CD47-SIRPα Controls ADCC Killing of Primary T Cells by PMN Through a Combination of Trogocytosis and NADPH Oxidase Activation.

Immunotherapies targeting the "don't eat me" myeloid checkpoint constituted by CD47 SIRPα interaction have promising clinical potential but are limited by toxicities associated with the destruction of non-tumor cells. These dose-limiting toxicities demonstrate the need to highlight the mechanisms of anti-CD47-SIRPα therapy effects on non-tumor CD47-bearing cells. Given the increased incidence of lymphopenia in patients receiving anti-CD47 antibodies and the strong ADCC (antibody-dependent cellular cytotoxicity) effector function of polymorphonuclear cells (PMNs), we investigated the behavior of primary PMNs cocultured with primary T cells in the presence of anti-CD47 mAbs. PMNs killed T cells in a CD47-mAb-dependent manner and at a remarkably potent PMN to T cell ratio of 1:1. The observed cytotoxicity was produced by a novel combination of both trogocytosis and a strong respiratory burst induced by classical ADCC and CD47-SIRPα checkpoint blockade. The complex effect of the CD47 blocking mAb could be recapitulated by combining its individual mechanistic elements: ADCC, SIRPα blockade, and ROS induction. Although previous studies had concluded that disruption of SIRPα signaling in PMNs was limited to trogocytosis-specific cytotoxicity, our results suggest that SIRPα also tightly controls activation of NADPH oxidase, a function demonstrated during differentiation of immature PMNs but not so far in mature PMNs. Together, our results highlight the need to integrate PMNs in the development of molecules targeting the CD47-SIRPα immune checkpoint and to design agents able to enhance myeloid cell function while limiting adverse effects on healthy cells able to participate in the anti-tumor immune response.

Neutrophil-Derived Tumor Necrosis Factor Drives Fungal Acute Lung Injury in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) results from deficiency of nicotinamide adenine dinucleotide phosphate(NADPH) oxidase and impaired reactive oxygen species (ROS) generation. This leads to impaired killing of Aspergillus and, independently, a pathologic hyperinflammatory response to the organism. We hypothesized that neutrophil-derived ROS inhibit the inflammatory response to Aspergillus and that acute lung injury in CGD is due to failure of this regulation. Mice with gp91phox deficiency, the most common CGD mutation, had more severe lung injury, increased neutrophilinfiltration, and increased lung tumor necrosis factor (TNF) after Aspergillus challenge compared with wild-types. Neutrophils were surprisingly the predominant source of TNF in gp91phox-deficient lungs. TNF neutralization inhibited neutrophil recruitment in gp91phox-deficient mice and protected from lung injury. We propose that, in normal hosts, Aspergillus stimulates TNF-dependent neutrophil recruitment to the lungs and neutrophil-derived ROS limit inflammation. In CGD, in contrast, recruited neutrophils are the dominant source of TNF, promoting further neutrophil recruitment in a pathologic positive-feedback cycle, resulting in progressive lung injury.

Sodium molybdate induces heterophil extracellular traps formation in chicken.

Molybdenum (Mo) is not only an important rare metal that is widely used in industrial production but also an essential trace element for plants and animals. Nevertheless, in Mo polluted areas, excess Mo intake will not only cause gout in humans but also cause diarrhea in livestock and growth inhibition of chickens. Heterophils extracellular traps (HETs) are an important way to clear pathogens in the innate immune system of the chicken. However, the effects of Mo on the innate immune responses of HETs formation in chicken, and the mechanism undergoing this phenomenon remain unknown. In the study, we firstly aim to investigate the effects of sodium molybdate (Na2MoO4) on chicken HETs formation in vitro, and further to explore its related metabolic requirements and molecular mechanisms. Chicken heterophils were cultured with Na2MoO4, and Na2MoO4-induced HETs structures were analyzed by confocal microscopy. Moreover, Na2MoO4-induced HETs were quantified by Quant-iT PicoGreen® dsDNA Assay kits and fluorescence microplate. It has been shown that Na2MoO4 truly triggered HETs-like structures that were composed of DNA decorated with citrullinated histone 3 (citH3) and elastase. The inhibitors of NADPH oxidase, ERK1/2 and p38 MAPK signaling pathway significantly reduced Na2MoO4-induced HETs formation. Further experiments on energy metabolism involving Na2MoO4-induced HETs formation showed that Na2MoO4-induced HETs release was relevant to glucose, and the inhibitors of glycolysis including 3PO, AZD23766 and 3-Bromopyuvic acid, the inhibitors of glucose transport including STF31 and Ritonavir and NSC23766 significantly decreased Na2MoO4-induced HETs formation. In summary, these results demonstrate that Mo does induce chicken HETs formation in vitro, and the formation of HETs is a process relying on glucose transport 1 (GLUT1)，glucose transport 4 (GLUT4), glycolysis, and ROS production depended on the activation of NADPH oxidase, ERK1/2 and p38 signaling pathways, which also reflects the early innate immune responses of chicken against excessive molybdenum intake.

