Helminth species specific expansion and increased TNF-alpha production of non-classical monocytes during active tuberculosis.

Both Mycobacterium tuberculosis infection and helminths may affect innate immune mechanisms such as differential effects on monocytes towards the non-classical and intermediate subsets that favor bacterial persistence. Our aim, was to investigate helminth species specific effects on the frequency and functional activity of monocyte subsets in patients with active tuberculosis and healthy subjects. HIV-negative patients with active pulmonary tuberculosis (PTB) and community controls (CCs) in Gondar, Ethiopia were screened for helminth infection by stool microscopy. Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and ex vivo stimulation with purified protein derivative (PPD) and helminth antigens were used to characterize the distribution of monocyte subsets and their function. A total of 74 PTB patients and 57 CCs with and without helminth infection were included. Non-classical monocytes were increased in PTB patients with Ascaris and hookworm infection but not in Schistosoma-infected patients. Ascaris had the strongest effect in increasing the frequency of non-classical monocytes in both PTB patients and CCs, whereas PTB without helminth infection did not affect the frequency of monocyte subsets. There was a helminth specific increase in the frequency of TNF-α producing non-classical monocytes in hookworm infected PTB patients, both with and without PPD-stimulation. Low-to-intermediate TB disease severity associated with increased frequency of non-classical monocytes only for helminth-positive PTB patients, and the frequency of TNF-α producing monocytes were significantly higher in intermediate and non-classical monocytes of helminth positive PTB patients with an intermediate disease score. Helminth infection affected the frequency of monocyte subsets and function both in TB patients and controls which was helminth species dependent in TB patients. The clinical role of this potential immunomodulatory effect needs further study and may affect the response and protection to tuberculosis in areas where helminth infections are endemic.

Helminth Antigen Exposure Enhances Early Immune Control of Mycobacterium tuberculosis in Monocytes and Macrophages.

Helminth and Mycobacterium tuberculosis (Mtb) coinfection is common and suggested to influence the risk of developing active tuberculosis (TB). It is known that helminths in contrast to TB induce a strong Th2 response in the host. However, the direct impact of helminth antigen exposure on host immunity against TB is largely unknown. Our aim was to explore the effects of helminth antigen exposure on the early immune control of Mtb in monocytes and macrophages. Ascaris lumbricoides (ASC) and Schistosoma mansoni (SM) protein antigens were used to study the immediate effect of helminth antigen exposure in monocytes, on monocyte-to-macrophage differentiation, or mature macrophages, in the control of virulent Mtb H37Rv. Pre-exposure of peripheral blood mononuclear cells reduced Mtb growth in monocytes, especially with SM, but no Th1/Th2 cytokines or activation markers indicated involvement of T cells. Monocytes exposed before maturing into macrophages reduced Mtb growth in macrophages (ASC), and pre-exposure of mature macrophages reduced (ASC) or kept Mtb growth at control levels (SM). This in vitro model shows how helminth infection directly affects the monocyte-macrophage axis at an early stage before cell-mediated immunity develops. During acute helminth coinfection or when helminth antigen concentration is elevated at the site of Mtb infection, these helminths provide an enhanced control and killing of Mtb owing to the direct stimulatory effect of helminth antigens on phagocytic cells.

Efferocytosis of Apoptotic Neutrophils Enhances Control of Mycobacterium tuberculosis in HIV-Coinfected Macrophages in a Myeloperoxidase-Dependent Manner.

Tuberculosis remains a big threat, with 1.6 million deaths in 2017, including 0.3 million deaths among patients with HIV. The risk of developing active disease increases considerably during an HIV coinfection. Alveolar macrophages are the first immune cells to encounter the causative agent Mycobacterium tuberculosis, but during the granuloma formation other cells are recruited in order to combat the bacteria. Here, we have investigated the effect of efferocytosis of apoptotic neutrophils by M. tuberculosis and HIV-coinfected macrophages in a human in vitro system. We found that the apo-ptotic neutrophils enhanced the control of M. tuberculosis in single and HIV-coinfected macrophages, and that this was dependent on myeloperoxidase (MPO) and reactive oxygen species in an autophagy-independent manner. We show that MPO remains active in the apoptotic neutrophils and can be harnessed by infected macrophages. In addition, MPO inhibition removed the suppression in M. tuberculosis growth caused by the apoptotic neutrophils. Antimycobacterial components from apoptotic neutrophils could thus increase the microbicidal activity of macrophages during an M. tuberculosis/HIV coinfection. This cooperation between innate immune cells could thereby be a way to compensate for the impaired adaptive immunity against M. tuberculosis seen during a concurrent HIV infection.

