Killing of Aspergillus spores depends on the anatomical source of the macrophage

To resolve the controversy over the capacity of macrophages to kill or inhibit germination of Aspergillus spores, we compared this function in peritoneal and alveolar macrophages. Alveolar macrophages from rabbits killed 82 to 90% and completely digested 72 to 82% of spores of Aspergillus fumigatus in 30 h. In contrast, peritoneal macrophages could not even inhibit the germination of ingested spores; more than 85% transformed into mycelia within 24 h. Killing by alveolar macrophages was delayed for 3 to 6 h after phagocytosis and was independent of oxidative killing mechanisms and immune activation. The ability of alveolar macrophages to kill Aspergillus spores without modulation by T lymphocytes or the generation of oxygen intermediates points out that concepts built on studies of peritoneal macrophages may be misleading and underscores the importance of studying the role of macrophages in immunity with cells from the appropriate anatomical site.     

Hyperoxia impairs antibacterial function of macrophages through effects on actin

Oxidative stress may impair alveolar macrophage function in patients with inflammatory lung diseases or those exposed to high concentrations of oxygen. We investigated putative mechanisms of injury to macrophages by oxidative stress, using RAW 264.7 cells exposed to 95% oxygen for 48 h. Hyperoxia-exposed macrophages were less able to phagocytose and kill Klebsiella pneumoniae than normoxic controls, despite increased production of nitric oxide, a free radical important in pathogen killing. Exposure of macrophages to hyperoxia had marked effects on the actin cytoskeleton, including increased actin polymerization, loss of cortical actin, formation of stress fibers, de novo synthesis of actin, and actin oxidation. Hyperoxia induced changes in cell morphology, with increased cell size and pseudopod formation. Exposure of macrophages to jasplakinolide, an agent that increases actin polymerization, also impaired their ability to phagocytose Klebsiella. Alveolar macrophages isolated from mice exposed to 100% oxygen for 84 h also demonstrated impaired phagocytic function, as well as similar effects on the actin cytoskeleton and cell morphology to macrophages exposed to hyperoxia in vitro. We conclude that oxidative stress in vitro and in vivo impairs macrophage antibacterial function through effects on actin.     

The volatile anaesthetic sevoflurane attenuates lipopolysaccharide‐induced injury in alveolar macrophages

Acute lung injury (ALI) is a well‐defined inflammation whereby alveolar macrophages play a crucial role as effector cells. As shown previously in numerous experimental approaches, volatile anaesthetics might reduce the degree of injury in pre‐ or post‐conditioning set‐ups. Therefore, we were interested to evaluate the effect of the application of the volatile anaesthetic sevoflurane on alveolar macrophages regarding the expression of inflammatory mediators upon lipopolysaccharide (LPS) stimulation in vitro. Alveolar macrophages were stimulated with LPS. Two hours later, cells were exposed additionally to air (control) or to sevoflurane‐containing air for 4, 6, 8, 12 or 24 h. Tumour necrosis factor (TNF)‐α, cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1), macrophage‐inflammatory protein‐2 (MIP‐2) and monocyte chemoattractant protein‐1 (MCP‐1) proteins were determined and chemotaxis assays were performed. To evaluate possible cellular signalling pathways phosphorylation of the kinases extracellular‐regulated kinase (ERK) and Akt was assessed. In the early phase of sevoflurane post‐conditioning expression of TNF‐α, CINC‐1, MIP‐2 and MCP‐1 was attenuated, leading to a diminished chemotaxis reaction for neutrophils. Phosphorylation of ERK seems to be a possible cellular mechanism in the sevoflurane‐induced protection in vitro. Pharmacological post‐conditioning of alveolar macrophages with sevoflurane immunmodulates the inflammatory response upon stimulation with endotoxin. This might be a possible option for a therapeutical approach in ALI.     