Independent and inter-dependent immunoregulatory effects of NCF1 and NOS2 in experimental autoimmune encephalomyelitis.

BACKGROUND: Increasing evidence has suggested that a single nucleotide polymorphism in the Ncf1 gene is associated with experimental autoimmune encephalomyelitis (EAE). However, the mechanisms of NCF1-induced immunoregulatory effects remain poorly understood. In this study, we focus on NCF1 deficiency-mediated effects on EAE in NOS2 dependent and independent ways. METHODS: To determine the effects of NCF1 and NOS2 during EAE development, we have established recombinant mouse strains deficient at NCF1 and/or NOS2 in a crossbreeding system. Different strains allow us to examine the entire course of the disease in the Nos2-null mice bearing a Ncf1 gene that encodes a mutated NCF1, deficient in triggering oxidative burst, after immunization with recombinant myelin oligodendrocyte glycoprotein (MOG)79-96 peptides. The peptide-induced innate and adaptive immune responses were analyzed by flow cytometry. RESULTS: NCF1-deficient mice developed a reduced susceptibility to EAE, whereas NCF1-NOS2 double-deficient mice developed an enhanced EAE, as compared with NOS2-deficient mice. Flow cytometry analyses show that double deficiencies resulted in an increase of neutrophils in the spleen, accompanied with higher release of interleukin-1ß in neutrophils prior to EAE onset. The additional deficiency in NCF1 had no added effect on either interleukin-17 or interferon-γ secretion of T cells during the priming phase. CONCLUSIONS: These studies show that NCF1 and NOS2 interact to regulate peptide-induced EAE.

Spatial Properties of Reactive Oxygen Species Govern Pathogen-Specific Immune System Responses.

Significance: Reactive oxygen species (ROS) are often considered to be undesirable toxic molecules that are generated under conditions of cellular stress, which can cause damage to critical macromolecules such as DNA. However, ROS can also contribute to the pathogenesis of cancer and many other chronic inflammatory disease conditions, including atherosclerosis, metabolic disease, chronic obstructive pulmonary disease, neurodegenerative disease, and autoimmune disease. Recent Advances: The field of ROS biology is expanding, with an emerging paradigm that these reactive species are not generated haphazardly, but instead produced in localized regions or in specific subcellular compartments, and this has important consequences for immune system function. Currently, there is evidence for ROS generation in extracellular spaces, in endosomal compartments, and within mitochondria. Intriguingly, the specific location of ROS production appears to be influenced by the type of invading pathogen (i.e., bacteria, virus, or fungus), the size of the invading pathogen, as well as the expression/subcellular action of pattern recognition receptors and their downstream signaling networks, which sense the presence of these invading pathogens. Critical Issues: ROS are deliberately generated by the immune system, using specific NADPH oxidases that are critically important for pathogen clearance. Professional phagocytic cells can sense a foreign bacterium, initiate phagocytosis, and then within the confines of the phagosome, deliver bursts of ROS to these pathogens. The importance of confining ROS to this specific location is the impetus for this perspective. Future Directions: There are specific knowledge gaps on the fate of the ROS generated by NADPH oxidases/mitochondria, how these ROS are confined to specific locations, as well as the identity of ROS-sensitive targets and how they regulate cellular signaling.

Maximum reproductive lifespan correlates with CD33rSIGLEC gene number: Implications for NADPH oxidase-derived reactive oxygen species in aging.

