Defective Macrophage Research Articles

Malacoplakia of the skin: overview of a rare clinical entity

Malacoplakia is a rare acquired, infection-related granulomatous disorder, that may affect many systems, but typically occurs in the urinary tract. Cutaneous involvement is less prevalent, and most commonly presents with a perianal or genital region localization. Cutaneous malacoplakia is believed to be caused by an acquired bactericidal defect of macrophages in the setting of chronic infections and immunocompromised states. A diagnosis of cutaneous malacoplakia should be considered when encountering non-specific granulomatous lesions that are refractory to treatment. Histologic findings are marked by the presence of foamy macrophages containing the pathognomonic Michaelis-Gutman bodies. The aim of this review is to discuss the current perspectives on the pathophysiology, clinical features, diagnosis, and treatment of this disease. We would also like to emphasize that the integration of clinical information, microscopic findings, and exclusion of other cutaneous granulomatous processes is necessary to accurately diagnose this exceedingly rare disease and provide opportunity for therapeutic intervention. Data for this work were collected from the published literature and textbooks. Combined surgical excision and protracted antibiotic courses appear to have the highest success rate. Antibiotics should be culture specific, but drugs that easily permeate the macrophages appear to be the best choice.

Bacterial load and defective monocyte-derived macrophage bacterial phagocytosis in biomass smoke-related COPD.

Lower airway colonisation with species of potentially pathogenic bacteria (PPB) is associated with defective bacterial phagocytosis, in monocyte-derived macrophages (MDMs) and alveolar macrophages, from tobacco smoke-associated chronic obstructive pulmonary disease (S-COPD) subjects. In the developing world, COPD among nonsmokers is largely due to biomass smoke (BMS) exposure; however, little is known about PPB colonisation and its association with impaired innate immunity in these subjects.We investigated the PPB load (Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Pseudomonas aeruginosa) in BMS-exposed COPD (BMS-COPD) subjects compared with S-COPD and spirometrically normal subjects. We also examined the association between PPB load and phagocytic activity of MDMs and lung function. Induced sputum and peripheral venous blood samples were collected from 18 healthy nonsmokers, 15 smokers without COPD, 16 BMS-exposed healthy subjects, 19 S-COPD subjects and 23 BMS-COPD subjects. PPB load in induced sputum and MDM phagocytic activity were determined using quantitative PCR and fluorimetry, respectively.Higher bacterial loads of S. pneumoniae, H. influenzae and P. aeruginosa were observed in BMS-COPD subjects. Increased PPB load in BMS-exposed subjects was significantly negatively associated with defective phagocytosis in MDMs and spirometric lung function indices (p<0.05).Increased PPB load in airways of BMS-COPD subjects is inversely associated with defective bacterial phagocytosis and lung function.

SREBP-1a–stimulated lipid synthesis is required for macrophage phagocytosis downstream of TLR4-directed mTORC1

There is a growing appreciation for a fundamental connection between lipid metabolism and the immune response. Macrophage phagocytosis is a signature innate immune response to pathogen exposure, and cytoplasmic membrane expansion is required to engulf the phagocytic target. The sterol regulatory element binding proteins (SREBPs) are key transcriptional regulatory proteins that sense the intracellular lipid environment and modulate expression of key genes of fatty acid and cholesterol metabolism to maintain lipid homeostasis. In this study, we show that TLR4-dependent stimulation of macrophage phagocytosis requires mTORC1-directed SREBP-1a-dependent lipid synthesis. We also show that the phagocytic defect in macrophages from SREBP-1a-deficient mice results from decreased interaction between membrane lipid rafts and the actin cytoskeleton, presumably due to reduced accumulation of newly synthesized fatty acyl chains within major membrane phospholipids. We show that mTORC1-deficient macrophages also have a phagocytic block downstream from TLR4 signaling, and, interestingly, the reduced level of phagocytosis in both SREBP-1a- and mTORC1-deficient macrophages can be restored by ectopic SREBP-1a expression. Taken together, these observations indicate SREBP-1a is a major downstream effector of TLR4-mTORC1 directed interactions between membrane lipid rafts and the actin cytoskeleton that are required for pathogen-stimulated phagocytosis in macrophages.