Alterations in the Bactericidal Ability of Rabbit Alveolar Macrophages as a Result of Tumbling Stress

Bacteriologic and electron microscopic studies support observations that the degradative phase of the phagocytic process in rabbit alveolar macrophages is altered by subjecting the animals to tumbling stress. Macrophages from control and stressed rabbits have similar ultrastructural features. In vitro incubation of macrophages from stressed animals with Pseudomonas aeruginosa showed that the process of bacterial ingestion was not impaired, nor were the numbers of bacteria ingested decreased as compared to control macrophages. However, the ability of macrophages from stressed animals to kill ingested bacteria was significantly decreased. Bacterial plate counts after 30 minutes incubation showed that approximately 70% of the ingested organisms had been killed by control macrophages, whereas there was practically no kill by macrophages from stressed animals even after 90 minutes. Electron microscopy demonstrated extensive degradation of Pseudomonas within phagocytic vacuoles of control macrophages. These changes consisted of cytoplasmic swelling, clumping of nuclear material and disruption of the bacterial cell wall. In contrast, bacterial degradation was not observed within macrophages from stressed animals at any of the intervals studied (up to 90 minutes after rotation). This form of stress thus appears to deprive the alveolar macrophages of the ability to destroy ingested microorganisms, a factor which may be important to the establishment of pulmonary infections.     

Activation of lung macrophage subpopulations in experimental acute pancreatitis

Pulmonary macrophages exist in two different anatomical compartments in the lower respiratory tract: alveolar macrophages in the alveoli and interstitial macrophages in the interstitium. Depending on the micro-environmental stimulation, macrophages follow different activation pathways. According to their inflammatory response pattern, activated macrophages have been characterized as pro-inflammatory (M1), wound-healing (M2a) and regulatory (M2b). Since acute pancreatitis occurs in parallel with acute lung injury, the profile of the different macrophage subpopulations could be relevant in the progression of the disease. The activation of lung alveolar and interstitial macrophages was assessed in an experimental model of severe acute pancreatitis induced in rats by intraductal infusion of 3.5% sodium taurocholate. Alveolar and interstitial macrophages were obtained and the expression of markers of different activations was evaluated. Activation of nuclear factors PPARγ and NF-κB, which are involved in the acquisition of different phenoytpes, was also measured. Alveolar macrophages acquired an early M1 phenotype characterized by the expression of inflammatory cytokines and NF-κB activation. In contrast, interstitial macrophages followed the inhibitory M2b pathway. In these macrophages, PPARγ became activated and the anti-inflammatory cytokine IL-10 was expressed. These results suggest that alveolar and interstitial macrophages play different roles in acute lung injury associated with acute pancreatitis. Alveolar macrophages promote an early inflammatory response, whereas interstitial macrophages help resolve inflammation.     

Effect of Influenza Virus Infection on Phagocytic and Cytopeptic Capacities of Guinea Pig Macrophages

Guinea pigs were exposed to influenza Al virus by aerosol. Peritoneal and alveolar macrophages were harvested three days after virus exposure and allowed to attach overnight to Leighton tubes. These macrophages were then challenged with a suspension of Klebsiella pneumoniae for two hours. The macrophages were washed free of extracellular bacteria and antibiotics were added to prevent extracellular multiplication. Plate counts were made at various time intervals on disrupted macrophages to determine the number of viable intracellular bacteria remaining. Alveolar macrophages that had been exposed to virus in vivo ingested the bacteria at a rate significantly greater (p <.05) than that of non-virus exposed control macrophages. However, virus exposed macrophages exhibited significantly reduced intracellular killing (cytopepsis) (p <.01) as compared to controls. In vitro virus exposed macrophages exhibited no significant difference in the rate of phagocytosis or cytopepsis. The data support the hypothesis that virus infection reduces host resistance to bacterial infection by interfering in vivo with cytopepsis of the ingested bacteria.     