Humans and orcas are among the very rare species that have a prolonged post-reproductive lifespan (PRLS), during which the aging process continues. Reactive oxygen species (ROS) derived from mitochondria and from the NADPH oxidase (NOX) enzymes of innate immune cells are known to contribute to aging, with the former thought to be dominant. CD33-related-Siglecs are immune receptors that recognize self-associated-molecular-patterns and modulate NOX-derived-ROS. We herewith demonstrate a strong correlation of lifespan with CD33rSIGLEC gene number in 26 species, independent of body weight or phylogeny. The correlation is stronger when considering total CD33rSIGLEC gene number rather than those encoding inhibitory and activating subsets, suggesting that lifetime balancing of ROS is important. Combining independent lines of evidence including the short half-life and spontaneous activation of neutrophils, we calculate that even without inter-current inflammation, a major source of lifetime ROS exposure may actually be neutrophil NOX-derived. However, genomes of human supercentenarians (>110 years) do not harbor a significantly higher number of functional CD33rSIGLEC genes. Instead, lifespan correlation with CD33rSIGLEC gene number was markedly strengthened by excluding the post-reproductive lifespan of humans and orcas (R2  = 0.83; P < .0001). Thus, CD33rSIGLEC modulation of ROS likely contributes to maximum reproductive lifespan, but other unknown mechanisms could be important to PRLS.

Thioredoxin-Interacting Protein Promotes Phagosomal Acidification Upon Exposure to Escherichia coli Through Inflammasome-Mediated Caspase-1 Activation in Macrophages.

In host defense, it is crucial to maintain the acidity of the macrophage phagosome for effective bacterial clearance. However, the mechanisms governing phagosomal acidification upon exposure to gram-negative bacteria have not been fully elucidated. In this study, we demonstrate that in macrophages exposed to Escherichia coli, the thioredoxin-interacting protein (TXNIP)-associated inflammasome plays a role in pH modulation through the activated caspase-1-mediated inhibition of NADPH oxidase. While there was no difference in early-phase bacterial engulfment between Txnip knockout (KO) macrophages and wild-type (WT) macrophages, Txnip KO macrophages were less efficient at destroying intracellular bacteria in the late phase, and their phagosomes failed to undergo appropriate acidification. These phenomena were associated with reactive oxygen species production and were reversed by treatment with an NADPH oxidase inhibitor or a caspase inhibitor. In line with these results, Txnip KO mice were more susceptible to both intraperitoneally administered E. coli and sepsis induced by cecum ligation and puncture than WT mice. Taken together, this study suggests that the TXNIP-associated inflammasome-caspase-1 axis regulates NADPH oxidase to modulate the pH of the phagosome, controlling bacterial clearance by macrophages.

Ochratoxin A-Triggered Chicken Heterophil Extracellular Traps Release through Reactive Oxygen Species Production Dependent on Activation of NADPH Oxidase, ERK, and p38 MAPK Signaling Pathways.

Ochratoxin A (OTA) is a mycotoxin which could cause strong immunosuppressive toxicological effects in animals and humans. Heterophil extracellular traps (HETs) as a novel defense of chicken heterophils play an important role against pathogen infection. It has been reported that OTA can weaken the phagocytosis function of neutrophils. However, whether or not OTA shows immunosuppressive effects on HET release remains unclear. In the present study, we aim to first investigate the effects of OTA on HET release and then try to clarify the mechanisms in this process. OTA-induced HET structures were observed and analyzed by fluorescence confocal microscopy. The quantitative determination of OTA-induced HETs was measured by PicoGreen and a fluorescence microplate. The results clearly showed that OTA obviously induced the release of HET-like structures in heterophils, and these extracellular networks were composed by chromatin decorated with histones and neutrophil elastase. Reactive oxygen species (ROS) production was also increased in the process of OTA-induced HET formation. Furthermore, the inhibitors of NADPH oxidase, ERK [Formula: see text], and p38 MAPK signaling pathways significantly decreased OTA-induced HET formation. The abovementioned results suggest that OTA-induced HET formation is related to ROS production dependent on the activation of NADPH oxidase, ERK [Formula: see text], and p38 MAPK signaling pathways. Taken together, this study first shows that OTA possesses the ability to trigger HET formation, which provides our understanding of the host that continuously suffered OTA exposure leading to the hyporeactivity of the immune system against infection.

Does Pioglitazone Lead to Neutrophil Extracellular Traps Formation in Chronic Granulomatous Disease Patients?

