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-1β 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.     

Immunohistological characterization of leukocytes in the lungs of healthy mice and after bacterial intratracheal infection

Leukocytes in the peripheral lung parenchyma of mice have not been characterized histologically during bacterial infection. The aim of this study was to investigate (a) the immunohistological characteristics of healthy murine lungs and (b) the cell kinetics during acute inflammation. BALB/c and MF1 mice were examined; as well as transgenic mice with the gene defect of cystic fibrosis (CF) in the airways as an animal model for this disease. MF1 mice served as controls for the transgenic animals. Lavaged and perfused lungs were snap frozen. B and T lymphocytes, CD4+ and CD8+ cells, dendritic cells, neutrophils and a subset of macrophages were enumerated on cryostat lung sections. The lung tissue and bronchoalveolar lavage (BAL) of BALB/c mice, infected intratracheally with Haemophilus influenzae type b (Hib), were studied at different time points after infection. In the lungs of healthy mice, including CF mice, the largest population was that of T cells, CD4+ cells being always more frequent than CD8+ cells. During acute inflammation the number of neutrophils in the lung parenchyma and BAL increased strongly within the first hours after bacterial instillation and reached baseline levels within one week. This study provides a semi-quantitative analysis of immunocompetent cells in normal and infected murine lung tissue. Differences in cell numbers are found between different strains. Moreover, the cellular reaction during Hib infection in mouse lungs is dominated by neutrophils, as expected in a primary immune response. In uninfected CF mice the numbers and distribution of immune cells in the lung tissue are normal, indicating that the cellular defense is adequate.     

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

Background

Intracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.

Methodology/Principal Findings

We could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.

Conclusions/Significance

Taken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.     

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.     

Apoptotic cell clearance of Leishmania major-infected neutrophils by dendritic cells inhibits CD8+ T-cell priming in vitro by Mer tyrosine kinase-dependent signaling

Neutrophils are the predominant recruited and infected cells during the early stages of Leishmania major infection in the skin, and depletion of neutrophils promotes immunity to infection transmitted by sand fly bite. In order to better understand how the acute neutrophilic response suppresses immunity, we assessed the consequences of the interaction between neutrophils recovered from the skin-inoculation site and bone marrow-derived dendritic cells (DCs) in vitro. The capture of infected, apoptotic neutrophils by the DCs completely inhibited their cross-presentation function that was dependent on engagement of the receptor tyrosine kinase Mer on the DCs. The capture of uninfected neutrophils, or neutrophils infected with Toxoplasma gondii, had only slight immunomodulatory effects. These studies define the clearance of infected, apoptotic neutrophils by DCs and Mer receptor signaling as central to the early immune evasion strategies of L. major, with relevance to other vector-borne pathogens delivered by bite to the skin.The phagocytosis of dying cells in the absence of inflammation was described over 100 years ago by Metchnikoff in the context of the removal of regressing tissue during amphibian morphogenesis.^{1, 2} Many studies have since addressed how dying cells that are continuously generated as a consequence of normal tissue turnover signal for their clearance by dendritic cells (DCs) or macrophages in a manner that maintains homeostasis. Phagocytosis of apoptotic cells by macrophages and DCs can inhibit their production of proinflammatory mediators while leading to the production of TGF-β and to the generation of regulatory T cells.^{3, 4, 5} Under steady-state conditions, the capture of apoptotic cells by DCs is thought to contribute to the maintenance of peripheral tolerance.⁶ In infection-driven inflammatory settings, the ingestion of apoptotic cells by macrophages and DCs may be exploited by pathogens to promote infection by inhibiting components of the antimicrobial response. For example, Trypanosoma cruzi grows better in macrophages that have ingested apoptotic lymphocytes;⁷ the killing of Streptococcus pneumonia by alveolar macrophages is suppressed following their uptake of apoptotic cells;⁸ and Mycobacterium tuberculosis-induced activation of human DCs for T-cell priming is inhibited by their co-culture with apoptotic neutrophils.⁹During infection, PMNs and DCs, which are normally located in distinct anatomical compartments, may colocalize at sites of inflammation. As neutrophils are short-lived cells that must be targeted for orderly removal, the function of DCs as antigen-presenting cells (APCs) in these infectious foci may become subordinate to their role in the clearance of dying neutrophils. For microbes that gain entry into the skin via the bite of an arthropod vector, the inflammatory signals that direct the recruitment and colocalization of neutrophils and DCs can be especially pronounced as they are driven not only by the microbial stimuli, but also by salivary constituents and by the tissue injury associated with the bite itself. In this context, we have previously reported that the inoculation of Leishmania major into the skin by sand fly bite or by needle results in the rapid recruitment of large numbers of neutrophils that phagocytose the parasite and constitute the predominant initial infected cell in the site.^{10, 11, 12} Although the majority of the infected neutrophils die and release viable parasites that are taken up by inflammatory monocytes in the skin, a significant proportion appear to be engulfed by DCs, with most infected DCs acquiring their early infections via this process.¹¹ As neutrophil depletion just before infection augments the development of immunity to sand fly-transmitted infection and of a Leishmania-specific T-cell response, the capture of infected neutrophils by DCs in the skin was suggested as a key mechanism to inhibit their APC function and to delay the development of acquired resistance. The current studies were designed to model these cellular interactions in vitro, and to directly explore the immunologic consequences of the capture of L. major-infected neutrophils by DCs. The findings reveal a profound impairment of the capacity of DCs for T-cell priming following their uptake of infected neutrophils that is mediated by Mer tyrosine kinase-dependent signaling.     

Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection

During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils.     

Induction of polymorphonuclear leukocyte response by human cytomegalovirus

Neutrophils are important in the defense against bacterial infections, by ingesting and killing invading microorganisms. Because of the higher incidence of bacterial infections in patients with active human cytomegalovirus (HCMV) infections, we hypothesized that HCMV-infected neutrophils were inefficient in eliminating the bacteria. Therefore, we mock infected or infected neutrophils with HCMV by contact with HCMV-infected human pulmonary artery endothelial cells. We found that HCMV infection without N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation increased the surface expression of CD11b to the same extent as fMLP stimulation of mock infected cells. Also, HCMV-infected neutrophils became more efficient in phagocytosing serum opsonized yeast particles than mock infected cells. Furthermore, we observed an increase in intracellular free calcium and chemiluminescence in HCMV-infected cells, in response to fMLP compared to fMLP-treated mock cells. We also found that apoptosis was significantly inhibited in HCMV-infected neutrophils. In conclusion, our results suggest that neutrophils become more effective in performing their effector functions when infected with HCMV. Thus, the higher incidence of bacterial infections in HCMV patients might not be due directly to a dysfunction in the neutrophils. Instead, the fact that apoptosis is inhibited may cause over-reactive neutrophils to remain in the tissues, where they will start leaking their contents, damaging the tissues and contributing to inflammatory processes.     

Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.     

Neutrophil Elastase Causes Tissue Damage That Decreases Host Tolerance to Lung Infection with Burkholderia Species

Two distinct defense strategies can protect the host from infection: resistance is the ability to destroy the infectious agent, and tolerance is the ability to withstand infection by minimizing the negative impact it has on the host''s health without directly affecting pathogen burden. Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and causes melioidosis. We have recently shown that inflammasome-triggered pyroptosis and IL-18 are equally important for resistance to B. pseudomallei, whereas IL-1β is deleterious. Here we show that the detrimental role of IL-1β during infection with B. pseudomallei (and closely related B. thailandensis) is due to excessive recruitment of neutrophils to the lung and consequent tissue damage. Mice deficient in the potentially damaging enzyme neutrophil elastase were less susceptible than the wild type C57BL/6J mice to infection, although the bacterial burdens in organs and the extent of inflammation were comparable between C57BL/6J and elastase-deficient mice. In contrast, lung tissue damage and vascular leakage were drastically reduced in elastase-deficient mice compared to controls. Bradykinin levels were higher in C57BL/6 than in elastase-deficient mice; administration of a bradykinin antagonist protected mice from infection, suggesting that increased vascular permeability mediated by bradykinin is one of the mechanisms through which elastase decreases host tolerance to melioidosis. Collectively, these results demonstrate that absence of neutrophil elastase increases host tolerance, rather than resistance, to infection by minimizing host tissue damage.     

