Search

Background: Malaria remains a major global health priority, and monoclonal antibodies (mAbs) are emerging as potential new tools to support efforts to control the disease. Recent data suggest that Fc-dependent mechanisms of immunity are important mediators of protection against the blood stages of the infection, but few studies have investigated this in the context of mAbs. We aimed to isolate mAbs agnostic to cognate antigens that target whole merozoites and simultaneously induce potent neutrophil activity measured by the level of reactive oxygen species (ROS) production using an antibody-dependent respiratory burst (ADRB) assay., Methods: We used samples from semi-immune adults living in coastal Kenya to isolate mAbs that induce merozoite-specific ADRB activity. We then tested whether modifying the expressed IgG1 isotype to an IgG-IgA Fc region chimera would enhance the level of ADRB activity., Results: We isolated a panel of nine mAbs with specificity to whole merozoites. mAb J31 induced ADRB activity in a dose-dependent fashion. Compared to IgG1, our modified antibody IgG-IgA bi-isotype induced higher ADRB activity across all concentrations tested. Further, we observed a negative hook effect at high IgG1 mAb concentrations (i.e., >200 µg/mL), but this was reversed by Fc modification. We identified MSP3.5 as the potential cognate target of mAb J31., Conclusions: We demonstrate an approach to engineer mAbs with enhanced ADRB potency against blood-stage parasites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ogwang, Murugu, Nkumama, Nyamako, Kai, Mwai, Murungi, Idro, Bejon, Tuju, Kinyanjui and Osier.)

Background: Trauma increases susceptibility to secondary bacterial infections. The events suppressing antimicrobial immunity are unclear. Polymorphonuclear neutrophils (PMNs) migrate toward bacteria using chemotaxis, trap them in extracellular neutrophil extracellular traps, and kill them using respiratory burst (RB). We hypothesized that plasma and wound fluids from trauma patients alter PMN function., Methods: Volunteer PMNs were incubated in plasma or wound fluids from trauma patients (days 0 and 1, days 2 and 3), and their functions were compared with PMNs incubated in volunteer plasma. Chemotaxis was assessed in transwells. Luminometry assessed total and intracellular RB responses to receptor-dependent and independent stimulants. Neutrophil extracellular trap formation was assessed using elastase assays. The role of tissue necrosis in creating functionally suppressive systemic PMN environments was assessed using a novel pig model where PMNs were incubated in uninjured pig plasma or plasma from pigs undergoing intraperitoneal instillation of liver slurry., Results: Both plasma and wound fluids from trauma patients markedly suppress total PMN RB. Intracellular RB is unchanged, implicating suppression of extracellular RB. Wound fluids are more suppressive than plasma. Biofluids suppressed RB maximally early after injury and their effects decayed with time. Chemotaxis and neutrophil extracellular trap formation were suppressed by biofluids similarly. Lastly, plasma from pigs undergoing abdominal liver slurry instillation suppressed PMN RB, paralleling suppression by human trauma biofluids., Conclusion: Trauma plasma and wound fluids suppress RB and other key PMNs antimicrobial functions. Circulating suppressive signals can be derived from injured or necrotic tissue at wound sites, suggesting a key mechanism by which tissue injuries can put the host at risk for infection., (Copyright © 2021 American Association for the Surgery of Trauma.)

In the lumen of blood vessels, there are large numbers of erythrocytes, which are approximately 95% of the total blood cells. Although the function of erythrocytes is to transport oxygen in the organism, recent studies have shown that mammalian and teleost erythrocytes are involved in the immune response against bacterial infections. However, the immune mechanisms used by avian erythrocytes are not yet clear. Here, we demonstrated that erythrocytes from goose have the ability to phagocytose as well as conduct antimicrobial activity. Firstly, we revealed the phagocytosis or adhesion activity of goose erythrocytes for latex beads 0.1-1.0 μm in diameter by fluorescence microscopy, and scanning and transmission electron microscopy. The low cytometry results also proved that goose erythrocytes had a wide range of phagocytic or adhesion activity for different bacteria. Followed, the low cytometry analysis data further explored that the goose erythrocytes contain the ability to produce reactive oxygen species (ROS) and inducible nitric oxide synthase (iNOS) in response to bacterial stimulation, and also up-regulated the expression of NOX family includes NOX1 and NOX5. Finally, we also found that goose erythrocytes showed a powerful antibacterial activity against all the three bacteria, meanwhile the stimulation of three kinds of bacteria up-regulated the expression of inflammatory factors, and increased the production of antioxidant enzymes to protect the cells from oxidative damage. Herein, our results demonstrate that goose Erythrocytes possess a certain phagocytic capacity and antioxidant system, and that the antimicrobial activity of erythrocytes can occurred through the production of unique respiratory burst against foreign pathogenic bacteria, which provides new clues to the interaction between bacteria and avian erythrocytes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Chen, Lu, Xu, Li, Yang, Li, Lin and Qin.)