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme complex responsible for reactive oxygen species (ROS) production, is defective in chronic granulomatous disease (CGD) patients. This enzyme helps in antimicrobial host defense by phagocytes. CGD patients are unable to form neutrophil extracellular traps (NETs), which are composed of granule-derived proteins from neutrophils decorated with decondensed chromatin. Mitochondria have gained attention, being a rich source of flavochrome enzymes due to the presence of several sites for superoxide production. Recently, PPARγ agonists, a mitochondrial ROS inducer, induce mitochondrial ROS formation post-treatment in murine NADPH oxidase knockout models. Mitochondrial ROS is also essential for NOX-independent NETosis. Our study for the first time detects induction of NETosis independent of NADPH oxidase post-treatment with agonists such as pioglitazone and rosiglitazone in CGD subjects. Neutrophils isolated from CGD subjects were treated with pioglitazone and rosiglitazone. After treatment, qualitative analysis of NET formation was done using confocal microscopy after staining with DAPI. Quantitative estimation of extracellular DNA was performed using Sytox green. Mitochondrial ROS production with PPARγ agonist-treated/untreated neutrophils was detected using MitoSOX red. Pioglitazone and rosiglitazone induce significant NET formation in CGD patients. Our data clearly signify the effect of PPARγ agonists in induction of NET formation in CGD cases. Apart from the proposed experimental studies regarding the detailed mechanism of action, controlled trials could provide valuable information regarding the clinical use of pioglitazone in CGD patients as curative HSCT remains challenging in developing countries.

Staphylococcus aureus Lipoic Acid Synthesis Limits Macrophage Reactive Oxygen and Nitrogen Species Production To Promote Survival during Infection.

Macrophages are critical mediators of innate immunity and must be overcome for bacterial pathogens to cause disease. The Gram-positive bacterium Staphylococcus aureus produces virulence factors that impede macrophages and other immune cells. We previously determined that production of the metabolic cofactor lipoic acid by the lipoic acid synthetase, LipA, blunts macrophage activation. A ΔlipA mutant was attenuated during infection and was more readily cleared from the host. We hypothesized that bacterial lipoic acid synthesis perturbs macrophage antimicrobial functions and therefore hinders the clearance of S. aureus Here, we found that enhanced innate immune cell activation after infection with a ΔlipA mutant was central to attenuation in vivo, whereas a growth defect imparted by the lipA mutation made a negligible contribution to overall clearance. Macrophages recruited to the site of infection with the ΔlipA mutant produced larger amounts of bactericidal reactive oxygen species (ROS) and reactive nitrogen species (RNS) than those recruited to the site of infection with the wild-type strain or the mutant strain complemented with lipA ROS derived from the NADPH phagocyte oxidase complex and RNS derived from the inducible nitric oxide synthetase, but not mitochondrial ROS, were critical for the restriction of bacterial growth under these conditions. Despite enhanced antimicrobial immunity upon primary infection with the ΔlipA mutant, we found that the host failed to mount an improved recall response to secondary infection. Our data suggest that lipoic acid synthesis in S. aureus promotes bacterial persistence during infection through limitation of ROS and RNS generation by macrophages. Broadly, this work furthers our understanding of the intersections between bacterial metabolism and immune responses to infection.

Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus Is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin.

Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

Neutrophil Cytosolic Factor 1 in Dendritic Cells Promotes Autoreactive CD8+ T Cell Activation via Cross-Presentation in Type 1 Diabetes.

Aims: Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in non-obese diabetic (NOD) mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of Ncf1 (the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC). Results:Ncf1-mutant DCs exhibit reduced ability to activate autoreactive CD8+ T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen, Ncf1-mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules. Innovation: This study demonstrates that Ncf1 (p47phox) is required for activation and effector function of CD8+ T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells. Conclusion: ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8+ T cells in individuals at-risk for developing T1D.

Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

Inflammatory consequences of inherited disorders affecting neutrophil function.

Primary immunodeficiencies affecting the function of neutrophils and other phagocytic leukocytes are notable for an increased susceptibility to bacterial and fungal infections as a result of impaired leukocyte recruitment, ingestion, and/or killing of microbes. The underlying molecular defects can also impact other innate immune responses to infectious and inflammatory stimuli, leading to inflammatory and autoimmune complications that are not always directly related to infection. This review will provide an update on congenital disorders affecting neutrophil function in which a combination of host defense and inflammatory complications are prominent, including nicotinamide dinucleotide phosphate oxidase defects in chronic granulomatous disease and ß2 integrin defects in leukocyte adhesion deficiency.

The roles of NADPH oxidase in modulating neutrophil effector responses.

Neutrophils are phagocytic innate immune cells essential for killing bacteria via activation of a wide variety of effector responses and generation of large amounts of reactive oxygen species (ROS). Majority of the ROS in neutrophils is generated by activation of the superoxide-generating enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Independent of their anti-microbial function, NADPH oxidase-derived ROS have emerged as key regulators of host immune responses and neutrophilic inflammation. Data from patients with inherited defects in the NADPH oxidase subunit alleles that ablate its enzyme function as well as mouse models demonstrate profound dysregulation of host inflammatory responses, neutrophil hyper-activation and tissue damage in response to microbial ligands or tissue trauma. A large body of literature now demonstrates how oxidants function as essential signaling molecules that are essential for the regulation of neutrophil responses during priming, degranulation, neutrophil extracellular trap formation, and apoptosis, independent of their role in microbial killing. In this review we summarize how NADPH oxidase-derived oxidants modulate neutrophil function in a cell intrinsic manner and regulate host inflammatory responses. In addition, we summarize studies that have elucidated possible roles of oxidants in neutrophilic responses within the oral mucosa and periodontal disease.

