Intracellular Killing Research Articles

Neutrophil phenotypes in health and disease.

Neutrophils are one of the most important effector cells of the innate immune response (1). They are traditionally seen as a homogenous population of short‐lived cells mainly involved in the defence against extracellular microorganisms by phagocytosis and intracellular killing (1,2). The cells contain a large armamentarium that aids in this function and ranges from the production of reactive oxygen species by a membrane‐bound NADPH oxidase to cytotoxic proteins and peptides residing in the different granules present in the cytoplasm (3). Recently, the view of neutrophils belonging to a homogenous population of cells has been challenged, and several neutrophil phenotypes have been described that exhibit specialized functions, such as involvement in tissue repair, tumour killing and immune regulation (4). It is not clear whether these cells belong to separate parallel lineages originating from the bone marrow or that neutrophils become instructed in the distant tissues, thus changing their phenotypes. In addition, functional heterogeneity in a phenotypically homogenous population of neutrophils adds to the complexity of neutrophil phenotypes(5). This article will review the current literature describing the heterogeneity within the neutrophil compartment with respect to both phenotype and function in health and disease.

Concentration-dependent protein loading of extracellular vesicles released by Histoplasma capsulatum after antibody treatment and its modulatory action upon macrophages

Diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. We recently demonstrated that the binding of monoclonal antibodies (mAb) modulates how Histoplasma capsulatum load and releases its extracellular vesicles (EV). In the present paper, we addressed a concentration-dependent impact on the fungus’ EV loading and release with different mAb, as well as the pathophysiological role of these EV during the host-pathogen interaction. We found that the mAbs differentially regulate EV content in concentration-dependent and independent manners. Enzymatic assays demonstrated that laccase activity in EV from H. capsulatum opsonized with 6B7 was reduced, but urease activity was not altered. The uptake of H. capsulatum by macrophages pre-treated with EV, presented an antibody concentration-dependent phenotype. The intracellular killing of yeast cells was potently inhibited in macrophages pre-treated with EV from 7B6 (non-protective) mAb-opsonized H. capsulatum and this inhibition was associated with a decrease in the reactive-oxygen species generated by these macrophages. In summary, our findings show that opsonization quantitatively and qualitatively modifies H. capsulatum EV load and secretion leading to distinct effects on the host’s immune effector mechanisms, supporting the hypothesis that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.

THE NEW LOOK AT NEUTROPHILIC GRANULOCYTES: RETHINKING OLD DOGMAS. PART 2

Numerous modern basic research done undeniable fact that neutrophilic granulocytes (NG) are key effector and regulatory circuits both innate and adaptive immunity, and play a crucial role in the pathogenesis of a wide range of diseases. NG have potent receptor repertoire, providing a connection between them, cells of the immune system, as well as communication with endothelial cells, epithelial and other tissues. NG inducing stimuli activate and promote the translocation of cytoplasmic granules and vesicles surface molecules on the cytoplasmic membrane the secretion of a large spectrum of pro-and anti-inflammatory, immunoregulatory cytokines, colony, angiogenic factors and fibrogenic, TNF superfamily members, chemokines, regulatory protein, etc. Chromatin nuclei NG capable of restructuring under the influence of inducing stimuli, which is associated with the expression of multiple cytokine genes. NG receiving complex cytokine influence not only acquire new features, but also in various stages of activation and differentiation processes involved in intracellular intraphagosomalis degranulation and killing of implementing elimination microorganisms and extracellular neutrophil degranulation in the formation neutrophil extracellular traps (NET), while this dying through NETosis. Features NG phenotype and their functional properties, demonstrate the existence of subpopulations of NG with different capabilities: equipment of different receptor, the ability to restructure chromatin expressing cytokine genes and secrete cytokines to implement the contents of the granular system, produce reactive oxygen species, implement cytotoxicity form NET. In our opinion, there subpopulation NG: regulatory; suppressor; proinflammatory — initiating an inflammatory response; inflammation with a positive potential microbicidal (antibacterial, antiviral, antifungal); inflammatory cytotoxic potential of the negative — «aggressive»; anti-inflammation regulating regression; antitumoral — TAN1; pro-tumoral — TAN2; hybrid, combining the characteristics of NG and dendritic cells. The absence of adequate response, or hyperactivation blockade NG functions leads to the development of low-intensity infectious and inflammatory diseases, do not respond to conventional therapy of autoimmune diseases/chronic immune-dependent processes. Remo deling dysfunctions NG — the key to new immunotherapeutic strategies.