Rapid recruitment of neutrophils to an inflamed site is one of the hallmarks of an effective host defense mechanism. The main pathway through which this happens is by the innate immune response. Neutrophils, which play an important part in innate immune defense, migrate into lungs through the modulation actions of chemokines to execute a variety of pro-inflammatory functions. Despite the importance of chemokines in host immunity, little has been discussed on their roles in host immunity. A holistic understanding of neutrophil recruitment, pattern recognition pathways, the roles of chemokines and the pathophysiological roles of neutrophils in host immunity may allow for new approaches in the treatment of infectious and inflammatory disease of the lung. Herein, this review aims at highlighting some of the developments in lung neutrophil-immunity by focusing on the functions and roles of CXC/CC chemokines and pattern recognition receptors in neutrophil immunity during pulmonary inflammations. The pathophysiological roles of neutrophils in COVID-19 and thromboembolism have also been summarized. We finally summarized various neutrophil biomarkers that can be utilized as prognostic molecules in pulmonary inflammations and discussed various neutrophil-targeted therapies for neutrophil-driven pulmonary inflammatory diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Effah, Drokow, Agboyibor, Ding, He, Liu, Akorli, Nuamah, Sun, Zhou, Liu, Xu, Feng, Wu and Zhang.)

Introduction: Inherited phagocyte defects are one of the subgroups of primary immunodeficiency diseases (PIDs) with various clinical manifestations. As oral manifestations are common at the early ages, oral practitioners can have a special role in the early diagnosis., Materials and Methods: A comprehensive search was conducted in this systematic review study and data of included studies were categorized into four subgroups of phagocyte defects, including congenital neutropenia, defects of motility, defects of respiratory burst, and other non-lymphoid defects., Results: Among all phagocyte defects, 12 disorders had reported data for oral manifestations in published articles. A total of 987 cases were included in this study. Periodontitis is one of the most common oral manifestations., Conclusion: There is a need to organize better collaboration between medical doctors and dentists to diagnose and treat patients with phagocyte defects. Regular dental visits and professional oral health care are recommended from the time of the first primary teeth eruption in newborns., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Hibernation consists of alternating periods of reduced metabolism (torpor) with brief periods of metabolism similar to summer euthermia (arousal). The function of the innate immune system is reduced during hibernation, of which the underlying mechanisms are incompletely understood. Here, we studied neutrophil functionality during hibernation in Syrian hamsters. The inflammatory response to LPS-induced endotoxemia is inhibited in hibernation, partly mediated by reduced IL-6 production in early arousal. Furthermore, neutrophil pathogen binding, phagocytosis and oxidative burst is profoundly reduced in early arousal. Functionality of both summer and early arousal neutrophils was repressed in plasma from early arousal and mixed plasma from early arousal and summer euthermic, but restored by summer euthermic plasma, signifying that a plasma factor in early arousal inhibits TLR-recognition. Identification of the inhibiting factor may offer a target to modulate neutrophil function with relevance to (auto-)inflammatory diseases., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2., Objective: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells., Methods: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34 + cells were then isolated, phenotyped, and assessed functionally., Results: Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34 + cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later., Conclusions: This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Aspergillus fumigatus causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti- Aspergillus defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require ex vivo differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an A. fumigatus lysate and two A. fumigatus  proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both A. fumigatus proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1β, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4 + and CD8 + T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with A. fumigatus germ tubes. Taken together, our in vitro data suggest that ex vivo CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Page, Wallstabe, Lother, Bauser, Kniemeyer, Strobel, Voltersen, Teutschbein, Hortschansky, Morton, Brakhage, Topp, Einsele, Wurster and Loeffler.)