Stimulating Respiratory Activity Primes Anaerobically Grown Listeria monocytogenes for Subsequent Intracellular Infections.

Listeria monocytogenes (L. monocytogenes) is a Gram-positive, enteric pathogen and the causative agent of listeriosis. During transition through the gastrointestinal tract, L. monocytogenes routinely encounters suboxic conditions. However, how the exposure to the low oxygen environment affects subsequent pathogenesis is not completely understood. Our lab previously reported that anaerobically grown L. monocytogenes exhibited an intracellular growth defect in macrophages even though the infection took place under aerobic conditions. This phenotype suggests that prior growth conditions have a prolonged effect on the outcome of subsequent intracellular infection. In this study, to further investigate the mechanisms that contribute to the compromised intracellular growth after anaerobic exposure, we hypothesized that the lack of respiratory activity under anaerobic conditions prevented anaerobically grown L. monocytogenes to establish subsequent intracellular growth under aerobic conditions. To test this hypothesis, respiratory activity in anaerobically grown L. monocytogenes was stimulated by exogenous fumarate and subsequent intracellular pathogenesis was assessed. The results showed that fumarate supplementation significantly increased the respiratory activity of anaerobically grown L. monocytogenes and rescued the subsequent intracellular growth defect, likely through promoting the production of listeriolysin O, phagosomal escape, and cell-cell spread. This study highlights the importance of respiratory activity in L. monocytogenes in modulating the outcome of subsequent intracellular infections.