Investigating the antibacterial effect of Sca1+ lung mesenchymal stem cells on multidrug resistant bacterial species

Abstract Rationale Although bone marrow-derived mesenchymal stem cells have been extensively studied in host protection against bacterial pneumonia, the role of lung stem/mesenchymal stem cells in pneumonia caused by the multidrug-resistant bacteria remains elusive. Methods We have compared the extracellular (using 10 MOI) and intracellular (using 1 MOI) killing effects of stem cell antigen 1+ lung mesenchymal stem cells (Sca1+ LMSC), bone marrow-derived neutrophils (BMDN), and bone marrow-derived macrophages (BMDM) by infecting them with Gram positive methicillin-resistant Staphylococcus aureus (MRSA) and Gram negative carbapenem-resistant Klebsiella pneumoniae (CRKP) at different time points. We evaluated the extracellular and intracellular killing at each time point by plating them on Tryptic soy agar and MacConkey agar plates, respectively. We also have examined expression of inflammasomes or inflammasome-associated proteins in MRSA (20 MOI for 6 h) infected LMSC using Western blotting. Results Sca1+ LMSC is potent in killing (extracellular) both MRSA and CRKP at 3 h post-infection. However, BMDN did not show significant extracellular killing on MRSA and CRKP at all time points examined. Interestingly, Sca1+ LMSC showed a remarkably increased intracellular killing of MRSA than BMDM. Western blotting revealed the increased level of inflammasome and inflammasome-associated proteins (NLRP3, NLRP6 and ASC) in MRSA infected LMSC. Conclusions These findings have solidified the potential of LMSC-based therapies for pneumonia caused by MRSA and CRKP. However, in vivo (animal) studies are required to determine the effectiveness of LMSC in the clearance of these bacteria and modulation of innate immune responses in the host.

Dual roles of the bacterial siderophore enterobactin by inducing apoptosis in macrophages and promoting survival advantages to Salmonella typhimurium

Abstract Iron, an essential transition metal ion, is required to facilitate the antimicrobial and redox activity of heme proteins expressed in macrophages (Mφs). In this study, we investigated whether the chelation of iron by enterobactin (Ent; a prototypical bacterial siderophore) can affect the immune responses of macrophages. Herein, we demonstrated that exogenous Ent confers protection to the intracellular pathogen Salmonella enterica serovar Typhimurium from Mφs by promoting M1 to M2 polarization, reducing the expression of pro-inflammatory cytokines and impairing their intracellular killing. Accordingly, WT Salmonella survived better compared to Ent-deficient strain (ΔentB) in Mφs. The addition of apo-Ent (iron-free), but not ferric-Ent, rescued both strains, thus suggesting that the immuno-modulatory effect of Ent could be likely mediated via chelation of Mφs cellular iron. To determine whether the effects of Ent on Mφs could be due to altered cell viability, we next assessed the Mφs for markers of apoptosis via flow cytometry. Interestingly, we observed that apo-Ent, but not ferric-Ent, promoted a time-dependent apoptosis in Mφs, which can be detected as early as 12h after Ent treatment. These findings corroborated with the time-dependent release of lactate dehydrogenase (cytotoxicity marker) from Ent-treated Mφs. Collectively, our findings demonstrate that Ent not only facilitates bacterial iron uptake but also serves to safeguard bacteria against Mφ innate immune responses, which could be mediated, in part, by inducing delayed cellular apoptosis.