Macrophage-related diseases of the gut: a pathologist's perspective

The resident macrophages of the gastrointestinal tract represent the largest population of macrophages in the human body and are usually located in the subepithelial lamina propria. This strategic location guarantees a first-line defense to the huge numbers of potentially harmful bacteria and antigenic stimuli that are present in the intestinal lumen. In non-inflamed mucosa, macrophages phagocytose and kill microbes in the absence of an inflammatory response. However, in the event of an epithelial breach and/or microbial invasion, new circulating monocytes and lymphocytes will be recruited to the damaged area of the gut, which will result in the secretion of proinflammatory mediators and engage a protective inflammatory response. Although macrophages are usually not conspicuous in normal mucosal samples of the gut, they can easily be detected when they accumulate exogenous particulate material or endogenous substances or when they become very numerous. These events will mostly occur in pathologic conditions, and this review presents an overview of the diseases which are either mediated by or affecting the resident macrophages of the gut.     

Macrophage tolerance in the gut: It is in the epigenome!

Intestinal macrophages are functionally specialized to phagocytose bacteria, including harmful and invading pathogens, without releasing pro‐inflammatory mediators. This unique functional combination guarantees an optimal defense against millions of gut bacteria while at the same time protecting the host from inflammation‐related tissue damage. IL‐10 seems to be a critical tissue factor involved in this balance and deficiencies in IL‐10 in humans and mice have been shown to deteriorate this fine balance, leading to chronic inflammation and diseases such as Crohn's disease. However, the molecular mechanisms downstream of IL‐10 are poorly understood. In this issue of European Journal of Immunology, Simon et al. [Eur. J. Immunol. 2016. 46: 1912–1925] demonstrate that the chromatin landscape in gut macrophages derived from mice lacking IL‐10 is characteristic of inflammatory macrophages, even in the absence of bacteria. The observed changes of the chromatin landscape represent an important initiating event in chronic intestinal inflammation. These observations are not only an excellent example linking a classical loss‐of‐function experiment to a molecular mechanism responsible for the induction of chronic inflammation; they also open up new avenues for targeting chromatin and remodeling enzymes in Crohn's disease.     

Caspase‐11 non‐canonical inflammasome: a critical sensor of intracellular lipopolysaccharide in macrophage‐mediated inflammatory responses

Inflammatory responses mediated by macrophages are part of the innate immune system, whose role is to protect against invading pathogens. Lipopolysaccharide (LPS) found in the outer membrane of Gram‐negative bacteria stimulates an inflammatory response by macrophages. During the inflammatory response, extracellular LPS is recognized by Toll‐like receptor 4, one of the pattern recognition receptors that activates inflammatory signalling pathways and leads to the production of inflammatory mediators. The innate immune response is also triggered by intracellular inflammasomes, and inflammasome activation induces pyroptosis and the secretion of pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) and IL‐18 by macrophages. Cysteine‐aspartic protease (caspase)‐11 and the human orthologues caspase‐4/caspase‐5 were recently identified as components of the ‘non‐canonical inflammasome’ that senses intracellular LPS derived from Gram‐negative bacteria during macrophage‐mediated inflammatory responses. Direct recognition of intracellular LPS facilitates the rapid oligomerization of caspase‐11/4/5, which results in pyroptosis and the secretion of IL‐1β and IL‐18. LPS is released into the cytoplasm from Gram‐negative bacterium‐containing vacuoles by small interferon‐inducible guanylate‐binding proteins encoded on chromosome 3 (GBP^chr3)‐mediated lysis of the vacuoles. In vivo studies have clearly shown that caspase‐11^−/− mice are more resistant to endotoxic septic shock by excessive LPS challenge. Given the evidence, activation of caspase‐11 non‐canonical inflammasomes by intracellular LPS is distinct from canonical inflammasome activation and provides a new paradigm in macrophage‐mediated inflammatory responses.     

Phagocytosis and Killing of Streptococcus pyogenes by Human Alveolar Macrophages

In contrast to human polymorphonuclear leukocytes and monocytes, alveolar macrophages were able to readily phagocytose and kill an M protein-positive Streptococcus pyogenes strain after opsonization in normal human serum.     