Role of galectin-3 in leukocyte recruitment in a murine model of lung infection by Streptococcus pneumoniae

Pneumonia can be caused by a variety of pathogens, among which Streptococcus pneumoniae causes one of the most common forms of community-acquired pneumonia. Depending on the invading pathogen, the elements of the immune response triggered will vary. For most pathogens, such as Escherichia coli, neutrophil recruitment involves a well-described family of adhesion molecules, beta(2)-integrins. In the case of streptococcal pneumonia, however, neutrophil recruitment occurs mainly through a beta(2)-integrin-independent pathway. Despite decades of research on this issue, the adhesion molecules involved in neutrophil recruitment during lung infection by S. pneumoniae have not been identified. We have previously shown that galectin-3, a soluble mammalian lectin, can be found in lungs infected by S. pneumoniae, but not by E. coli, and can mediate the adhesion of neutrophils on the endothelial cell layer, implying its role in the recruitment of neutrophils to lungs infected with S. pneumoniae. In this study, using galectin-3 null mice, we report further evidence of the involvement of this soluble lectin in the recruitment of neutrophils to S. pneumonia-infected lungs. Indeed, in the absence of galectin-3, lower numbers of leukocytes, mainly neutrophils, were recruited to the infected lungs during infection by S. pneumoniae. In the case of beta(2)-integrin-dependent recruitment induced by lung infection with E. coli, the number of recruited neutrophils was not reduced. Thus, taken together, our data suggest that galectin-3 plays a role as a soluble adhesion molecule in the recruitment of neutrophils to lungs infected by S. pneumoniae, which induces beta(2)-integrin-independent migration.     

Guinea pig neutrophil–macrophage interactions during infection with Mycobacterium tuberculosis

We examined the ability of recombinant guinea pig IL-8 (CXCL8) to activate neutrophils upon infection with virulent Mycobacterium tuberculosis. Using a Transwell insert culture system, contact-independent cell cultures were studied in which rgpIL-8-treated neutrophils were infected with virulent M. tuberculosis in the upper well, and AM were cultured in the lower well. IL-1β and TNF-α mRNA expression was significantly upregulated by AM. Neutralizing anti-rgpTNF-α polyclonal antibody abrogated the response of AM to supernatants from the rgpIL-8-treated, infected neutrophils, while an anti-rgpIL-8 polyclonal antibody had no effect. This suggests that TNF-α produced by rgpIL-8 treated, infected neutrophils may play an important role in the activation of AM in the early response of the host against M. tuberculosis infection. Significant induction of apoptosis in M. tuberculosis-infected neutrophils was observed as compared to the uninfected neutrophils. Feeding of infected, apoptotic neutrophils to AM induced a significant up-regulation of TNF-α and IL-1β mRNA compared to AM exposed to staurosporine-treated apoptotic neutrophils. Suppressed intracellular mycobacterial growth was also seen in AM fed with infected, apoptotic neutrophils as compared to the AM infected with M. tuberculosis H37Rv alone. Taken together, these data suggest that neutrophil–macrophage interactions may contribute to host defense against M. tuberculosis infection.     

Role of innate immune cells and their products in lung immunopathology

The lung, with its enormous surface area, is literally 'bathed in a sea' of potential toxins that include pathogenic microorganisms, allergens, and pollutants. To preserve homeostasis and protect itself from injury, the lung has evolved intricate defense systems that guard it from these injurious agents. This chapter will focus on the innate component of the immune system that represents the first line of defense against microbial pathogens and pollutants. The innate immune system of the lung is diverse and includes structural cells such as epithelial cells and fibroblasts as well as itinerant leukocytes such as neutrophils, monocytes, and macrophages. Dendritic cells and mast cells, although of hematopoietic origin, are resident in the lung and help sense and orchestrate immune responses in the lung. Cells of the innate immune system secrete various soluble factors that are directly or indirectly microbicidal and/or modulate the inflammatory response. Among these soluble factors, proteinases and anti-proteinases factor prominently and exert both physiological and pathological effects on the function of diverse cell types in the lung. In concert with the adaptive immune system, the innate immune system of the lung is highly effective in combating invading microbial pathogens as evidenced by the rarity with which healthy humans succumb to lung infections.     

Exposure to Leishmania braziliensis Triggers Neutrophil Activation and Apoptosis

BackgroundNeutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate.ConclusionsWe show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection.     

Efficient Capture of Infected Neutrophils by Dendritic Cells in the Skin Inhibits the Early Anti-Leishmania Response

Neutrophils and dendritic cells (DCs) converge at localized sites of acute inflammation in the skin following pathogen deposition by the bites of arthropod vectors or by needle injection. Prior studies in mice have shown that neutrophils are the predominant recruited and infected cells during the earliest stage of Leishmania major infection in the skin, and that neutrophil depletion promotes host resistance to sand fly transmitted infection. How the massive influx of neutrophils aimed at wound repair and sterilization might modulate the function of DCs in the skin has not been previously addressed. The infected neutrophils recovered from the skin expressed elevated apoptotic markers compared to uninfected neutrophils, and were preferentially captured by dermal DCs when injected back into the mouse ear dermis. Following challenge with L. major directly, the majority of the infected DCs recovered from the skin at 24 hr stained positive for neutrophil markers, indicating that they acquired their parasites via uptake of infected neutrophils. When infected, dermal DCs were recovered from neutrophil depleted mice, their expression of activation markers was markedly enhanced, as was their capacity to present Leishmania antigens ex vivo. Neutrophil depletion also enhanced the priming of L. major specific CD4⁺ T cells in vivo. The findings suggest that following their rapid uptake by neutrophils in the skin, L. major exploits the immunosuppressive effects associated with the apoptotic cell clearance function of DCs to inhibit the development of acquired resistance until the acute neutrophilic response is resolved.     