In This Issue

HomeCirculation ResearchVol. 123, No. 11In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published8 Nov 2018https://doi.org/10.1161/RES.0000000000000240Circulation Research. 2018;123:1177is related toGDF11 Decreases Pressure Overload–Induced Hypertrophy, but Can Cause Severe Cachexia and Premature Deathis related toMacrophage Inflammation, Erythrophagocytosis, and Accelerated Atherosclerosis in Jak2V617F MiceA Miniaturized, Programmable Pacemaker for Long-Term Studies in the MouseProgrammable Mouse Pacemaker (p 1208)Download figureDownload PowerPointHulsmans et al have created a tiny programmable pacemaker for mice.Implantable electronic pacemakers can be lifesaving. They improve quality of life for patients with arrhythmia due to heart failure or other causes. However, not all patients respond equally well, and sometimes pacemakers can even cause cardiomyopathy. Therefore, a better understanding of the effects of pacemakers during different cardiac pathologies is needed. Although several mouse models for heart failure and arrhythmias are available, there is a lack of long-term pacing technologies that could be used in small animals. Therefore, Hulsmans and colleagues built a programmable, battery-powered pacemaker for freely moving mice that could remain operational for months. Adapted from the smallest clinical pacemaker available, the team’s new device, once surgically implanted, can be used to both monitor the effects of pacing on a diseased heart and to analyze pacing-induced myopathy. Using this device, the team could successfully treat mice with complete atrioventricular block. They also found that rapid ventricular pacing in healthy animals could lead to myocardial fibrosis, inflammation, and myopathy. The new device could enable researchers to study the positive and negative outcomes of pacemakers, which could lead to further clinical optimization for use in patients with arrhythmias.Jak2V617F Promotes Atherosclerosis (p e35)Download figureDownload PowerPointMutation to Janus kinase 2 prompts clonal hematopoiesis and acceleration of atherosclerosis in model mice, report Wang et al.Clonal hematopoiesis (CH)—the clonal expansion of certain subpopulations of blood cells—is associated with both myeloproliferative neoplasms (blood cancers) and an increased risk of athero-thrombotic disease. Commonly observed in elderly patients, CH can be caused by mutations to any one of a handful of genes, including JAK2—the gene encoding cell signaling factor Janus kinase 2. To examine the mechanisms linking CH to athero-thrombosis, Wang and colleagues studied atherosclerosis-prone mice in which bone marrow cells carried a known CH-associated JAK2 mutation. After several weeks on a Western diet, the mutation-carrying mice showed accelerated atherosclerosis with advanced lesions that displayed increased necrotic core size and reduced clearance of dead cells. Macrophages of the mutant mice had increased expression of proinflammatory cytokines, whereas, their red blood cells were more susceptible to phagocytosis by macrophages. These defects in erythrocytes and macrophages could, in part, be responsible for the accumulation of iron in plaques, defective clearance of dead cells, and increased inflammation in the lesions. Together, the team’s work confirms the role of the JAK2 mutation in CH-linked atherogenesis and provides a model system for further CH studies.GDF11 Decreases Hypertrophy but Causes Cachexia (p 1220)Download figureDownload PowerPointBoosting GDFII improves hypertrophy but causes severe muscle wasting, say Harper et al.Cardiac hypertrophy—the abnormal enlargement of the heart—can lead to heart failure and death. Current therapies treat the symptoms of hypertrophy, but do not reverse tissue pathology. Thus, the development of strategies to restore cardiac function is a desirable goal. Previous work has suggested that treatment with growth differentiation factor II (GDFII) could be one such approach. However, while some studies indicate that raising GDFII levels in aged mice leads to rejuvenation of hypertrophic hearts and skeletal muscle, others purport that over-expression of GDF11 prompts cardiac and skeletal muscle wasting. In an attempt to resolve these disparate findings, Harper and colleagues performed a blinded dose-ranging study in which they treated both healthy mice and those with hypertrophy (from aortic constriction) for 2 weeks with daily doses of GDFII. They found that in mice with aortic constriction, high doses of GDFII reduced signs of hypertrophy, such as fibrosis, and improved pump function, but these doses were also associated with severe skeletal muscle wasting, weight loss and premature death in both groups. Such negative effects, the authors say, are likely to preclude clinical translation of GDFII-boosting treatments. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesGDF11 Decreases Pressure Overload–Induced Hypertrophy, but Can Cause Severe Cachexia and Premature DeathShavonn C. Harper, et al. Circulation Research. 2018;123:1220-1231Macrophage Inflammation, Erythrophagocytosis, and Accelerated Atherosclerosis in Jak2V617F MiceWei Wang, et al. Circulation Research. 2018;123:e35-e47A Miniaturized, Programmable Pacemaker for Long-Term Studies in the MouseMaarten Hulsmans, et al. Circulation Research. 2018;123:1208-1219 November 9, 2018Vol 123, Issue 11 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000240PMID: 30571476 Originally publishedNovember 8, 2018 PDF download Advertisement

Macrophage Inflammation, Erythrophagocytosis, and Accelerated Atherosclerosis in Jak2 V617F Mice.

The mechanisms driving atherothrombotic risk in individuals with JAK2 V617F ( Jak2 VF) positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. The goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2 VF expression. Irradiated low-density lipoprotein receptor knockout ( Ldlr-/-) mice were transplanted with bone marrow from wild-type or Jak2 VF mice and fed a high-fat high-cholesterol Western diet. Hematopoietic functions and atherosclerosis were characterized. After 7 weeks of Western diet, Jak2 VF mice showed increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophil adhesion and content, correlating with lesion size. After 12 weeks of Western diet, Jak2 VF lesions showed increased complexity, with larger necrotic cores, defective efferocytosis, prominent iron deposition, and costaining of erythrocytes and macrophages, suggesting erythrophagocytosis. Jak2 VF erythrocytes were more susceptible to phagocytosis by wild-type macrophages and showed decreased surface expression of CD47, a "don't-eat-me" signal. Human JAK2VF erythrocytes were also more susceptible to erythrophagocytosis. Jak2 VF macrophages displayed increased expression and production of proinflammatory cytokines and chemokines, prominent inflammasome activation, increased p38 MAPK (mitogen-activated protein kinase) signaling, and reduced levels of MerTK (c-Mer tyrosine kinase), a key molecule mediating efferocytosis. Increased erythrophagocytosis also suppressed efferocytosis. Hematopoietic Jak2 VF expression promotes early lesion formation and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, Jak2 VF caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes promote accumulation of iron in plaques and increased necrotic core formation which, together with exacerbated proinflammatory responses, likely contribute to plaque instability.