Novel, Broadly Reactive Anticapsular Antibodies against Carbapenem-Resistant Klebsiella pneumoniae Protect from Infection.

ABSTRACTCarbapenem-resistant (CR) sequence type 258 (ST258) Klebsiella pneumoniae has become an urgent health care threat, causing an increasing number of high-mortality infections. Its resistance to numerous antibiotics and threat to immunocompromised patients necessitate finding new therapies to combat these infections. Previous successes in the laboratory, as well as the conservation of capsular polysaccharide (CPS) among the members of the ST258 clone, suggest that monoclonal antibody (MAb) therapy targeting the outer polysaccharide capsule of K. pneumoniae could serve as a valuable treatment alternative for afflicted patients. Here, we isolated several IgG antibodies from mice inoculated with a mixture of CR K. pneumoniae CPS conjugated to anthrax protective antigen. Two of these MAbs, 17H12 and 8F12, bind whole and oligosaccharide epitopes of the CPS of clade 2 ST258 CR K. pneumoniae, which is responsible for the most virulent CR K. pneumoniae infections in the United States. These antibodies were shown to agglutinate all clade 2 strains and were also shown to promote extracellular processes killing these bacteria, including biofilm inhibition, complement deposition, and deployment of neutrophil extracellular traps. Additionally, they promoted opsonophagocytosis and intracellular killing of CR K. pneumoniae by human-derived neutrophils and cultured murine macrophages. Finally, when mice were intratracheally infected with preopsonized clade 2 CR K. pneumoniae, these MAbs reduced bacterial dissemination to organs. Our data suggest that broadly reactive anticapsular antibodies and vaccines against clade 2 ST258 CR K. pneumoniae are possible. Such MAbs and vaccines would benefit those susceptible populations at risk of infection with this group of multidrug-resistant bacteria.

Structure/Function Analysis of a Novel Myeloperoxidase Inhibitor from Staphylococcus delphini

Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. We recently discovered a highly conserved S.aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that specifically binds and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils [de Jong et al., PNAS, 2017]. S. aureus cells deleted for SPIN gene showed decreased survival compared to wild‐type bacteria after phagocytosis by neutrophils. A crystal structure of SPIN bound to MPO suggests that it acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. To further our understanding of the structure/function relationships of SPIN proteins, we cloned, overexpressed, purified, and characterized a series of SPIN orthologs of Staphylococcal origin.We found that SPIN originating from S. delphini although shares only ~53% identity to SPIN‐aureus, it still retained a strong affinity and inhibitory capacity for human MPO. A 2.4 Å crystal structure of SPIN‐delphini bound to human MPO provided a basis for comparing the similarities and differences between SPIN‐aureus and SPIN‐delphini. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO, and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors.Support or Funding InformationNIH Grants AI111203 and GM121511This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

HIV and the Macrophage: From Cell Reservoirs to Drug Delivery to Viral Eradication

Macrophages serve as host cells, inflammatory disease drivers and drug runners for human immunodeficiency virus infection and treatments. Low-level viral persistence continues in these cells in the absence of macrophage death. However, the cellular microenvironment changes as a consequence of viral infection with aberrant production of pro-inflammatory factors and promotion of oxidative stress. These herald viral spread from macrophages to neighboring CD4+ T cells and end organ damage. Virus replicates in tissue reservoir sites that include the nervous, pulmonary, cardiovascular, gut, and renal organs. However, each of these events are held in check by antiretroviral therapy. A hidden and often overlooked resource of the macrophage rests in its high cytoplasmic nuclear ratios that allow the cell to sense its environment and rid it of the cellular waste products and microbial pathogens it encounters. These phagocytic and intracellular killing sensing mechanisms can also be used in service as macrophages serve as cellular carriage depots for antiretroviral nanoparticles and are able to deliver medicines to infectious disease sites with improved therapeutic outcomes. These undiscovered cellular functions can lead to reductions in persistent infection and may potentially facilitate the eradication of residual virus to eliminate disease.

