Macrophage Antimicrobial Activity Research Articles

Desaminotyrosine is a redox-active microbial metabolite that bolsters macrophage antimicrobial functions while attenuating IL-6 production.

Intestinal microbiota contributes to host defense against pathogens while avoiding the induction of inflammation in homeostatic conditions, but the mechanism is not fully understood. To investigate the potential role of the bacterial metabolite desaminotyrosine (DAT) in regulating host defense and inflammation, we pretreated mouse bone marrow-derived macrophages (BMDMs) with DAT for 12 hours and then challenged with bacterial lipopolysaccharide (LPS). We found that DAT priming-enhanced type I interferon response while selectively inhibiting proinflammatory interleukin (IL)-6 production after exposure to LPS. This is related to the fact that DAT is a natural antioxidant determined by radical scavenging assay in a cell-free system. DAT-primed cells had increased levels of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) upon LPS stimulation. Countering the increased NADPH by supplementing extra oxidized NADP+ to cells reversed DAT's effect on LPS-induced Il-6 and interferon-stimulated gene expressions. DAT-primed cells also were more resistant to oxidative stress-induced generation of reactive oxygen species and cell death. DAT promoted the production of antimicrobial effector nitric oxide in a cellular redox-dependent manner, leading to enhanced macrophage antimicrobial activity during Salmonella enterica infection. Our data suggest that DAT acts as a host-microbiota crosstalk signal in shaping host immune defense and inflammatory response.

Iron regulatory protein 2 contributes to antimicrobial immunity by preserving lysosomal function in macrophages

Colorectal cancer and Crohn's disease patients develop pyogenic liver abscesses due to failures of immune cells to fight off bacterial infections. Here, we show that mice lacking iron regulatory protein 2 (Irp2), globally (Irp2-/-) or myeloid cell lineage (Lysozyme 2 promoter-driven, LysM)-specifically (Irp2ΔLysM), are highly susceptible to liver abscesses when the intestinal tissue was injured with dextran sodium sulfate treatment. Further studies demonstrated that Irp2 is required for lysosomal acidification and biogenesis, both of which are crucial for bacterial clearance. In Irp2-deficient liver tissue or macrophages, the nuclear location of transcription factor EB (Tfeb) was remarkably reduced, leading to the downregulation of Tfeb target genes that encode critical components for lysosomal biogenesis. Tfeb mislocalization was reversed by hypoxia-inducible factor 2 inhibitor PT2385 and, independently, through inhibition of lactic acid production. These experimental findings were confirmed clinically in patients with Crohn's disease and through bioinformatic searches in databases from Crohn's disease or ulcerative colitis biopsies showing loss of IRP2 and transcription factor EB (TFEB)-dependent lysosomal gene expression. Overall, our study highlights a mechanism whereby Irp2 supports nuclear translocation of Tfeb and lysosomal function, preserving macrophage antimicrobial activity and protecting the liver against invading bacteria during intestinal inflammation.

An isoform quantitative trait locus in SBNO2 links genetic susceptibility to Crohn’s disease with defective antimicrobial activity

Despite major advances in linking single genetic variants to single causal genes, the significance of genetic variation on transcript-level regulation of expression, transcript-specific functions, and relevance to human disease has been poorly investigated. Strawberry notch homolog 2 (SBNO2) is a candidate gene in a susceptibility locus with different variants associated with Crohn’s disease and bone mineral density. The SBNO2 locus is also differentially methylated in Crohn’s disease but the functional mechanisms are unknown. Here we show that the isoforms of SBNO2 are differentially regulated by lipopolysaccharide and IL-10. We identify Crohn’s disease associated isoform quantitative trait loci that negatively regulate the expression of the noncanonical isoform 2 corresponding with the methylation signals at the isoform 2 promoter in IBD and CD. The two isoforms of SBNO2 drive differential gene networks with isoform 2 dominantly impacting antimicrobial activity in macrophages. Our data highlight the role of isoform quantitative trait loci to understand disease susceptibility and resolve underlying mechanisms of disease.

Simultaneous Increases in Intracellular Sodium and Tonicity Boost Antimicrobial Activity of Macrophages.