Dynamic G protein alpha signaling in Arabidopsis innate immunity.

Heterotrimeric G proteins composed of Gα, Gß and Gγ subunits are evolutionarily conserved signaling modules involved in diverse biological processes in plants and animals. The role and action of Gα remain largely enigmatic in plant innate immunity. We have recently demonstrated that Arabidopsis Gα (GPA1) is a key component of a new immune signaling pathway activated by bacteria-secreted proteases. Here we show that GPA1 is also involved in the signaling network of Arabidopsis in response to the bacterial flagellin epitope flg22. Specifically, GPA1 plays a pivotal role in an immune pathway involving the flg22 receptor FLS2, co-receptor BAK1, Regulator of G Signaling 1 (RGS1), and Arabidopsis Gß (AGB1), in which flg22 elicits GPA1/AGB1 dissociation from the FLS2/BAK1/RGS1 receptor complex. Consequently, we observed flg22-induced degradation of FLS2, BAK1 and RGS1 but not GPA1 or AGB1. We also found that GPA1 constitutively interacts with the NADPH oxidase RbohD to potentiate flg22-induced ROS burst independently of the central cytoplasmic kinase BIK1. Taken together, our work sheds multiple novel insights into the functions and regulatory mechanisms of GPA1 in Arabidopsis innate immunity.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Park 7: A Novel Therapeutic Target for Macrophages in Sepsis-Induced Immunosuppression.

Sepsis remains a serious and life-threatening condition with high morbidity and mortality due to uncontrolled inflammation together with immunosuppression with few therapeutic options. Macrophages are recognized to play essential roles throughout all phases of sepsis and affect both immune homeostasis and inflammatory processes, and macrophage dysfunction is considered to be one of the major causes for sepsis-induced immunosuppression. Currently, Parkinson disease protein 7 (Park 7) is known to play an important role in regulating the production of reactive oxygen species (ROS) through interaction with p47phox, a subunit of NADPH oxidase. ROS are key mediators in initiating toll-like receptor (TLR) signaling pathways to activate macrophages. Emerging evidence has strongly implicated Park 7 as an antagonist for sepsis-induced immunosuppression, which suggests that Park 7 may be a novel therapeutic target for reversing immunosuppression compromised by sepsis. Here, we review the main characteristics of sepsis-induced immunosuppression caused by macrophages and provide a detailed mechanism for how Park 7 antagonizes sepsis-induced immunosuppression initiated by the macrophage inflammatory response. Finally, we further discuss the most promising approach to develop innovative drugs that target Park 7 in patients whose initial presentation is at the late stage of sepsis.

A Label-Free Quantitative Proteomic Analysis of Mouse Neutrophil Extracellular Trap Formation Induced by Streptococcus suis or Phorbol Myristate Acetate (PMA).

Streptococcus suis (S. suis) ranks among the five most important porcine pathogens worldwide and occasionally threatens human health, particularly in people who come into close contact with pigs or pork products. An S. suis infection induces the formation of neutrophil extracellular traps (NETs) in vitro and in vivo, and the NET structure plays an essential role in S. suis clearance. However, the signaling pathway by which S. suis induces NET formation remains to be elucidated. In the present study, we used a label-free quantitative proteomic analysis of mouse NET formation induced by S. suis or phorbol myristate acetate (PMA), a robust NET inducer. Greater than 50% of the differentially expressed proteins in neutrophils infected by S. suis showed similar changes as observed following PMA stimulation, and PKC, NADPH oxidase, and MPO were required for NET formation induced by both stimuli. Because PMA induced robust NET formation while S. suis (MOI = 2) induced only weak NET formation, the association between the inducer and NET formation was worth considering. Interestingly, proteins involved in peptidase activity showed significant differential changes in response to each inducer. Of these peptidases, MMP-8 expression was obviously decreased in response to PMA, but it was not significantly changed in response to S. suis. A subsequent study further confirmed that MMP-8 activity was inversely correlated with NET formation induced by both stimuli. Therefore, the present study provides potentially important information about the manner by which neutrophils responded to the inducers to form NETs.