Identification and structural characterization of a novel myeloperoxidase inhibitor from Staphylococcus delphini

Staphylococcus aureus and related species are highly adapted to their hosts and have evolved numerous strategies to evade the immune system. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium evades intracellular killing mechanisms used by neutrophils. We recently discovered an S. aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that binds to and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. To allow for comparative studies between multiple SPIN sequences, we identified a panel of homologs from species closely related to S. aureus. Characterization of these proteins revealed that SPIN molecules from S. agnetis, S. delphini, S. schleiferi, and S. intermedius all bind human MPO with nanomolar affinities, and that those from S. delphini, S. schleiferi, and S. intermedius inhibit human MPO in a dose-dependent manner. A 2.4 Å resolution co-crystal structure of SPIN-delphini bound to recombinant human MPO allowed us to identify conserved structural features of SPIN proteins, and to propose sequence-dependent physical explanations for why SPIN-aureus binds human MPO with higher affinity than SPIN-delphini. Together, these studies expand our understanding of MPO binding and inhibition by a recently identified component of the staphylococcal innate immune evasion arsenal.

Tomatidine and analog FC04\u2013100 possess bactericidal activities against Listeria, Bacillus and Staphylococcus spp

BackgroundTomatidine (TO) is a plant steroidal alkaloid that possesses an antibacterial activity against the small colony variants (SCVs) of Staphylococcus aureus. We report here the spectrum of activity of TO against other species of the Bacillales and the improved antibacterial activity of a chemically-modified TO derivative (FC04–100) against Listeria monocytogenes and antibiotic multi-resistant S. aureus (MRSA), two notoriously difficult-to-kill microorganisms.MethodsBacillus and Listeria SCVs were isolated using a gentamicin selection pressure. Minimal inhibitory concentrations (MICs) of TO and FC04–100 were determined by a broth microdilution technique. The bactericidal activity of TO and FC04–100 used alone or in combination with an aminoglycoside against planktonic bacteria was determined in broth or against bacteria embedded in pre-formed biofilms by using the Calgary Biofilm Device. Killing of intracellular SCVs was determined in a model with polarized pulmonary cells.ResultsTO showed a bactericidal activity against SCVs of Staphylococcus aureus, Bacillus cereus, B. subtilis and Listeria monocytogenes with MICs of 0.03–0.12 μg/mL. The combination of an aminoglycoside and TO generated an antibacterial synergy against their normal phenotype. In contrast to TO, which has no relevant activity by itself against Bacillales of the normal phenotype (MIC > 64 μg/mL), the TO analog FC04–100 showed a MIC of 8–32 μg/mL. Furthermore, FC04–100 showed a strong bactericidal activity against L. monocytogenes SCVs in kill kinetics experiments, while TO did not. The addition of FC04–100 (4 μg/mL) to a cefalexin:kanamycin (3:2) combination improved the activity of the combination by 32 fold against cefalexin and kanamycin-resistant MRSA strains. In combination with gentamicin, FC04–100 also exhibited a strong bactericidal activity against biofilm-embedded S. aureus. Also, FC04–100 and TO showed comparable intracellular killing of S. aureus SCVs.ConclusionsChemical modifications of TO allowed improvement of its antibacterial activity against prototypical S. aureus and of its bactericidal activity against L. monocytogenes. Antibacterial activities against such prominent pathogens could be useful to prevent Listeria contamination in the food chain or as treatment for MRSA infections.

Beta-Defensin 2 and 3 Promote Bacterial Clearance of Pseudomonas aeruginosa by Inhibiting Macrophage Autophagy through Downregulation of Early Growth Response Gene-1 and c-FOS.