Salmonella transiently reside in luminal neutrophils in the inflamed gut

Background

Enteric pathogens need to grow efficiently in the gut lumen in order to cause disease and ensure transmission. The interior of the gut forms a complex environment comprising the mucosal surface area and the inner gut lumen with epithelial cell debris and food particles. Recruitment of neutrophils to the intestinal lumen is a hallmark of non-typhoidal Salmonella enterica infections in humans. Here, we analyzed the interaction of gut luminal neutrophils with S. enterica serovar Typhimurium (S. Tm) in a mouse colitis model.

Results

Upon S. Tm^wt infection, neutrophils transmigrate across the mucosa into the intestinal lumen. We detected a majority of pathogens associated with luminal neutrophils 20 hours after infection. Neutrophils are viable and actively engulf S. Tm, as demonstrated by live microscopy. Using S. Tm mutant strains defective in tissue invasion we show that pathogens are mostly taken up in the gut lumen at the epithelial barrier by luminal neutrophils. In these luminal neutrophils, S. Tm induces expression of genes typically required for its intracellular lifestyle such as siderophore production iroBCDE and the Salmonella pathogenicity island 2 encoded type three secretion system (TTSS-2). This shows that S. Tm at least transiently survives and responds to engulfment by gut luminal neutrophils. Gentamicin protection experiments suggest that the life-span of luminal neutrophils is limited and that S. Tm is subsequently released into the gut lumen. This “fast cycling” through the intracellular compartment of gut luminal neutrophils would explain the high fraction of TTSS-2 and iroBCDE expressing intra- and extracellular bacteria in the lumen of the infected gut.

Conclusion

In conclusion, live neutrophils recruited during acute S. Tm colitis engulf pathogens in the gut lumen and may thus actively engage in shaping the environment of pathogens and commensals in the inflamed gut.     

Type I Interferon Induction during Influenza Virus Infection Increases Susceptibility to Secondary Streptococcus pneumoniae Infection by Negative Regulation of γδ T Cells

The majority of deaths following influenza virus infection result from secondary bacterial superinfection, most commonly caused by Streptococcus pneumoniae. Several models have been proposed to explain how primary respiratory viral infections exacerbate secondary bacterial disease, but the mechanistic explanations have been contradictory. In this study, mice were infected with S. pneumoniae at different days after primary influenza A (X31) virus infection. Our findings show that the induction of type I interferons (IFNs) during a primary nonlethal influenza virus infection is sufficient to promote a deadly S. pneumoniae secondary infection. Moreover, mice deficient in type I interferon receptor (IFNAR knockout [KO] mice) effectively cleared the secondary bacterial infection from their lungs, increased the recruitment of neutrophils, and demonstrated an enhanced innate expression of interleukin-17 (IL-17) relative to wild-type (WT) mice. Lung γδ T cells were responsible for almost all IL-17 production, and their function is compromised during secondary S. pneumoniae infection of WT but not IFNAR KO mice. Adoptive transfer of γδ T cells from IFNAR KO mice reduced the susceptibility to secondary S. pneumoniae infection in the lung of WT mice. Altogether, our study highlights the importance of type I interferon as a key master regulator that is exploited by opportunistic pathogens such as S. pneumoniae. Our findings may be utilized to design effective preventive and therapeutic strategies that may be beneficial for coinfected patients during influenza epidemics.     

T cell mediated immunity to Mycobacterium tuberculosis

Recent advances in the characterization of the protective immune response to mycobacteria have highlighted the central role of phenotypically and functionally distinct subsets of T cells. These T cell subsets not only contribute to host defense by the secretion of macrophage-activating cytokines, but also by lysing the host cell. Besides releasing intracellular pathogens, which can then be taken up and killed by newly recruited macrophages, it has now been demonstrated that lysis of infected targets by one subset of cytolytic T cells can directly kill Mycobacterium tuberculosis.