During enteric salmonellosis, neutrophil-generated reactive oxygen species alter the gut microenvironment, favoring survival of Salmonella Typhimurium. While type 3 secretion system 1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone is responsible for stimulation of a maximal respiratory burst. To identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in coculture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single-gene deletion mutants representing particular regions under selection. A subset of single-gene deletion mutants was selected for further investigation. Mutants in four genes, STM1696 ( sapF ), STM2201 ( yeiE ), STM2112 ( wcaD ), and STM2441 ( cysA ), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (Δ STM1696 and Δ STM2201 ). The Δ STM2441 mutant, defective for sulfate transport, formed aggregates in minimal medium and adhered to surfaces in rich medium, suggesting a role for sulfur homeostasis in the regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the Δ STM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how S almonella may alter human neutrophil antimicrobial defenses., (Copyright © 2021 American Society for Microbiology.)

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Temming, Tammes Buirs, Bentlage, Treffers, Feringa, de Taeye, Kuijpers, Nagelkerke, Brasser, Mok, van Esch, van den Berg, Rispens, van der Schoot and Vidarsson.)

Background and Aims: Acute-on-chronic liver failure (ACLF) is characterized by systemic inflammation accompanied by defective anti-bacterial immunity. The role of neutrophils in immune derangement of ACLF has not been fully elucidated. This study is aimed to characterize the role of circulating neutrophils in HBV-related ACLF patients., Methods: Quantitative, phenotypic, transcriptomic, and functional alterations of circulating neutrophils were compared in ACLF and non-ACLF subjects and analyzed for associations with short-term outcomes. Interventional experiments were performed to test the impact on ACLF-patient neutrophil function in vitro ., Results: Circulating absolute neutrophil count was significantly increased in patients with ACLF and was an independent risk factor for 28-day mortality. ACLF-patient neutrophils differentially expressed a panel of surface markers (include TLR-1, TLR-2, TLR-4, CEACAM-1 and FPR1), as well as a distinct transcriptomic signature. ACLF-neutrophils displayed significantly impaired phagocytosis but an increased capacity to form neutrophil extracellular traps (NETs), which was more pronounced in patients with poor outcome. Healthy neutrophils mimicked functional characteristics of ACLF counterpart after co-cultured with plasma from ACLF patients. The oxidative burst and cytokine production capacities remained unchanged. Plasma GM-CSF, IL-6, IL-8, IL-10, and IP-10 levels, as well as lipopolysaccharide (LPS) concentration, were markedly elevated in ACLF patients but not DAMP molecules HMGB-1 and HSP70. Finally, a glycolysis inhibitor, 2-deoxy-glucose, reduced NET formation of ACLF patients' neutrophils., Conclusions: Circulating ACLF-patient neutrophils exhibit alterations in number, phenotype, gene expression and function, which was associated with poor outcome and shaped by the ACLF circulatory environment. Inhibiting glycolysis can reverse neutrophil dysfunction in ACLF patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Sun, Wang, Zhao, Ren, Li, Zhao, Zhang, Sheng, Chen and Shi.)

A wide variety of environmental contaminants has been shown to disrupt immune functions of fish and may compromise their defense capability against pathogens. Immunotoxic effects, however, are rarely considered in ecotoxicological testing strategies. The aim of this study was to systematically evaluate the suitability of an in vitro immuno-assay using selected fish immune parameters to screen for chemicals with known immunotoxic potential and to differentiate them from non-immunotoxicants. Non-stimulated and lipopolysaccharide-stimulated head kidney leukocytes of rainbow trout (Oncorhynchus mykiss) were exposed for 3 h or 19 h to chemicals with different modes of action. As immune parameters, phagocytosis activity, oxidative burst activity and cytokine transcription (IL-1β, TNFα, IL-10) were examined, accompanied by in silico modelling. The immunotoxicants dexamethasone, benzo(a)pyrene, ethinylestradiol and bisphenol A significantly altered the immune parameters at non-cytotoxic concentrations whereas diclofenac had only weak effects. However, the two baseline chemicals with no known immunotoxic potential, butanol and ethylene glycol, caused significant effects, too. From our results it appears that the in vitro fish leukocyte assay as performed in the present study has only a limited capacity for discriminating between immunotoxicants and non-immunotoxicants.