Alterations to chromatin in intestinal macrophages link IL‐10 deficiency to inappropriate inflammatory responses

Intestinal macrophages (IMs) are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL‐10 maintains the macrophage anti‐inflammatory response such that loss of IL‐10 results in chronic intestinal inflammation. To investigate how IL‐10‐deficiency alters IM programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone‐marrow, from both wild‐type and IL‐10‐deficient (Il10^?/?) mice. We identified chromatin accessibility changes associated with bacterial exposure and IL‐10 deficiency in both bone marrow derived macrophages and IMs. Surprisingly, Il10^?/? IMs adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, when recombinant IL‐10 was added to Il10^?/? cells, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL‐10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model in which IL‐10‐deficiency leads to chromatin alterations that contribute to a loss of IM tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation.     

Role of lysozyme in the microbicidal activity of rat alveolar macrophages

Lysozyme release from alveolar macrophages is stimulated by exposure to particles, such as latex and zymosan, and to bacteria. Rat alveolar marcophages contain 10-fold-greater intracellualr concentrations of lysozyme and release more lysozyme after stimulation than rat blood neutrophils. During 30 min of incubation in vitro, alveolar macrophages kill more than 99% of Micrococcus lysodeikticus in the incubation mixture, whereas neutrophils kill approximately 50% of the bacteria. The bactericidal capacity of alveolar macrophages for M. lysodeikticus exceeds that of neutrophils at all bacteria-to-cell ratios tested. This bacterial killing by alveolar macrophages is inhibited when specific rabbit antirat lysozyme serum is added to the incubation mixture. Electron microscopy studies indicate that bacterial killing occurs extracellularly. Initial degradation of bacteria occurs within 5 min, and lysis is complete by 25 to 30 min. Phagocytosis of lysed bacteria is maximum after 25 to 30 min. The greater quantities of lysozyme, both intracellularly and released into the extracellular environment by alveolar macrophages, suggest that this factor may be a mechanism by which alveolar macrophages contribute to pulmonary defense.     

Pulmonary Chlamydia muridarum challenge activates lung interstitial macrophages which correlate with IFN‐γ production and infection control in mice

Protective immunity to the pathogen Chlamydia is dependent on a robust IFN‐γ response generated by innate and adaptive lymphocytes. Here we assess the role of the macrophage in orchestrating a protective response in vivo to the murine pathogen, Chlamydia muridarum. During acute pulmonary and peritoneal infection, resident macrophages in both sites are infected with C. muridarum and adopt an inflammatory phenotype. In the lung, this activation is restricted to interstitial macrophages, which harbor higher levels of C. muridarum 16sRNA than alveolar macrophages. We examined innate and adaptive lymphocyte activation in the peritoneal cavity with macrophage depletion and with adoptive transfer of infected macrophages. These experiments demonstrate macrophage activation correlates with a protective IFN‐γ response and effective control of C. muridarum. These studies suggest that a quantitative or qualitative alteration in macrophages may play a key role in the development of Chlamydia‐associated diseases.     

Toll‐like receptor signalling in regenerative myogenesis: friend and foe

Skeletal muscle regeneration in normal and diseased muscle is regulated by multiple factors and cells present in the injured muscle micro‐environment. In addition to muscle progenitor cells, several immunocytes participate in the regenerative response. Among them, macrophages are one of the most important components of the immune response that governs the step‐wise progression of muscle regeneration. The initial role of macrophages is to phagocytose muscle cell debris and later, through their transition to an anti‐inflammatory phenotype, they promote regeneration. However, in several genetic muscle disorders, continuous muscle injury disrupts the balance between pro‐inflammatory and anti‐inflammatory macrophages, leading to an overall inflammatory milieu and inhibition of muscle regeneration. Accumulating evidence suggests that Toll‐like receptor (TLR)‐mediated signalling plays an important role in the regulation of macrophage phenotypes during regenerative myogenesis in response to both acute and chronic muscle injury. Here, we discuss the role of TLR signalling in regulating macrophage phenotypes and skeletal muscle regeneration in healthy and diseased muscle. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Pulmonary surfactant protein A enhances the host-defense mechanism of rat alveolar macrophages