Polymorphisms in CARD8 and NLRP3 are associated with extrapulmonary TB and poor clinical outcome in active TB in Ethiopia.

Innate immunity is a first line defense against Mycobacterium tuberculosis infection where inflammasome activation and secretion of the pro-inflammatory cytokine IL-1beta, plays a major role. Thus, genetic polymorphisms in innate immunity-related genes such as CARD8 and NLRP3 may contribute to the understanding of why most exposed individuals do not develop infection. Our aim was to investigate the association between polymorphisms in CARD8 and NLRP3 and active tuberculosis (TB) as well as their relationship to treatment outcome in a high-endemic setting for TB. Polymorphisms in CARD8 (C10X) and NLRP3 (Q705K) were analysed in 1190 TB patients and 1990 healthy donors (HD). There was a significant association between homozygotes in the CARD8 polymorphism and extrapulmonary TB (EPTB), which was not the case for pulmonary TB or HDs. Among TB-patients, there was an association between poor treatment outcome and the NLRP3 (Q705K) polymorphism. Our study shows that inflammasome polymorphisms are associated with EPTB and poor clinical outcome in active TB in Ethiopia. The practical implications and determining causal relationships on a mechanistic level needs further study.

HIV Interferes with the Dendritic Cell-T Cell Axis of Macrophage Activation by Shifting Mycobacterium tuberculosis-Specific CD4 T Cells into a Dysfunctional Phenotype.

HIV coinfection is the greatest risk factor for transition of latent Mycobacterium tuberculosis infection into active tuberculosis (TB). Epidemiological data reveal both the reduction and the impairment of M. tuberculosis-specific CD4 T cells, although the cellular link and actual mechanisms resulting in immune impairment/suppression need further characterization. M. tuberculosis-specific CD4 T cells play a central role in development of protective immunity against TB, in which they participate in the activation of macrophages through the dendritic cell (DC)-T cell axis. Using an in vitro priming system for generating Ag-specific T cells, we explored if HIV-M. tuberculosis-infected (coinfected) human DCs can dysregulate the M. tuberculosis-specific CD4 T cell phenotype and functionality and subsequently mediate the failure to control M. tuberculosis infection in macrophages. After coculture with coinfected DCs, M. tuberculosis Ag-specific CD4 T cells lost their ability to enhance control of M. tuberculosis infection in infected macrophages. Coinfection of DCs reduced proliferation of M. tuberculosis Ag-specific CD4 T cells without affecting their viability, led to increased expression of coinhibitory factors CTLA-4, PD-1, and Blimp-1, and decreased expression of costimulatory molecules CD40L, CD28, and ICOS on the T cells. Expression of the regulatory T cell markers FOXP3 and CD25, together with the immunosuppressive cytokines TGF-ß and IL-10, was also significantly increased by coinfection compared with M. tuberculosis single infection. Our data suggest a pattern in which HIV, through its effect on DCs, impairs the ability of M. tuberculosis-specific CD4 T cells to maintain a latent TB within human macrophages, which could play an early role in the subsequent development of TB.

Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages.

BACKGROUND: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. METHOD: Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. RESULTS: When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. CONCLUSION: In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.

HIV Interferes with Mycobacterium tuberculosis Antigen Presentation in Human Dendritic Cells.

HIV coinfection is the most prominent risk factor for progression of Mycobacterium tuberculosis (Mtb) infection into active tuberculosis (TB) disease. The mechanisms behind the increased transition from latent to active TB in coinfected individuals have not been well elucidated at the cellular level. We hypothesized that HIV infection contributes to Mtb pathogenesis by interfering with the dendritic cell (DC)-mediated immune control. Mtb-antigen processing and presentation are key events in the immune response against TB. Human immature DCs coinfected with HIV/Mtb had decreased expression of human leukocyte antigen antigen D related and the costimulatory molecules CD40, CD80, and CD86. In addition, Mtb-infected DCs triggered a significant release of the proinflammatory cytokines IL-6, IL-1ß, and tumor necrosis factor-α, whereas coinfected DCs did not. To assess the DC antigen presentation capacity, we measured interferon-γ from co-cultures of DCs and autologous Mtb antigen-specific CD4+ T cells. Interferon-γ release was significantly reduced when purified protein derivative- and Ag85B-specific CD4+ T cells had been activated with coinfected DCs compared to Mtb-infected DCs, and this effect was attributed to Mtb antigen processing rather than peptide-major histocompatibility complex class II loading. Evaluating autophagy as a measure of vesicular processing and maturation further revealed that HIV efficiently blocks initiation of this pathway during coinfection. Overall, our results demonstrate that HIV impairs Mtb antigen presentation in DCs, thereby suppressing an important cell linking innate and adaptive immune response in TB.

Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages.

Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1ß signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.

Human gene variants linked to enhanced NLRP3 activity limit intramacrophage growth of Mycobacterium tuberculosis.

Activation of the NLRP3 inflammasome and subsequent generation of interleukin 1ß is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequent infection of the cells with virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.

Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages.

Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-α, IL-1ß and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.

Vitamin D enhances IL-1ß secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1ß and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

Common genetic variations in the NALP3 inflammasome are associated with delayed apoptosis of human neutrophils.

BACKGROUND: Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1ß production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages. METHODS AND FINDINGS: Patients carrying the Q705K/C10X polymorphisms displayed significantly delayed spontaneous as well as microbe-induced apoptosis compared to matched controls. Western blotting revealed increased levels and phosphorylation of Akt and Mcl-1 in the patients' neutrophils. In contrast to macrophages from healthy controls, macrophages from the patients produced lower amounts of TNF; suggesting impaired macrophage clearance response. CONCLUSIONS: The Q705K/C10X polymorphisms are associated with delayed apoptosis of neutrophils. These findings are explained by altered involvement of different regulators of apoptosis, resulting in an anti-apoptotic profile. Moreover, the macrophage response to ingestion of microbe-induced apoptotic neutrophils is altered in the patients. Taken together, the patients display impaired turnover and clearance of apoptotic neutrophils, pointing towards a dysregulated innate immune response that influences the resolution of inflammation. The future challenge is to understand how microbes affect the activation of inflammasomes, and why this interaction will develop into severe inflammatory disease in certain individuals.

Human macrophages infected with a high burden of ESAT-6-expressing M. tuberculosis undergo caspase-1- and cathepsin B-independent necrosis.

Mycobacterium tuberculosis (Mtb) infects lung macrophages, which instead of killing the pathogen can be manipulated by the bacilli, creating an environment suitable for intracellular replication and spread to adjacent cells. The role of host cell death during Mtb infection is debated because the bacilli have been shown to be both anti-apoptotic, keeping the host cell alive to avoid the antimicrobial effects of apoptosis, and pro-necrotic, killing the host macrophage to allow infection of neighboring cells. Since mycobacteria activate the NLRP3 inflammasome in macrophages, we investigated whether Mtb could induce one of the recently described inflammasome-linked cell death modes pyroptosis and pyronecrosis. These are mediated through caspase-1 and cathepsin-B, respectively. Human monocyte-derived macrophages were infected with virulent (H37Rv) Mtb at a multiplicity of infection (MOI) of 1 or 10. The higher MOI resulted in strongly enhanced release of IL-1ß, while a low MOI gave no IL-1ß response. The infected macrophages were collected and cell viability in terms of the integrity of DNA, mitochondria and the plasma membrane was determined. We found that infection with H37Rv at MOI 10, but not MOI 1, over two days led to extensive DNA fragmentation, loss of mitochondrial membrane potential, loss of plasma membrane integrity, and HMGB1 release. Although we observed plasma membrane permeabilization and IL-1ß release from infected cells, the cell death induced by Mtb was not dependent on caspase-1 or cathepsin B. It was, however, dependent on mycobacterial expression of ESAT-6. We conclude that as virulent Mtb reaches a threshold number of bacilli inside the human macrophage, ESAT-6-dependent necrosis occurs, activating caspase-1 in the process.

Importance of phagosomal functionality for growth restriction of Mycobacterium tuberculosis in primary human macrophages.