Beta-defensins 2 and 3 (BD2 and BD3) are inducible peptides present at the sites of infection, and they are well characterized for their antimicrobial activities and immune-regulatory functions. However, no study has thoroughly investigated their immunomodulatory effects on macrophage-mediated immune responses against Pseudomonas aeruginosa (PA). Here, we use THP-1 and RAW264.7 cell lines and demonstrate that BD2 and BD3 suppressed macrophage autophagy but enhanced the engulfment of PA and Zymosan bioparticles as well as the formation of phagolysosomes, using immunofluorescence staining and confocal microscopy. Plate count assay showed that macrophage-mediated phagocytosis and intracellular killing of PA were promoted by BD2 and BD3. Furthermore, microarray and real-time PCR showed that the expression of two genes, early growth response gene-1 (EGR1) and c-FOS, was attenuated by BD2 and BD3. Western blot revealed that BD2 and BD3 inhibited the expression and nuclear translocation of EGR1 and c-FOS. Knockdown of EGR1 and c-FOS by siRNA transfection suppressed macrophage autophagy before and after PA infection; while overexpression of these two transcription factors enhanced autophagy but reversed the role of BD2 and BD3 on macrophage-mediated PA eradication. Together, these results demonstrate a novel immune defense activity of BD2 and BD3, which promotes clearance of PA by inhibiting macrophage autophagy through downregulation of EGR1 and c-FOS.

Comparative studies evaluating macrophage activity of pulmonary tuberculosis patients with and without diabetes mellitus (tb-dm and non tb-dm)

Background: Macrophages, as the first defense mechanism in Mycobacterium tuberculosis infection, play the important role in pulmonary TB pathogenesis. The increasing prevalence of pulmonary TB is followed by the increasing prevalence of diabetes mellitus (DM). DM patients have 4,7 times higher risk to develop pulmonary TB compared to patients without DM, since DM can increase the frequency and severity of an infection, including pulmonary TB.Aim: To analyze macrophage activity (phagocytosis, intracellular killing, and IFN-γsynthesis) of TBDM and TB non-DM patient.Method: This experimental study used a PBMC cultured sample from TB-DM and TB non-DM patient’s which undergo observation of macrophage activity (phagocytic, intracellular killing and IFN-γsynthesis). The data were taken from microscopic observation of TB-DM and TB non-DM patients, colony growth of viable M. tuberculosis and the IFN-γlevel secreted by macrophages.Result: The result showed that macrophages of TB-DM patient’s were less amount of phagocytosedM. tuberculosis, a little amount of formed vacuoles and giant cells, secrete low level of IFN-γ, and more viable M. tuberculosis(from subculture).Conclusions: Macrophages of TB-DM patients are reduced phagocytic activity toward M. tuberculosis which is this macrophages are less activated.Bangladesh Journal of Medical Science Vol.17(1) 2018 p.107-111

P092 PHYSIOLOGIC HYPOXIA AND LIPID PEROXIDATION PRODUCTS MODULATE GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR-DEPENDENT NEUTROPHIL BACTERIAL KILLING IN PEDIATRIC CROHN’S DISEASE

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) enhances neutrophil regulation of gut bacteria. Crohn’s Disease (CD) patients with reduced neutrophil GM-CSF signaling are more likely to experience stricturing complications requiring surgery. We aimed to determine whether bacterial killing by GM-CSF primed neutrophils is enhanced by physiologic hypoxia and suppressed by lipid peroxidation products including 4-hydroxynonenal (4-HNE). HL-60 human promyelocytic leukemia cells were differentiated to a neutrophil phenotype via 7-day incubation in DMSO. Primary human control or CD patient neutrophils or differentiated HL-60 cells were exposed to GM-CSF and/or 4-HNE, at 5% O2 (physiologic distal gut hypoxia) and atmospheric O2, exposed to S. Aureus, and the frequency of bacterial killing was determined via light microscopy. The frequency of intra-cellular bacterial killing increased in CD patient primary neutrophils when exposed to hypoxia (99(3)) vs. atmospheric oxygen tension (92(6), p<0.0001) as well as in non-IBD control primary neutrophils when exposed to hypoxia (99.75(1)) vs. atmospheric oxygen tension (93(3), p<0.0001). Bacterial killing also increased in HL-60 cells when exposed to hypoxia (88(11)) vs. atmospheric oxygen tension (80(10), p=0.0089) and when primed with GM-CSF (80(4)) vs. basal conditions (73(8), p=0.0446). Bacterial killing was markedly suppressed in HL-60 cells stimulated with GM-CSF under hypoxic conditions in the presence of 4-HNE (68(7)) vs. HL-60 cells stimulated with GM-CSF under hypoxic conditions in the absence of 4-HNE (84(4), p=0.0171). Physiologic hypoxia in the distal gut boosts GM-CSF primed neutrophil bacterial killing which is in turn inhibited by lipid peroxidation products. These data have implications for therapeutic approaches to boost neutrophil function in CD patients with reduced GM-CSF signaling and stricturing complications.