Inflamed and infected tissues can display increased local sodium (Na+) levels, which can have various effects on immune cells. In macrophages, high salt (HS) leads to a Na+/Ca2+-exchanger 1 (NCX1)-dependent increase in intracellular Na+ levels. This results in augmented osmoprotective signaling and enhanced proinflammatory activation, such as enhanced expression of type 2 nitric oxide synthase and antimicrobial function. In this study, the role of elevated intracellular Na+ levels in macrophages was investigated. Therefore, the Na+/K+-ATPase (NKA) was pharmacologically inhibited with two cardiac glycosides (CGs), ouabain (OUA) and digoxin (DIG), to raise intracellular Na+ without increasing extracellular Na+ levels. Exposure to HS conditions and treatment with both inhibitors resulted in intracellular Na+ accumulation and subsequent phosphorylation of p38/MAPK. The CGs had different effects on intracellular Ca2+ and K+ compared to HS stimulation. Moreover, the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) was not upregulated on RNA and protein levels upon OUA and DIG treatment. Accordingly, OUA and DIG did not boost nitric oxide (NO) production and showed heterogeneous effects toward eliminating intracellular bacteria. While HS environments cause hypertonic stress and ionic perturbations, cardiac glycosides only induce the latter. Cotreatment of macrophages with OUA and non-ionic osmolyte mannitol (MAN) partially mimicked the HS-boosted antimicrobial macrophage activity. These findings suggest that intracellular Na+ accumulation and hypertonic stress are required but not sufficient to mimic boosted macrophage function induced by increased extracellular sodium availability.

Cell painting assays for profiling responses in Mycobacterium tuberculosis-infected macrophages after metabolic perturbation

Abstract Tuberculosis (TB) is the leading infectious disease killer in the world, and a lack of accessible, easy treatments contributes to its growing burden. Repurposing approved treatments for other conditions, including metabolic inhibitors under consideration for use in cancer therapy, as host-directed therapies may improve TB treatments. Detailed preliminary studies to identify mechanisms that underly drug function need to be performed before these drugs can be used in patients but there is a lack of tools to investigate metabolic perturbations in target cells. This problem is particularly acute for treatments that have uncharacterized effects on host cells or host-pathogen interactions, or for adherent cells where cell morphology and migration patterns may be factors that are modulated by treatments. Cell painting assays are microscopy-based analyses that use quantitative imaging data to generate insight into how drugs and infections perturb host cells. Here, we sought to optimize a cell painting assay to investigate changes in macrophage morphology and antimicrobial activity after metabolic perturbation and Mycobacterium tuberculosis(Mtb) infection. Our objective was to apply the quantification and analysis pipelines to macrophages that were infected with fluorescent Mtb reporter strains to identify cellular responses after inhibition of fatty acid oxidation, oxidative phosphorylation, or glycolysis. Moreover, we compared inhibitors that target different points in the glycolysis pathway to define how these points differentially affect macrophage antimicrobial activity. Our work provides a framework for using cell painting assays as tools for dissecting host-pathogen interactions in metabolically perturbed cells. Supported by grants from NIH grants AI167710 and #LINK#AI164970#ENDLINK#

Impact of IFN-y and CD40 signalling on Toxoplasma gondii cyst formation in differentiated Neuro-2a neuroblastoma cells.

Signalling by IFN-y and CD40 is known to trigger anti-microbial activity in macrophages infected with Toxoplasma gondii, but their effects on infected neurons are less well known. Here, we compared how stimulation with IFN-y and an agonistic anti-CD40 mAb impacts infection and cyst formation in the mouse neuroblastoma cell line Neuro-2a relative to bone marrow-derived macrophages. Both IFN-y and CD40 mAb decreased cyst emergence in Neuro-2a cells. In macrophages, these stimuli decreased infection, but had no impact on infection in the neuroblastoma cell line. Resistance to killing in Neuro-2a cells may explain why neurons preferentially harbour parasites during chronic infection in the brain.

Sepsis expands a CD39+ plasmablast population that promotes immunosuppression via adenosine-mediated inhibition of macrophage antimicrobial activity

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.