Currently, assessment of the potential immunotoxicity of a given agent involves a tiered approach for hazard identification and mechanistic studies, including observational studies, evaluation of immune function, and measurement of susceptibility to infectious and neoplastic diseases. These studies generally use costly low-throughput mammalian models. Zebrafish, however, offer an excellent alternative due to their rapid development, ease of maintenance, and homology to mammalian immune system function and development. Larval zebrafish also are a convenient model to study the innate immune system with no interference from the adaptive immune system. In this study, a respiratory burst assay (RBA) was utilized to measure reactive oxygen species (ROS) production after developmental xenobiotic exposure. Embryos were exposed to non-teratogenic doses of chemicals and at 96 h post-fertilization, the ability to produce ROS was measured. Using the RBA, 12 compounds with varying immune-suppressive properties were screened. Seven compounds neither suppressed nor enhanced the respiratory burst; five reproducibly suppressed global ROS production, but with varying potencies: benzo[a]pyrene, 17β-estradiol, lead acetate, methoxychlor, and phenanthrene. These five compounds have all previously been reported as immunosuppressive in mammalian innate immunity assays. To evaluate whether the suppression of ROS by these compounds was a result of decreased immune cell numbers, flow cytometry with transgenic zebrafish larvae was used to count the numbers of neutrophils and macrophages after chemical exposure. With this assay, benzo[a]pyrene was found to be the only chemical that induced a change in the number of immune cells by increasing macrophage but not neutrophil numbers. Taken together, this work demonstrates the utility of zebrafish larvae as a vertebrate model for identifying compounds that impact innate immune function at non-teratogenic levels and validates measuring ROS production and phagocyte numbers as metrics for monitoring how xenobiotic exposure alters the innate immune system.

Hyaluronan (HA) is a glycosaminoglycan that in its natural, high molecular mass (HMM) form, promotes tissue repair and homeostasis. With inflammation, HA metabolism and HMM HA fragmentation to low molecular mass (LMM) forms is greatly enhanced. Considerable evidence suggests that LMM HA may act as a damage-associated molecular pattern to initiate innate immune responses. However, the responsiveness of myeloid cells to LMM HA is controversial and largely unknown for neutrophils. Peripheral blood cells from healthy donors were incubated ex vivo with pharmaceutical grade HA of different molecular mass (HMM, LMM, and HA fragments <10 kDa). Key innate immune functions were assessed, namely production of cytokines and reactive oxygen species release (ROS), granule mobilization, and apoptosis. None of the tested sizes of HA altered cytokine production by PBMC and neutrophils. Also, HA had no effect on neutrophil granule mobilization and apoptosis. In contrast, HA primed neutrophils for rapid and robust release of ROS in response to a secondary stimulus (N-formyl-methionyl-leucyl phenylalanine). Priming occurred within 20 min of exposure to HA and was similar for all tested molecular mass. The observed effect was independent of granule mobilization and associated with the activation of intracellular signaling pathways involving Src family kinases, glycogen synthase kinase-3, and the proline-rich Akt substrate of 40 kDa. Our findings provide new evidence that HA, irrespective of molecular mass, is a specific priming agent of the neutrophil oxidative burst, which is a critical, early component of an innate immune response., (©2020 Society for Leukocyte Biology.)