Priming of innate antimycobacterial immunity by heat-killed Listeria monocytogenes induces sterilizing response in the adult zebrafish tuberculosis model.

ABSTRACTMycobacterium tuberculosis remains one of the most problematic infectious agents, owing to its highly developed mechanisms to evade host immune responses combined with the increasing emergence of antibiotic resistance. Host-directed therapies aiming to optimize immune responses to improve bacterial eradication or to limit excessive inflammation are a new strategy for the treatment of tuberculosis. In this study, we have established a zebrafish-Mycobacterium marinum natural host-pathogen model system to study induced protective immune responses in mycobacterial infection. We show that priming adult zebrafish with heat-killed Listeria monocytogenes (HKLm) at 1 day prior to M. marinum infection leads to significantly decreased mycobacterial loads in the infected zebrafish. Using rag1−/− fish, we show that the protective immunity conferred by HKLm priming can be induced through innate immunity alone. At 24 h post-infection, HKLm priming leads to a significant increase in the expression levels of macrophage-expressed gene 1 (mpeg1), tumor necrosis factor α (tnfa) and nitric oxide synthase 2b (nos2b), whereas superoxide dismutase 2 (sod2) expression is downregulated, implying that HKLm priming increases the number of macrophages and boosts intracellular killing mechanisms. The protective effects of HKLm are abolished when the injected material is pretreated with nucleases or proteinase K. Importantly, HKLm priming significantly increases the frequency of clearance of M. marinum infection by evoking sterilizing immunity (25 vs 3.7%, P=0.0021). In this study, immune priming is successfully used to induce sterilizing immunity against mycobacterial infection. This model provides a promising new platform for elucidating the mechanisms underlying sterilizing immunity and to develop host-directed treatment or prevention strategies against tuberculosis.This article has an associated First Person interview with the first author of the paper.

Mycobacterium tuberculosis Modulates miR-106b-5p to Control Cathepsin S Expression Resulting in Higher Pathogen Survival and Poor T-Cell Activation.

The success of tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), relies on the ability to survive in host cells and escape to immune surveillance and activation. We recently demonstrated that Mtb manipulation of host lysosomal cathepsins in macrophages leads to decreased enzymatic activity and pathogen survival. In addition, while searching for microRNAs (miRNAs) involved in posttranscriptional gene regulation during mycobacteria infection of human macrophages, we found that selected miRNAs such as miR-106b-5p were specifically upregulated by pathogenic mycobacteria. Here, we show that miR-106b-5p is actively manipulated by Mtb to ensure its survival in macrophages. Using an in silico prediction approach, we identified miR-106b-5p with a potential binding to the 3′-untranslated region of cathepsin S (CtsS) mRNA. We demonstrated by luminescence-based methods that miR-106b-5p indeed targets CTSS mRNA resulting in protein translation silencing. Moreover, miR-106b-5p gain-of-function experiments lead to a decreased CtsS expression favoring Mtb intracellular survival. By contrast, miR-106b-5p loss-of-function in infected cells was concomitant with increased CtsS expression, with significant intracellular killing of Mtb and T-cell activation. Modulation of miR-106b-5p did not impact necrosis, apoptosis or autophagy arguing that miR-106b-5p directly targeted CtsS expression as a way for Mtb to avoid exposure to degradative enzymes in the endocytic pathway. Altogether, our data suggest that manipulation of miR-106b-5p as a potential target for host-directed therapy for Mtb infection.