Impaired HDL (High-Density Lipoprotein)-Mediated Macrophage Cholesterol Efflux in Patients With Abdominal Aortic Aneurysm-Brief Report.

Objective- The ability of HDL (high-density lipoprotein) to promote macrophage cholesterol efflux is considered the main HDL cardioprotective function. Abdominal aortic aneurysm (AAA) is usually characterized by cholesterol accumulation and macrophage infiltration in the aortic wall. Here, we aim to evaluate the composition of circulating HDL particles and their potential for promoting macrophage cholesterol efflux in AAA subjects. Approach and Results- First, we randomly selected AAA and control subjects from Spain. The AAA patients in the Spanish cohort showed lower plasma apoA-I levels concomitantly associated with low levels of plasma HDL cholesterol and the amount of preβ-HDL particles. We determined macrophage cholesterol efflux to apoB-depleted plasma, which contains mature HDL, preβ-HDL particles and HDL regulatory proteins. ApoB-depleted plasma from AAA patients displayed an impaired ability to promote macrophage cholesterol efflux. Next, we replicated the experiments with AAA and control subjects derived from Danish cohort. Danish AAA patients also showed lower apoA-I levels and a defective HDL-mediated macrophage cholesterol efflux. Conclusions- AAA patients show impaired HDL-facilitated cholesterol removal from macrophages, which could be mechanistically linked to AAA.

The deficiency of macrophage erythropoietin signaling contributes to delayed acute inflammation resolution in diet-induced obese mice

Obesity has been linked with altered acute inflammation resolution which contributes to obesity-related clinical complications; however, the mechanisms that contribute to obesity-related unresolved inflammation are not fully known. Here we demonstrated that the deficiency of macrophage erythropoietin (EPO) signaling contributed to delayed acute inflammation resolution in diet-induced obese mice. In zymosan-induced acute peritonitis, in line with the delayed resolution of inflammation, the induction of macrophage EPO signaling was significantly reduced in obese mice relative to normal mice. Exogenous EPO induced macrophage EPO signaling and promoted acute inflammation resolution in obese mice. Efferocytosis of apoptotic cells by macrophages which is central in inflammation resolution was impaired in obese mice and restored by exogenous EPO. Mechanistically, macrophage peroxisome proliferator-activated receptor-γ (PPARγ) was greatly reduced in obese mice and EPO increased macrophage PPARγ to promote efferocytosis in obese mice. Together, our results identify an important mechanism underlying aberrant acute inflammation resolution in obesity, with important implications for regulating unresolved acute inflammation and normalizing macrophage defects in obese and diabetic individuals.

Differential expression of genes and receptors in monocytes from patients with cystic fibrosis

IntroductionWe previously reported defective alternative polarization (M2) of macrophages and early expression of classically polarized (M1) macrophage markers in unpolarized monocyte-derived macrophages (MDMs) in patients with cystic fibrosis (CF). The present study assessed whether the mechanism(s) underlying defective macrophage polarization resided in circulating monocytes. MethodsMonocyte subsets (classical, intermediate and non-classical), markers for monocyte activation (CD163) and recruitment (CD195), receptors/genes associated with macrophage differentiation and polarization were analyzed in CF and compared with healthy individuals. ResultsNo differences were observed in the monocyte subsets or in the expression of CD163 or CD195. Expression of the M-CSF receptor, TLR4, γC, IL-4Rα, IL-13Rα1, TIMP-1 and Cox-2 were higher in CF monocytes, albeit at low levels, whereas, LRP1, MMP9, MMP28 were downregulated compared to mooncytes from healthy individuals. ConclusionsOur data suggest that differences in CF monocytes may contribute to the reported CFTR-dependent defect in macrophage differentiation, polarization and function.