Scientific and practical aspects of the use of prebiotics in the process of feeding ruminants

The review article presents current literature data on the classification of prebiotics. Information on the characteristics of the most common and used in the feeding of ruminants prebiotic drugs is presented. Based on literature, attention is drawn to the fact that one of the main functions of mannan oligosaccharides is their competitive binding to gram-negative bacteria of the pancreas and cecum in ruminants and inhibition of pathogenic microbiota growth in these departments of gastrointestinal tract. Fructooligosaccharides are not digested by animals, starting from the oral cavity and ending with the intestines. They are readily available substrates for the microflora of the ruminant pancreas and large intestine of animals. As for galactosyloligosaccharides, experiments have convincingly proven that they stimulate the growth and development of bifidobacteria and lactobacilli, enterobacteria and streptococci in the digestive tract of animals. Lactulose and lactiol as prebiotics have a positive effect on feed intake in young animals, changing the microbial balance and biochemical composition of the contents of the cecum. These prebiotics promote the reproduction of gram-positive bacteria in the digestive tract of animals and inhibit the growth and development of clostridia. Lactulose and lactiol activate the formation of short-chain fatty acids by the microbiota of the cecum of animals, as well as increase the permeability of the intestinal mucosa and the solubility of minerals in the colon. High molecular weight beta-glucans enhance the phagocytic, cytotoxic and antimicrobial activity of macrophages. They help to produce reactive intermediates of oxygen and nitrogen and clean the tissues of apoptotic cells. Furthermore, stimulating innate immune responses, beta-glucans increase the production of anti-inflammatory cytokines and chemokines. Inulin in ruminants has a pronounced activating effect on the immune system, as well as activates the production of short-chain fatty acids in the pancreas and large intestine. The biological mechanism of action in the digestive tract of ruminants of such prebiotics as: mannan oligosaccharides, fructooligosaccharides, galactooligosaccharides, lactulose, lactiol, beta-glucans, inulin is described. The productive effect of prebiotics when using their additives in the diets of young and adult ruminants is characterized. It is shown that the use of the above prebiotic drugs in the feeding of ruminant species selectively stimulates the metabolic processes of the symbiotic microflora of the pancreas and cecum, activates their vital functions and growth. The use of prebiotic supplements in the diets of cows stimulates milk productivity and improves milk quality. In fattening cattle, the stabilization of the pH of the scar content through the use of prebiotics increases the average daily gain and live weight of animals and the efficiency of assimilation of nutrients in feed.

Potentiation of Cytokine-Mediated Restriction of Legionella Intracellular Replication by a Dot/Icm-Translocated Effector.

Legionella pneumophila is ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires' disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires' disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression of legC4 was sufficient to restrict Legionella longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionella spp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction of Legionella by macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

Low Admission Plasma Gelsolin Concentrations Identify Community-acquired Pneumonia Patients at High Risk for Severe Outcomes.

Plasma gelsolin (pGSN) is an abundant circulating protein that neutralizes actin exposed by damaged cells, modulates inflammatory responses, and enhances alveolar macrophage antimicrobial activity. We investigated whether adults with low pGSN at hospital admission for community-acquired pneumonia (CAP) were at high risk for severe outcomes. Admission pGSN concentrations in 455 adults hospitalized with CAP were measured using enzyme-linked immunosorbent assay. Patients were grouped into the following 4 hierarchical, mutually exclusive categories based on maximum clinical severity experienced during their hospitalization: general floor care without intensive care unit (ICU) admission, invasive respiratory or vasopressor support (IRVS), or death; ICU care without IRVS or death; IRVS without death; or death. Admission pGSN concentrations were compared across these discrete outcome categories. Additionally, outcomes among patients in the lowest quartile of pGSN concentration were compared to those in the upper 3 quartiles. Overall, median (interquartile range) pGSN concentration was 38.1 (32.1, 45.7) μg/mL. Patients with more severe outcomes had lower pGSN concentrations (P = .0001); median values were 40.3 μg/mL for floor patients, 36.7 μg/mL for ICU patients, 36.5 μg/mL for patients receiving IRVS, and 25.7 μg/mL for patients who died. Compared to patients with higher pGSN concentrations, patients in the lowest quartile (pGSN ≤ 32.1 μg/mL) more often required IRVS (21.2% vs 11.7%, P = .0114) and died (8.8% vs 0.9%, P < .0001). Among adults hospitalized with CAP, lower pGSN concentrations were associated with more severe clinical outcomes. Future studies are planned to investigate possible therapeutic benefits of recombinant human pGSN in this population.