The effects of surfactant, surfactant lipids, and surfactant protein A (SP-A) on the surface phagocytosis of [3H]thymidine-labeled Staphylococcus aureus (SAE) by rat alveolar macrophages were studied. Alveolar macrophages only ingest SAE when the bacteria are opsonized with rat serum prior to incubation with alveolar macrophages. Preincubation or "opsonization" of the bacteria with surfactant did not result in phagocytosis by the macrophages. However, preincubation of the macrophages with surfactant increased the phagocytosis of rat serum-opsonized bacteria by approximately 70% when compared to the control macrophages. The factor present in surfactant causing the stimulation of the phagocytosis is probably SP-A. Preincubation of macrophages with human SP-A enhanced the phagocytosis to the same extent as whole surfactant, whereas preincubation with surfactant lipids had no effect on the phagocytosis. The SP-A-induced enhancement of the phagocytosis is time, temperature, and concentration dependent. Phagocytosis of opsonized SAE by alveolar macrophages was maximal after 15 min of incubation and at an SP-A concentration of 1 micrograms/ml. No phagocytosis occurred at 0 degrees C. In addition, whole surfactant and SP-A induce a lucigenin-dependent chemiluminescence response in alveolar macrophages. The chemiluminescence response is initiated after 15 min of incubation and reaches a maximum after 30 min. The concentration of SP-A needed for an optimal response is in the same order of magnitude as the concentration needed for maximal enhancement of the phagocytosis of SAE by alveolar macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucocorticoid sensitivity of lipopolysaccharide‐stimulated chronic obstructive pulmonary disease alveolar macrophages

It has been reported that alveolar macrophages from patients with chronic obstructive pulmonary disease (COPD) display glucocorticoid (Gc) resistance. The Gc sensitivity of inflammatory mediators released by COPD macrophages may vary. The objective of this study was to identify Gc‐insensitive inflammatory mediators produced by lipopolysaccharide (LPS)‐stimulated alveolar macrophages from COPD patients. LPS‐stimulated alveolar macrophages from 15 COPD patients, nine smokers (S) and nine healthy non‐smokers (HNS) were stimulated with LPS with or without dexamethasone (100 and 1000 nM). Luminex and enzyme‐linked immunosorbent assay were used to measure 23 inflammatory mediators. After LPS stimulation there were lower levels of inflammatory mediators in COPD patients and S compared to HNS. There was no difference between groups for the effects of dexamethasone at either concentration (P > 0·05 for all comparisons). Tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and growth‐related oncogene (GRO)‐α displayed the greatest sensitivity to dexamethasone in COPD patients, while IL‐8, granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and granulocyte colony‐stimulating factor (G‐CSF) were the least sensitive. COPD macrophages have a reduced response to LPS. Gc sensitivity was similar in COPD macrophages compared to controls. We identify some Gc‐insensitive cytokines, including GM‐CSF, G‐CSF and IL‐8, that may be involved in the progression of airway inflammation in COPD patients.     

Inactivation of TMEM106A promotes lipopolysaccharide‐induced inflammation via the MAPK and NF‐κB signaling pathways in macrophages

Pattern recognition receptors, such as Toll‐like receptors (TLRs), play an important role in the host defense against invading microbial pathogens. Their activation must be precisely regulated, as inappropriate activation or overactivation of TLR signaling pathways may result in inflammatory disorders, such as septic shock or autoimmune diseases. TMEM106A is a type II transmembrane protein constitutively expressed in macrophages. Our current study demonstrated that TMEM106A levels were increased in macrophages upon lipopolysaccharide (LPS) stimulation, as well as in the peripheral monocytes of patients with sepsis. Tmem106a knockout mice were more sensitive to lipopolysaccharide (LPS)‐induced septic shock than wild‐type mice. Further experiments indicated that Tmem106a ablation enhanced the expression of CD80, CD86 and major histocompatibility complex (MHC)‐II in mouse macrophages upon LPS stimulation, accompanied with up‐regulation of tumor necrosis factor (TNF)‐α, interleukin (IL)‐6, interferon (IFN)‐β and inducible nitric oxide synthase (iNOS), indicating the activation of macrophages and polarization towards the M1 inflammatory phenotype. Moreover, elevated mitogen‐activated protein kinase (MAPK) and nuclear factor kappa B (NF‐κB) signaling were found to be involved in the LPS‐induced inflammatory response in Tmem106a^?/? macrophages. However, this effect was largely abrogated by macrophage deletion in Tmem106a^?/? mice. Therefore, deficiency of Tmem106a in macrophages may enhance the M1 polarization in mice, resulting in inflammation. This suggests that TMEM106A plays an important regulatory role in maintaining macrophage homeostasis.     