Defective HDL remodeling and macrophage cholesterol efflux in adult and adolescent familial hypercholesterolemic patients

Defective HDL remodeling and macrophage cholesterol efflux in adult and adolescent familial hypercholesterolemic patients

Pharmacological inactivation of the PI3K p110\u03b4 prevents breast tumour progression by targeting cancer cells and macrophages

Patient selection for PI3K-targeted solid cancer treatment was based on the PIK3CA/PTEN mutational status. However, it is increasingly clear that this is not a good predictor of the response of breast cancer cells to the anti-proliferative effect of PI3K inhibitors, indicating that isoform(s) other than p110α may modulate cancer cells sensitivity to PI3K inhibition. Surprisingly, we found that although no mutations in the p110δ subunit have been detected thus far in breast cancer, the expression of p110δ becomes gradually elevated during human breast cancer progression from grade I to grade III. Moreover, pharmacological inactivation of p110δ in mice abrogated the formation of tumours and the recruitment of macrophages to tumour sites and strongly affected the survival, proliferation and apoptosis of grafted tumour cells. Pharmacological inactivation of p110δ in mice with defective macrophages or in mice with normal macrophages but grafted with p110δ-lacking tumours suppressed only partly tumour growth, indicating a requisite role of p110δ in both macrophages and cancer cells in tumour progression. Adoptive transfer of δD910A/D910A macrophages into mice with defected macrophages suppressed tumour growth, eliminated the recruitment of macrophages to tumour sites and prevented metastasis compared with mice that received WT macrophages further establishing that inactivation of p110δ in macrophage prevents tumour progression. Our work provides the first in vivo evidence for a critical role of p110δ in cancer cells and macrophages during solid tumour growth and may pave the way for the use of p110δ inhibitors in breast cancer treatment.

GvmR - A Novel LysR-Type Transcriptional Regulator Involved in Virulence and Primary and Secondary Metabolism of Burkholderia pseudomallei.

Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.

Abstract 040: Accelerated Atherosclerosis and Thrombosis in Jak2v617f Mice

The mechanisms driving increased athero-thrombotic risk in individuals with JAK2V617F positive clonal hematopoiesis or myeloproliferative neoplasms are poorly understood. This study was to assess atherosclerosis and thrombosis and associated mechanisms in hypercholesterolemic mice with hematopoietic JAK2V617F expression. Irradiated Ldlr -/- mice were transplanted with bone marrow from WT or JAK2V617F mice and fed a high fat high cholesterol diet (WD). After 7 weeks of WD JAK2V617F mice showed increased hematopoiesis, platelet activation, accelerated thrombosis and increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophils, correlating with lesion size and in association with increased rolling and adhesion of neutrophils on endothelial cells in carotid artery. After 12 weeks of WD JAK2V617F lesions were slightly larger than controls but showed additional complexity, with increased necrotic core area, prominent iron deposition and co-staining of erythrocytes and macrophages suggesting erythrophagocytosis. JAK2V617F erythrocytes were more susceptible to phagocytosis by WT macrophages and showed decreased surface expression of CD47, a “don’t eat me” signal. Human JAK2V617F erythrocytes were also more susceptible to erythrophagocytosis. JAK2V617F macrophages displayed increased expression of pro-inflammatory cytokines and chemokines, increased p38 map kinase signaling and increased cleavage and reduced levels of MerTK, a key molecule mediating phagocytosis of apoptotic cells (efferocytosis) in atherosclerotic lesions. Erythrophagocytosis also suppressed efferocytosis. As a result, JAK2V617F lesional efferocytosis was reduced in advanced lesions. In conclusion, JAK2V617F promotes early lesion formation, arterial thrombosis and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, JAK2V617F caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes appear to promote accumulation of iron and increased necrotic core formation which, together with exacerbated pro-inflammatory responses, may contribute to plaque instability.