Calcitriol increases nitric oxide production and modulates microbicidal capacity against Mycobacterium bovis in bovine macrophages.

Bovine tuberculosis, a re-emerging infectious disease caused by Mycobacterium bovis, can be transmitted to humans. Global prevalence of M. bovis in humans is underestimated and represents a serious public health risk in developing countries. In light of this situation, it is important to note that our understanding of the immunopathogenesis of human tuberculosis can be improved by studying this disease in the bovine model. Stimulation of the bovine innate immune system with calcitriol (1,25(OH)2D3) leads to an increase in bactericidal molecules involved in macrophage antimicrobial activity. It is unknown, however, if calcitriol´s effect on bovine macrophages impacts intracellular bacterial replication. With these considerations in mind, this study sought to investigate the specific role of calcitriol in tuberculosis control in bovine macrophages, in the hopes of uncovering information applicable to human tuberculosis. As such, infection with M. bovis was shown to induce expression of CYP27B1 and VDR genes in macrophages. Moreover, addition of 1,25(OH)2D3 to cultures of macrophages previously infected with mycobacteria and/or activated by LPS triggered cellular expression of nitric oxide synthase (NOS2) and increased nitrite concentrations, both indicators of nitric oxide (NO) production. By means of a microbicidal assay, addition of 1,25(OH)2D3 was seen to increase macrophage phagocytosis and to decrease mycobacterial intracellular replication. Thus, taken together, our results show that calcitriol can help stimulate the innate immune system of bovines by increasing phagocytosis and decreasing intracellular replication of microorganisms, such as M. bovis, in macrophages, through the VDR pathway.

Type I and II IFNs differentially regulate IFNGR1 to tune IFNγ responsiveness in myeloid cells

Abstract Interferons (IFNs) act on macrophages to shape immune responses against microbial infections and tumors. Type I and II IFNs signal to induce transcription of IFN-stimulated genes (ISGs). Binding of the type II IFN (IFNγ) to its receptor, IFNGR, induces ISGs that enhance macrophage inflammatory and anti-microbial activity critical for bacterial infection. Conversely, when type I IFNs bind their receptor (IFNAR), they reduce expression of myeloid cell surface IFNGR and dampen macrophage responsiveness to IFNγ. Macrophages stimulated with IFNγ also reduce cell surface IFNGR, but this was previously attributed to ligand-induced receptor internalization. Here, we confirm the ability of IFNγ stimulation to rapidly reduce cell surface IFNGR in cultured mouse and human macrophages. However, we find this ligand-induced reduction in IFNGR is associated with reduced ifngr1 transcript abundance. Unlike IFNβ, IFNγ did not impede de novo transcription from an ifngr1 promoter-driven reporter construct, suggesting IFNγ reduces ifngr1 transcript abundance via a distinct mechanism. BMDMs pulsed with IFNγ transiently became refractory to a secondary IFNγ treatment as measured by impaired STAT1 phosphorylation. As shown previously, type I IFNs cause a reduction in myeloid cell surface IFNGR1 seen 72 hpi with systemic L. monocytogenes (Lm), and this reduction was abrogated by myeloid cell expression of a transgenic IFNGR1 in fGR1 mice. In contrast, IFNγ was responsible for a reduction in IFNGR on myeloid cells early after systemic Lm infection (24 hip), and this was not abrogated in fGR1 mice. Together, our results demonstrate that type I and II IFNs act via distinct mechanisms to prevent hyper-activation of myeloid cells following exposure to IFNγ.

Down regulation of macrophage IFNGR1 exacerbates systemic L. monocytogenes infection.

Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.

Solubilized extracellular matrix bioscaffolds derived from diverse source tissues differentially influence macrophage phenotype.