Innate IFN‐γ Production by Subsets of Natural Killer Cells,Natural Killer T Cells and γδ T Cells in Response to Dying Bacterial‐Infected Macrophages

Interferon‐γ (IFN‐γ) activation of macrophages is a crucial step in the early innate defence against bacterial infection. This innate IFN‐γ is thought to be produced mainly by natural killer (NK) cells through activation with interleukin (IL)‐12p70 secreted by macrophages and dendritic cells (DCs) that have sensed bacterial products. However, a number of reports have shown that bacterial stimuli are unable to induce macrophages and/or DCs to produce sufficient amounts of IL‐12p70 unless these cells are primed by IFN‐γ. It remains, therefore, unsettled how initial IFN‐γ is produced. In a previous study, we reported a novel IFN‐γ production pathway that was associated with cell death in macrophages caused by intracellular bacteria like Listeria monocytogenes (LM) and Shigella flexneri. In this study, we showed that cell death of bone‐marrow‐derived macrophage (BMM) cells following in vitro infection with Staphylococcus aureus (SA), an extracellular bacterium, can also stimulate this IFN‐γ production pathway. We also unequivocally demonstrated by using BMM cells from IL‐12‐deficient mice that the bacterial‐infected macrophage cell death‐mediated IFN‐γ production can occur without IL‐12 although the magnitude of the response is much smaller than that in the presence of IL‐12. The enhancing effect of IL‐12 on this response proved to be attributable to the negligible amounts (0.5～1.5 pg/ml) of IL‐12p70 but not to the large amounts of IL‐12p40 that were both secreted by SA‐ and LM‐infected macrophages. Taken all together, we propose that macrophage cell death caused by bacteria may trigger the initial IFN‐γ production at an early stage of bacterial infection.     

Scavenger receptor A dampens induction of inflammation in response to the fungal pathogen Pneumocystis carinii

Alveolar macrophages are the effector cells largely responsible for clearance of Pneumocystis carinii from the lungs. Binding of organisms to beta-glucan and mannose receptors has been shown to stimulate phagocytosis of the organisms. To further define the mechanisms used by alveolar macrophages for clearance of P. carinii, mice deficient in the expression of scavenger receptor A (SRA) were infected with P. carinii, and clearance of organisms was monitored over time. SRA-deficient (SRAKO) mice consistently cleared P. carinii faster than did wild-type control mice. Expedited clearance corresponded to elevated numbers of activated CD4(+) T cells in the alveolar spaces of SRAKO mice compared to wild-type mice. Alveolar macrophages from SRAKO mice had increased expression of CD11b on their surfaces, consistent with an activated phenotype. However, they were not more phagocytic than macrophages expressing SRA, as measured by an in vivo phagocytosis assay. SRAKO alveolar macrophages produced significantly more tumor necrosis factor alpha (TNF-alpha) than wild-type macrophages when stimulated with lipopolysaccharide in vitro but less TNF-alpha in response to P. carinii in vitro. However, upon in vivo stimulation, SRAKO mice produced significantly more TNF-alpha, interleukin 12 (IL-12), and IL-18 in response to P. carinii infection than did wild-type mice. Together, these data indicate that SRA controls inflammatory cytokines produced by alveolar macrophages in the context of P. carinii infection.