Ssu72 phosphatase is involved in immunometabolism

Abstract Reversible protein phosphorylation is the primary type of various post-translational modifications in multiple cellular signaling pathways of eukaryotic cell. Dynamic alterations of phosphorylation state of many cellular signaling-mediated proteins including adipogenesis and thermogenesis regulate their molecular and cellular fates. Ssu72 is dual protein phosphatase that can act upon tyrosine or serine/threonine residues and transcription/RNA-processing factor. Recently, we found that Ssu72 expressed in adipose tissue, especially strongly in BAT, indicating that the potential involvement of Ssu72 in regulating BAT function. In this aspect, we generated conditional knock out mice which Ssu72 is deleted specifically in adipose tissue and found that the deficiency of Ssu72 leads to BAT dysfunction and abnormal thermoregulation compared to wild type mice. Moreover, we observed not only dramatically reduction of macrophage population but also defective M2 macrophage generation in Ssu72-deficient BAT. Notably, it has been reported that IL-4-induced M2 activation of macrophages is required for adaptive thermogenesis in mice. Thus, we further generated Ssu72-deleted macrophage mouse model. This study includes the physiological relevance of Ssu72 loss-of-function in macrophage during metabolic and thermogenic responses.

Abstract 637: Genome-wide Crispr Screen in Primary Macrophages for Identification of a Regulatory Network for Macrophage Efferocytosis

Defective macrophage efferocytosis drives important diseases including atherosclerosis, whereas enhancing efferocytosis has potential therapeutic benefits. A systematic approach to identify regulators of efferocytosis in an unbiased manner holds promise for novel molecular mechanisms and targets. This study establishes an experimental framework using a novel CRISPR knockout screen to systematically map the regulatory networks of macrophage efferocytosis. We infected bone marrow-derived macrophages (BMDM) isolated from Cas9 transgenic mice with a pooled, genome-wide lentiviral library with 78,637 gRNAs, and induced efferocytosis by fluorescently-labeled apoptotic Jurkat cells. We used FACS to isolate BMDM that engulfed multiple apoptotic cells (enriched in gRNAs targeting negative regulators) or did not engulf apoptotic cells (enriched in gRNAs targeting positive regulators), and determined gRNA abundance by deep sequencing. The top-ranked 1,000 genes analyzed by two published methods MAGeCK and PBNPA showed substantial overlap (53% for positive regulators, 36% for negative regulators). We recovered many of the known regulators. e.g., Actr2 , Actr3 and Arpc3 , key components of the Arp2/3 complex regulating actin polymerization, were ranked 2, 8 and 4; Atp6v0b and Atp6v0c , subunits of vacuolar ATPase responsible for acidification of intracellular organelles, were ranked 22 and 27. Cd36 , a well-known efferocytosis receptor, was ranked 5. The top ranked positive regulators were enriched in GO terms “positive regulation of response to stimulus”, “Fcγ receptor signaling pathway” and “phagocytosis” (FDR-adjusted P = 2.19E-14, 1.10E-12 and 4.08E-11, respectively). Individual gRNAs verified a number of top hits from the pooled screen. In summary, we have developed a functional screen that has defined number of genes essential for macrophage efferocytosis. By focusing on a functional phenotype beyond cell viability and growth, we illustrate the versatility of CRISPR screens and provide a general strategy for systematically identifying gene of interest and uncovering novel regulators of complex macrophage functions.