Characterization of the Phospholipid Platelet-Activating Factor As a Mediator of Inflammation in Chickens.

Lipid mediators are known to play important roles in the onset and resolution phases of the inflammatory response in mammals. The phospholipid platelet-activating factor (PAF) is a pro-inflammatory lipid mediator which participates in vascular- and innate immunity-associated processes by increasing vascular permeability, by facilitating leukocyte adhesion to the endothelium, and by contributing to phagocyte activation. PAF exerts its function upon binding to its specific receptor, PAF receptor (PAFR), which is abundantly expressed in leukocytes and endothelial cells (ECs). In chickens, lipid mediators and their functions are still poorly characterized, and the role of PAF as an inflammatory mediator has not yet been investigated. In the present study we demonstrate that primary chicken macrophages express PAFR and lysophosphatidylcholine acyltransferase 2 (LPCAT2), the latter being essential to PAF biosynthesis during inflammation. Also, exogenous PAF treatment induces intracellular calcium increase, reactive oxygen species release, and increased phagocytosis by primary chicken macrophages in a PAFR-dependent manner. We also show that PAF contributes to the Escherichia coli lipopolysaccharide (LPS)-induced pro-inflammatory response and boosts the macrophage response to E. coli LPS via phosphatidylinositol 3-kinase/Akt- and calmodulin kinase II-mediated intracellular signaling pathways. Exogenous PAF treatment also increases avian pathogenic E. coli intracellular killing by chicken macrophages, and PAFR and LPCAT2 are upregulated in chicken lungs and liver during experimental pulmonary colibacillosis. Finally, exogenous PAF treatment increases cell permeability and upregulates the expression of genes coding for proteins involved in leukocyte adhesion to the endothelium in primary chicken endothelial cells (chAEC). In addition to these vascular phenomena, PAF boosts the chAEC inflammatory response to bacteria-associated molecular patterns in a PAFR-dependent manner. In conclusion, we identified PAF as an inflammation amplifier in chicken macrophages and ECs, which suggests that PAF could play important roles in the endothelium-innate immunity interface in birds during major bacterial infectious diseases such as colibacillosis.

Impaired IFNγ-Signaling and Mycobacterial Clearance in IFNγR1-Deficient Human iPSC-Derived Macrophages.

SummaryMendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of interferon gamma (IFNγ) immunity and is characterized by severe infections by weakly virulent mycobacteria. Although IFNγ is the macrophage-activating factor, macrophages from these patients have never been studied. We demonstrate the generation of heterozygous and compound heterozygous (iMSMD-cohet) induced pluripotent stem cells (iPSCs) from a single chimeric patient, who suffered from complete autosomal recessive IFNγR1 deficiency and received bone-marrow transplantation. Loss of IFNγR1 expression had no influence on the macrophage differentiation potential of patient-specific iPSCs. In contrast, lack of IFNγR1 in iMSMD-cohet macrophages abolished IFNγ-dependent phosphorylation of STAT1 and induction of IFNγ-downstream targets such as IRF-1, SOCS-3, and IDO. As a consequence, iMSMD-cohet macrophages show impaired upregulation of HLA-DR and reduced intracellular killing of Bacillus Calmette-Guérin. We provide a disease-modeling platform that might be suited to investigate novel treatment options for MSMD and to gain insights into IFNγ signaling in macrophages.

Filamin A Regulates Neutrophil Adhesion, Production of Reactive Oxygen Species, and Neutrophil Extracellular Trap Release.