The host response to biomaterials is a critical determinant of their success or failure in tissue-repair applications. Macrophages are among the first responders in the host response to biomaterials and have been shown to be predictors of downstream tissue remodeling events. Biomaterials composed of mammalian extracellular matrix (ECM) in particular have been shown to promote distinctive and constructive remodeling outcomes when compared to their synthetic counterparts, a property that has been largely attributed to their ability to modulate the host macrophage response. ECM bioscaffolds are prepared by decellularizing source tissues such as dermis and small intestinal submucosa. The differential ability of such scaffolds to influence macrophage behavior has not been determined. The present study determines the effects of ECM bioscaffolds derived from eight different source tissues upon macrophage surface marker expression, protein content, phagocytic capability, metabolism, and antimicrobial activity. The results show that macrophages exposed to small intestinal submucosa (SIS), urinary bladder matrix (UBM), brain ECM (bECM), esophageal ECM (eECM), and colonic ECM (coECM) express a predominant M2-like macrophage phenotype, which is pro-remodeling and anti-inflammatory (iNOS-/Fizz1+/CD206+). In contrast, macrophage exposure to dermal ECM resulted in a predominant M1-like, pro-inflammatory phenotype (iNOS+/Fizz1-/CD206-), whereas liver ECM (LECM) and skeletal muscle ECM (mECM) did not significantly change the expression of these markers. All solubilized ECM bioscaffold treatments resulted in an increased macrophage antimicrobial activity, but no differences were evident in macrophage phagocytic capabilities, and macrophage metabolism was decreased following exposure to UBM, bECM, mECM, coECM, and dECM. The present work could have important implications when considering the macrophage response following ECM implantation for site-appropriate tissue remodeling. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 138-147, 2017.

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

Mycobacterium leprae-induced Insulin-like Growth Factor I attenuates antimicrobial mechanisms, promoting bacterial survival in macrophages.

Mycobacterium leprae (ML), the etiologic agent of leprosy, can subvert macrophage antimicrobial activity by mechanisms that remain only partially understood. In the present study, the participation of hormone insulin-like growth factor I (IGF-I) in this phenomenum was investigated. Macrophages from the dermal lesions of the disseminated multibacillary lepromatous form (LL) of leprosy expressed higher levels of IGF-I than those from the self-limited paucibacillary tuberculoid form (BT). Higher levels of IGF-I secretion by ML-infected macrophages were confirmed in ex vivo and in vitro studies. Of note, the dampening of IGF-I signaling reverted the capacity of ML-infected human and murine macrophages to produce antimicrobial molecules and promoted bacterial killing. Moreover, IGF-I was shown to inhibit the JAK/STAT1-dependent signaling pathways triggered by both mycobacteria and IFN-γ most probably through its capacity to induce the suppressor of cytokine signaling-3 (SOCS3). Finally, these in vitro findings were corroborated by in vivo observations in which higher SOCS3 expression and lower phosphorylation of STAT1 levels were found in LL versus BT dermal lesions. Altogether, our data strongly suggest that IGF-I contributes to the maintenance of a functional program in infected macrophages that suits ML persistence in the host, reinforcing a key role for IGF-I in leprosy pathogenesis.

Physiologic stress molecules activate an acute phase response and enhance macrophage antimicrobial activity to Mycobacterium leprae (INM3P.411)

Abstract Leprosy is a disease caused by the pathogen Mycobacterium leprae (mLEP). Patients can present with the progressive, lepromatous form of the disease or the self-limited, tuberculoid form of the disease. Physiologic stress is thought to inhibit host defense mediated by adrenergic catecholamines and neuroendocrine glucocorticoids. We hypothesize that acute treatment with adrenergic agents or glucocorticoids would inhibit in vitro macrophage mediated antimicrobial activity. Human monocyte-derived macrophages or mouse bone marrow-derived macrophages were treated with the adrenergic agonist metaproterenol hemisulfate (MPH, 1μM) or the glucocorticoid dexamethasone (Dex, 100nM) for two hours followed by LPS (100ng/ml) or mLEP (MOI 5). RNASeq was performed at various time points or an mLEP viability measurement was performed at 24 hours following infection. Unexpectedly, when compared to vehicle (DMSO), pretreatment with MPH resulted in greatly enhanced antimicrobial activity against mLEP, similar to interferon-γ pretreatment, whereas Dex resulted in a more modest induction of antimicrobial activity. Both Dex and MPH did not reduce the expression of the antimicrobial peptide cathelicidin, but did induce a dramatic increase in expression of genes involved in the acute phase response. Our findings suggest that molecules involved in the physiological stress response may be acutely beneficial during initial encounter with mLEP.

ATP exhibits antimicrobial action by inhibiting bacterial utilization of ferric ions.

