of the unstructured PAGHG474L C-terminal region initiates the degradation of HSP65 by MMPs. This initial cleavage destroys the substrate-binding capacity of the HSP65 chaperone. Multiple additional cleavages of the unfolded HSP65 then follow. MMP-2, MMP-8, MMP-14, MMP-15 and MMP-16, in addition to MMP-9, generate the known highly immunogenic N-terminal peptide of HSP65. Based on our biochemical data, we now suspect that MMP proteolysis of HSP65 in vivo, including MMP-9 proteolysis, also results in the abundant generation of the N-terminal immunogenic peptide and that this peptide, in addition to intact HSP65, contributes to the complex immunomodulatory interplay in the course of TB infection.     

Mycobacterium abscessus activates the macrophage innate immune response via a physical and functional interaction between TLR2 and dectin-1

Mycobacterium abscessus (Mab) is an emerging and rapidly growing non-tuberculous mycobacterium (NTM). Compared with M. tuberculosis , which is responsible for tuberculosis, much less is known about NTM-induced innate immune mechanisms. Here we investigated the involvement of pattern-recognition receptors and associated signalling in Mab-mediated innate immune responses. Mab activated the extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinases (MAPKs), and induced the secretion of tumour necrosis factor-α, interleukin (IL)-6 and IL-12p40 in murine macrophages via Toll-like receptor (TLR) 2. Notably, the activation of ERK1/2, but not p38, was crucial for Mab-induced pro-inflammatory cytokine production. The ITAM-like motif of dectin-1 critically contributed to Mab internalization and cytokine secretion by macrophages. In addition, dectin-1, in cooperation with TLR2, was required for the efficient phagocytosis of Mab, ERK1/2 activation and pro-inflammatory cytokine secretion. Co-immunoprecipitation and confocal analysis showed the physical interaction and colocalization of dectin-1 with TLR2 following Mab stimulation. Moreover, dectin-1-induced Syk activation was essential for the production of inflammatory cytokines and the release of reactive oxygen species by Mab-infected macrophages. Collectively, these data demonstrate that Mab actively internalizes into and robustly activates innate immune responses in macrophages through a physical and functional interaction between TLR2 and dectin-1.     

Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guérin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guérin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.     

Mycobacterial growth inhibition by interferon-gamma-activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium tuberculosis

Growth inhibition of the intracellular bacterial pathogens Mycobacterium bovis and M. tuberculosis by lymphokine-activated murine bone marrow macrophages was studied. Mycobacterial growth was assessed by the uptake of 3H-uracil or by determination of colony-forming units. Stimulation of macrophages with recombinant interferon-gamma (r-IFN-gamma) or with IFN-gamma-containing supernatants from antigen- or mitogen-stimulated T cells markedly reduced growth of M. bovis strain BCG Phipps or M. tuberculosis strain H37Rv. In contrast, M. tuberculosis strain Middelburg proved resistant to lymphokine-stimulated macrophages, suggesting heterogeneous susceptibility toward lymphokine-activated macrophages among different M. tuberculosis strains. Stimulation could be blocked by anti-IFN-gamma antiserum, indicating that IFN-gamma was capable of activating antimycobacterial macrophage functions. Stimulation with r-IFN-gamma and subsequent phagocytosis of M. bovis did not lead to increased chemiluminescence responses by bone marrow macrophages, suggesting that mycobacterial growth inhibition was not paralleled by the release of reactive oxygen metabolites. We conclude that IFN-gamma-mediated macrophage activation represents a major step in acquired resistance against tuberculosis and that evasion from this mechanism contributes to mycobacterial virulence.     

Macrophage's proinflammatory response to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinase C, ERK1/2, and phosphatidylinositol 3-kinase

Previous studies have shown that the ability of Mycobacterium tuberculosis to block a Ca(2+) flux is an important step in its capacity to halt phagosome maturation. This affect on Ca(2+) release results from M. tuberculosis inhibition of sphingosine kinase (SPK) activity. However, these studies did not address the potential role of SPK and Ca(2+) in other aspects of macrophage activation including production of proinflammatory mediators. We previously showed that nonpathogenic Mycobacterium smegmatis and to a lesser extent pathogenic Mycobacterium avium, activate Ca(2+)-dependent calmodulin/calmodulin kinase and MAPK pathways in murine macrophages leading to TNF-alpha production. However, whether SPK functions in promoting MAPK activation upon mycobacterial infection was not defined in these studies. In the present work we found that SPK is required for ERK1/2 activation in murine macrophages infected with either M. avium or M. smegmatis. Phosphoinositide-specific phospholipase C (PI-PLC) and conventional protein kinase C (cPKC) were also important for ERK1/2 activation. Moreover, there was increased activation of cPKC and PI3K in macrophages infected with M. smegmatis compared with M. avium. This cPKC and PI3K activation was dependent on SPK and PI-PLC. Finally, in macrophages infected with M. smegmatis compared with M. avium, we observed enhanced secretion of TNF-alpha, IL-6, RANTES, and G-CSF and found production of these inflammatory mediators to be dependent on SPK, PI-PLC, cPKC, and PI3K. These studies are the first to show that the macrophage proinflammatory response following a mycobacterial infection is regulated by SPK/PI-PLC/PKC activation of ERK1/2 and PI3K pathways.     