A Coxiella burnetii phospholipase A homolog pldA is required for optimal growth in macrophages and developmental form lipid remodeling

BackgroundMany gram-negative bacteria produce an outer membrane phospholipase A (PldA) that plays an important role in outer membrane function and is associated with virulence.ResultsIn the current study, we characterized a pldA mutant of Coxiella burnetii, an intracellular gram-negative pathogen and the agent of human Q fever. The C. burnetti pldA open reading frame directs synthesis of a protein with conserved PldA active site residues. A C. burnetii ΔpldA deletion mutant had a significant growth defect in THP-1 macrophages, but not axenic medium, that was rescued by complementation. Thin layer chromatography was employed to assess whether pldA plays a role in remodeling membrane lipids during C. burnetii morphological differentiation. Extracted lipids were analyzed from replicating, logarithmic phase large cell variants (LCVs), non-replicating, stationary phase small cell variants (SCVs), and a mixture of LCVs and SCVs. Similar to Escherichia coli, all three forms contained cardiolipin (CL), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE). However, PE and PG were present in lower quantities in the SCV while three additional lipid species were present in higher quantities. Co-migration with standards tentatively identified two of the three SCV-enriched lipids as lyso-phosphatidylethanolamine, a breakdown product of PE, and free fatty acids, which are generally toxic to bacteria. Developmental form lipid modifications required the activity of PldA.ConclusionsCollectively, these results indicate developmentally-regulated lipid synthesis by C. burnetii contributes to colonization of macrophages and may contribute to the environmental stability and the distinct biological properties of the SCV.

Drosophila GATA Factor Serpent Establishes Phagocytic Ability of Embryonic Macrophages.

During Drosophila embryogenesis, a large number of apoptotic cells are efficiently engulfed and degraded by professional phagocytes, macrophages. Phagocytic receptors Six-Microns-Under (SIMU), Draper (Drpr) and Croquemort (Crq) are specifically expressed in embryonic macrophages and required for their phagocytic function. However, how this function is established during development remains unclear. Here we demonstrate that the key regulator of Drosophila embryonic hemocyte differentiation, the transcription factor Serpent (Srp), plays a central role in establishing macrophage phagocytic competence. Srp, a homolog of the mammalian GATA factors, is required and sufficient for the specific expression of SIMU, Drpr and Crq receptors in embryonic macrophages. Moreover, we show that each of these receptors can significantly rescue phagocytosis defects of macrophages in srp mutants, including their distribution in the embryo and engulfment of apoptotic cells. This reveals that the proficiency of macrophages to remove apoptotic cells relies on the expression of SIMU, Crq and/or Drpr. However, Glial Cells Missing (GCM) acting downstream of Srp in the differentiation of hemocytes, is dispensable for their phagocytic function during embryogenesis. Taken together, our study discloses the molecular mechanism underlying the development of macrophages as skilled phagocytes of apoptotic cells.

P037 Bacterial handling defect in macrophages of patients with defects in glucose-6-phosphate metabolism and Crohn’s disease like intestinal inflammation

P037 Bacterial handling defect in macrophages of patients with defects in glucose-6-phosphate metabolism and Crohn’s disease like intestinal inflammation

The Multiple Localized Glyceraldehyde-3-Phosphate Dehydrogenase Contributes to the Attenuation of the Francisella tularensis dsbA Deletion Mutant.

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.

MafB is a critical regulator of complement component C1q

The transcription factor MafB is expressed by monocytes and macrophages. Efferocytosis (apoptotic cell uptake) by macrophages is important for inhibiting the development of autoimmune diseases, and is greatly reduced in Mafb-deficient macrophages. Here, we show the expression of the first protein in the classical complement pathway C1q is important for mediating efferocytosis and is reduced in Mafb-deficient macrophages. The efferocytosis defect in Mafb-deficient macrophages can be rescued by adding serum from wild-type mice, but not by adding serum from C1q-deficient mice. By hemolysis assay we also show that activation of the classical complement pathway is decreased in Mafb-deficient mice. In addition, MafB overexpression induces C1q-dependent gene expression and signals that induce C1q genes are less effective in the absence of MafB. We also show that Mafb-deficiency can increase glomerular autoimmunity, including anti-nuclear antibody deposition. These results show that MafB is an important regulator of C1q.