The best characterized survival mechanism of Mycobacterium tuberculosis inside the macrophage is the inhibition of phagosomal maturation. Phagosomal maturation involves several steps including fusion with lysosomes and acidification. However, it has not been elucidated which components of phagosomal maturation correlate with growth restriction of virulent mycobacteria in human macrophages, and we aimed to study this. We infected human monocyte-derived macrophages with M. tuberculosis and assessed bacterial replication, translocation of CD63 to the phagosome, and phagosomal acidification. We found that unstimulated human macrophages were able to control infection with M. tuberculosis upon inoculation at a low multiplicity of infection (MOI) of 1, but not at a high MOI of 10. The low MOI resulted in a macrophage-controlled balance between host cells and bacteria. Both H37Rv and H37Ra infection, at high and low MOI, led to equally ineffective translocation of CD63 to the phagosome. On the other hand, acidification of mycobacterial phagosomes was more efficient at MOI 1 than 10 with both mycobacterial strains, consistent with a direct or indirect role for phagosomal acidification in restricting M. tuberculosis growth. Furthermore, inhibition of the vacuolar H(+)-ATPase as well as of cathepsin D led to enhanced mycobacterial replication inside the macrophage. This again shows the importance of phagosomal acidification for control of mycobacterial growth, through the activation of lysosomal hydrolases. We conclude that acidification and related functional aspects of the mature phagosome are important factors for restriction of M. tuberculosis replication in human macrophages.

Dendritic cell activation by sensing Mycobacterium tuberculosis-induced apoptotic neutrophils via DC-SIGN.

Mycobacterium tuberculosis (Mtb) manipulates cells of the innate immune system to provide the bacteria with a sustainable intracellular niche. Mtb spread through aerosol carrying them deep into the lungs, where they are internalized by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs), and macrophages. PMNs undergo accelerated apoptosis after interaction with the bacterium, and apoptotic cells are sequestered by neighboring phagocytes. Removal of aged apoptotic cells because of natural tissue turnover is described as an immunologically silent process facilitating resolution of inflammation and inhibition of DC maturation. Silencing of immune cells could be favorable for intracellular bacteria. The aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate whether this interaction follows the proposed anti-inflammatory pathway. In contrast to aged apoptotic cells, Mtb-induced apoptotic PMNs induced functional DC maturation. We found that the cell fraction from Mtb-induced apoptotic PMNs contained almost all stimulatory capacity, suggesting that cell-cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact-dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity involving the alpha(v)beta(5) but did not involve the scavenger receptor CD36. Taken together, this study demonstrates that the DCs can distinguish between normal and infected apoptotic PMNs via cellular crosstalk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.

Validation of a medium-throughput method for evaluation of intracellular growth of Mycobacterium tuberculosis.

Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.

Nitric oxide production in the exhaled air of patients with pulmonary tuberculosis in relation to HIV co-infection.

BACKGROUND: Nitric oxide (NO) is essential for host defense in rodents, but the role of NO during tuberculosis (TB) in man remains controversial. However, earlier observations that arginine supplementation facilitates anti-TB treatment, supports the hypothesis that NO is important in the host defense against TB. Local production of NO measured in fractional exhaled air (FeNO) in TB patients with and without HIV co-infection has not been reported previously. Thus, our aim was to investigate levels of FeNO in relation to clinical symptoms and urinary NO metabolites (uNO). METHODS: In a cross sectional study, FeNO and uNO were measured and clinical symptoms, chest x-ray, together with serum levels of arginine, tumor necrosis factor alpha (TNF-alpha) and interleukin 12 (IL-12) were evaluated in sputum smear positive TB patients (HIV+/TB, n = 36, HIV-/TB, n = 59), their household contacts (n = 17) and blood donors (n = 46) from Gondar University Hospital, Ethiopia. RESULTS: The proportion of HIV-/TB patients with an increased FeNO level (> 25 ppb) was significantly higher as compared to HIV+/TB patients, but HIV+/TB patients had significantly higher uNO than HIV-/TB patients. HIV+ and HIV-/TB patients both had lower levels of FeNO compared to blood donors and household contacts. The highest levels of both uNO and FeNO were found in household contacts. Less advanced findings on chest x-ray, as well as higher sedimentation rate were observed in HIV+/TB patients as compared to HIV-/TB patients. However, no significant correlation was found between FeNO and uNO, chest x-ray grading, clinical symptoms, TNF-alpha, IL-12, arginine levels or sedimentation rate. CONCLUSION: In both HIV negative and HIV co infected TB patients, low levels of exhaled NO compared to blood donors and household were observed. Future studies are needed to confirm whether low levels of exhaled NO could be a risk factor in acquiring TB and the relative importance of NO in human TB.

Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block.

Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor. LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase, contradicting some previous suggestions.

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.

Inactivation of Cdc42 is necessary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation.

Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.