ESX-1-dependent cytolysis in lysosome secretion and inflammasome activation during mycobacterial infection

Exocytosis of lysosomes from macrophages has been described as a response to microbial cytotoxins and haemolysins, as well as for releasing pro-inflammatory cytokines interleukin (IL)-1β and IL-18 during inflammasome activation. The mycobacterial ESX-1 secretion system, encoded in part by the Region of Difference-1, is a virulence factor necessary for phagosome escape and host cell lysis by a contact-dependent haemolysin in Mycobacterium marinum . Here we show that ESX-1 from M. marinum and M. tuberculosis is required for Ca²⁺-dependent induction of lysosome secretion from macrophages. Mycobacteria-induced lysosome secretion was concurrent to release of IL-1β and IL-18, dependent on phagocytosis of bacteria containing ESX-1. Synthesis but not release of IL-1β and IL-18 occurred in response to dead bacilli and bacteria lacking ESX-1, indicating that only cytokine release was regulated by ESX-1. Release of these cytokines and exocytosis of lysosomes were independent of intracellular mycobacterial growth, yet correlated with mycobacteria-encoded haemolytic activity, demonstrating a parallel pathway for the two responses. We further identified inflammasome components caspase-1, ASC and NALP3, but not Ipaf, required for release of IL-1β and IL-18. Collectively, these results reveal a role for ESX-1 in triggering secretion of lysosomes, as well as release of IL-1β and IL-18 during mycobacteria infection.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

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.     

Role of Mincle in Alveolar Macrophage-Dependent Innate Immunity against Mycobacterial Infections in Mice

The role of macrophage-inducible C-type lectin Mincle in lung innate immunity against mycobacterial infection is incompletely defined. In this study, we show that wild-type (WT) mice responded with a delayed Mincle induction on resident alveolar macrophages and newly immigrating exudate macrophages to infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), peaking by days 14-21 posttreatment. As compared with WT mice, Mincle knockout (KO) mice exhibited decreased proinflammatory mediator responses and leukocyte recruitment upon M. bovis BCG challenge, and they demonstrated increased mycobacterial loads in pulmonary and extrapulmonary organ systems. Secondary mycobacterial infection on day 14 after primary BCG challenge led to increased cytokine gene expression in sorted alveolar macrophages of WT mice, but not Mincle KO mice, resulting in substantially reduced alveolar neutrophil recruitment and increased mycobacterial loads in the lungs of Mincle KO mice. Collectively, these data show that WT mice respond with a relatively late Mincle expression on lung sentinel cells to M. bovis BCG infection. Moreover, M. bovis BCG-induced upregulation of C-type lectin Mincle on professional phagocytes critically shapes antimycobacterial responses in both pulmonary and extrapulmonary organ systems of mice, which may be important for elucidating the role of Mincle in the control of mycobacterial dissemination in mice.     

Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages

Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis.     

Mycobacterium tuberculosis-induced apoptotic neutrophils trigger a pro-inflammatory response in macrophages through release of heat shock protein 72, acting in synergy with the bacteria

Mycobacterium tuberculosis (Mtb) survive inside macrophages by manipulating microbicidal functions such as phago-lysosome fusion, production of reactive oxygen species and nitric oxide, and by rendering macrophages non-responsive to IFN-gamma. Mtb-infected lung tissue does however not only contain macrophages, but also significant numbers of infiltrating polymorphonuclear neutrophils (PMN). These are able to phagocytose and kill ingested Mtb, but are short-lived cells that constantly need to be removed from tissues to avoid tissue damage. Phagocytosis of aged or UV-induced apoptotic PMN by macrophages induce an anti-inflammatory response in macrophages. However, in the present study, we show that engulfment of Mtb-induced apoptotic PMN by macrophages initiates secretion of TNF-alpha from the macrophages, reflecting a pro-inflammatory response. Moreover, Mtb-induced apoptotic PMN up-regulate heat shock proteins 60 and 72 (Hsp60, Hsp72) intracellularly and also release Hsp72 extracellularly. We found that both recombinant Hsp72 and released Hsp72 enhanced the pro-inflammatory response to both Mtb-induced apoptotic PMN and Mtb. This stimulatory effect of the supernatant was abrogated by depleting the Hsp72 with immunoprecipitation. These findings indicate that released Hsp72 from Mtb-infected PMN can trigger macrophage activation during the early stage of Mtb infections, thereby creating a link between innate and adaptive immunity.