ATP up-regulates macrophage antimycobacterial activity in a P2X7-dependent manner, but little is known about whether ATP directly exhibits antimicrobial effects against intracellular mycobacteria. In this study, we found that ATP inhibited the growth of various bacteria, including Staphylococcus, Pseudomonas, and mycobacteria, without damaging bacterial surface structures. Using gene technology, we newly established an enterobactin-deficient (entB−) mutant from ATP-resistant Klebsiella pneumoniae, and found the recovery of ATP susceptibility in the enterobactin-deleted mutant. Therefore, ATP's antibacterial activity is attributable to its iron-chelating ability. Since ATP distributed in the cytosol of macrophages at high concentrations, ATP appears to augment macrophage's antimicrobial activity by directly attacking intracytosolic and intra-autophagosomal pathogens. Furthermore, ATP exhibited combined effects with some antimicrobials against methicillin-resistant S. aureus (MRSA) and M. intracellulare, suggesting its usefulness as an adjunctive drug in the chemotherapy of certain intractable infections.

IL-17A Produced by Neutrophils Protects against Pneumonic Plague through Orchestrating IFN-γ–Activated Macrophage Programming

Innate immune cells, including neutrophils and macrophages, are critically involved in host antimicrobial defense responses. Intrinsic regulatory mechanisms controlling neutrophil and macrophage activities are poorly defined. In this study, we found that IL-17A, a natural signal factor, could provide protection against early pneumonic plague inflammation by coordinating the functions of neutrophils and programming of macrophages. The IL-17A level is promptly increased during the initial infection. Importantly, abrogation of IL-17A or IL-17AR significantly aggravated the infection, but mIL-17A treatment could significantly alleviate inflammatory injury, revealing that IL-17A is a critical requirement for early protection of infection. We also demonstrated that IL-17A was predominantly produced by CD11b(+)Ly6G(+) neutrophils. Although IL-17A could not significantly affect the antimicrobial responses of neutrophils, it could target the proinflammatory macrophage (M1) programming and potentiate the M1's defense against pneumonic plague. Mechanistically, IFN-γ treatment or IFN-γ-activated M1 macrophage transfer could significantly mitigate the aggravated infection of IL-17A(-/-) mice. Finally, we showed that IL-17A and IFN-γ could synergistically promote macrophage anti-infection immunity. Thus, our findings identify a previously unrecognized function of IL-17A as an intrinsic regulator in coordinating neutrophil and macrophage antimicrobial activity to provide protection against acute pneumonic plague.

Macrophage activation and leishmanicidal activity by galactomannan and its oxovanadium (IV/V) complex in vitro

Compounds that activate macrophage antimicrobial activity are potential targets for treatment of leishmaniasis. The present study investigated the in vitro immunomodulatory effects of a galactomannan (GALMAN-A) isolated from seeds of Mimosa scabrella and its oxovanadium (IV/V) complex (GALMAN-A:VO2+/VO3+) on macrophage activity. GALMAN-A increased nitric oxide levels by ~33% at a concentration of 250μg/ml, while GALMAN-A:VO2+/VO3+ decreased nitric oxide levels by ~33% at a concentration of 50μg/ml. Furthermore, GALMAN-A increased interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) levels by 5.5 and 2.3 times, respectively, at a concentration of 25μg/ml; at the same concentration, GALMAN-A:VO2+/VO3+ promoted an increase in IL-1β and IL-6 production by 8 and 5.5 times, respectively. However, neither GALMAN-A nor GALMAN-A:VO2+/VO3+ affected tumor necrosis factor alpha (TNF-α) or interleukin-10 (IL-10) levels. Importantly, both GALMAN-A and GALMAN-A:VO2+/VO3+ exhibited leishmanicidal activity on amastigotes of Leishmania (L.) amazonensis, reaching ~60% activity at concentrations of 100 and 25μg/ml, respectively. These results indicate that GALMAN-A is three times more potent and its oxovanadium complex is twelve times more potent than Glucantime (300μg/ml), which is the drug of choice in leishmaniasis treatment. The IC50 value for GALMAN-A:VO2+/VO3+ was 74.4μg/ml (0.58μg/ml of vanadium). Thus, the significant activation of macrophages and the noted leishmanicidal effect demonstrate the need for further studies to clarify the mechanisms of action of these compounds.