Neutrophils are of fundamental importance in the early immune response and use various mechanisms to neutralize invading pathogens. They kill endocytosed pathogens by releasing reactive oxygen species in the phagosome and release neutrophil extracellular traps (NETs) into their surroundings to immobilize and kill invading micro-organisms. Filamin A (FlnA) is an important actin cross-linking protein that is required for cellular processes involving actin rearrangements, such cell migration. It has also been shown to negatively regulate integrin activation and adhesion. However, its role in the regulation of β2 integrin-dependent adhesion, as well as in other cellular functions in neutrophils, is poorly understood. Using a transgenic mouse model in which FlnA is selectively depleted in myeloid cells, such as neutrophils, we show that FlnA negatively regulates β2 integrin adhesion to complement component iC3b and ICAM-1 in shear-free, but not shear-flow, conditions. FlnA deletion does not affect phagocytosis of Escherichia coli or Staphylococcus aureus or their intracellular killing. However, FlnA negatively regulates production of reactive oxygen species upon cell activation. Conversely, neutrophil activation through TLR4, as well as through activation by the Gram-negative bacteria E. coli, results in reduced NET production in FlnA-depleted neutrophils. Thus, FlnA is a negative regulator of β2 integrin-dependent cell adhesion and reactive oxygen species production but is required for NET production in primary murine neutrophils.

The NOD2 receptor is crucial for immune responses towards New World Leishmania species

American Tegumentary Leishmaniasis is a chronic infection caused by Leishmania protozoan. It is not known whether genetic variances in NOD-like receptor (NLR) family members influence the immune response towards Leishmania parasites and modulate intracellular killing. Using functional genomics, we investigated whether genetic variants in NOD1 or NOD2 influence the production of cytokines by human PBMCs exposed to Leishmania. In addition, we examined whether recognition of Leishmania by NOD2 contributes to intracellular killing. Polymorphisms in the NOD2 gene decreased monocyte- and lymphocyte-derived cytokine production after stimulation with L. amazonensis or L. braziliensis compared to individuals with a functional NOD2 receptor. The phagolysosome formation is important for Leishmania-induced cytokine production and upregulation of NOD2 mRNA expression. NOD2 is crucial to control intracellular infection caused by Leishmania spp. NOD2 receptor is important for Leishmania recognition, the control of intracellular killing, and the induction of innate and adaptive immune responses.

Mesenchymal stem cells internalize Mycobacterium tuberculosis through scavenger receptors and restrict bacterial growth through autophagy

Human mesenchymal stem cells (MSCs) express scavenger receptors that internalize lipids, including oxidized low-density lipoprotein (oxLDL). We report that MSCs phagocytose Mycobacterium tuberculosis (Mtb) through two types of scavenger receptors (SRs; MARCO and SR-B1), as blockade of the receptors with antibodies or siRNA knockdown decreased the uptake of Mtb. MSCs also expressed mannose receptor (MR) that was found to endocytose rhodamine-labeled mannosylated BSA (rMBSA), though the receptor was not involved in the uptake of Mtb. Dil-oxLDL and rMBSA taken up into MSC endosomes colocalized with Mtb phagosomes, thus suggesting that the latter were fusion competent. Phagocytosed Mtb did not replicate within MSCs, thus suggesting an intrinsic control of bacterial growth. Indeed, MSCs exhibited intrinsic autophagy, which was up-regulated after activation with rapamycin. SiRNA knockdown of autophagy initiator beclin-1 enhanced Mtb survival, whereas rapamycin-induced autophagy increased intracellular killing of Mtb. In addition, MSCs secreted nitric oxide after Mtb infection, and inhibition of NO by N(G)-monomethyl-L-arginine enhanced intracellular survival of Mtb. MSCs can be grown in large numbers in vitro, and autologous MSCs transfused into tuberculosis patients have been found to be safe and improve lung immunity. Thus, MSCs are novel phagocytic cells with a potential for immunotherapy in treating multidrug-resistant tuberculosis.