Neutrophils are an essential cellular component of the innate immune system, responsible for multiple effector mechanisms and aspects of inflammation. Neutrophil priming results in a rapid elevation in antimicrobial activities and can be measured by reactive oxygen species production, bacterial endocytosis, and de-novo synthesis of components such as interleukins. Mannose binding lectin (MBL), a C-type lectin pathogen recognition receptor is associated with immune functions including complement activation, opsonization and modulating immune responses. Whether MBL opsonization of pathogen can induce neutrophil priming has not been studied so far. Hence, studies were performed using MBL and neutrophils of Capra hircus (domestic goat) to evaluate the effects of MBL + MASPs interactions on neutrophil functions. It was found that MBL + MASPs opsonization of zymosan stimulates neutrophil functions including increased oxidative burst, enhanced endocytosis and modulates the expression level of NCF4, XBP1, CCL2, and CR1 genes. The results suggest that MBL-MASP complex can regulate neutrophil functioning., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Background: Polymorphonuclear neutrophils (PMN) are the largest leukocyte population in the blood of most mammals including horses, and play an important defensive role in many infectious diseases. However, the mechanisms that increase PMN as one of the main cellular subsets in the defense against pathogens could also be involved in the pathophysiology of dysregulated inflammatory conditions. Mesenchymal stem/stromal cells (MSCs) are a heterogeneous population with a modulatory potential on the inflammatory response and are known to interact with nearly all cells of the immune system, including PMN. In this study, the in vitro modulation of equine bone marrow-derived MSCs on equine PMN phagocytosis, ROS production, and NETs generation was assessed., Results: In co-culture with MSCs, unstimulated PMN produce less ROS (2.88 % ± 1.43) than PMN in single culture (5.89 % ± 2.63) (p = 0.016). Moreover, PMN co-cultured with MSCs remain conditioned to produce fewer ROS after PMA stimulation in comparison to PMN in single culture (p < 0.05). Additionally, it was found that incubation with MSC supernatant strongly inhibited ROS production (83 % ± 6.35 less than control) without affecting phagocytosis or capacity for NETosis (p < 0.01)., Conclusions: These results suggest a modulatory effect of equine BM-derived MSCs on PMN respiratory burst, without impairing other important microbicidal functions. This supports the potential use of equine MSCs in excessive or persistent inflammatory conditions in which neutrophils are the main effector cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2019. Published by Elsevier B.V.)

Millions of people are infected with the liver fluke, Opisthorchis viverrini (OV), but only ~25% of those infected develop liver disease and even fewer develop cholangiocarcinoma. The reasons for these differential outcomes following infection are unknown but it has been proposed that differential immune responses to the parasite may play a role. We therefore measured granulocyte (neutrophil) function in OV-infected individuals, with and without advanced periductal fibrosis, to determine if these cells have a "pro-inflammatory" phenotype that may contribute to liver disease post-infection. A case-controlled study (n = 54 in each cohort) from endemic OV-infected areas of northeastern Thailand measured neutrophil functions in whole blood from non-infected (healthy controls) and OV-infected individuals with and without APF. We measured reactive oxygen species production, phagocytosis, receptor expression and apoptosis. Secreted products from OV cultures (obtained after in vitro culture of parasites) stimulated reactive oxygen species production in non-infected healthy controls, but levels were two-fold greater after OV infection (P < 0.0001); neutrophil reactive oxygen species production in individuals with APF was double that observed in those without APF (P < 0.0001). OV-infected neutrophils had elevated CD11b expression and greater phagocytic capacity, which was even three-fold higher in those with advanced periductal fibrosis (P < 0.0001). This "activated" phenotype of circulating neutrophils was further confirmed by the observation that isolated neutrophils had delayed apoptosis ex vivo. We believe this is the first study to show that circulating blood neutrophil function is enhanced following OV infection and is more activated in those with advanced periductal fibrosis. We propose that this activated phenotype could contribute to the pathology of liver disease. These data support the hypothesis of an activated innate inflammatory phenotype following OV infection and provide the first evidence for involvement of neutrophils in disease pathology., (Copyright © 2020 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Activation of professional phagocytes, potent microbial killers of our innate immune system, is associated with an increased cellular consumption of molecular oxygen (O 2 ). The O 2 molecules consumed are reduced by electrons delivered by a membrane localized NADPH-oxidase that initially generate one- and two electron reduced superoxide anions (O 2 - ) and hydrogen peroxide (H 2 O 2 ), respectively. These oxidants can then be processed into other highly reactive oxygen species (ROS) that can kill microbes, but that may also cause tissue destruction and drive other immune cells into apoptosis. The development of basic techniques to measure and quantify ROS generation by phagocytes is of great importance, and a large number of methods have been used for this purpose. A selection of methods (including chemiluminescence amplified by luminol or isoluminol, absorbance change following reduction of cytochrome c, and fluorescence increase upon oxidation of PHPA) are described in detail in this chapter with special emphasis on how to distinguish between ROS that are released extracellularly, and those that are retained within intracellular organelles. These techniques can be valuable tools in research spanning from basic phagocyte biology to diagnosis of diseases linked to the NADPH-oxidase and more clinically oriented research on innate immune mechanisms and inflammation.

Two critical functional responses of neutrophils are chemotaxis, a response driven by concentration gradients of chemokines released by infected or inflamed tissues, and production of reactive oxygen species (ROS), molecules essential to their capacity to kill pathogens. Assays to accurately test each response have been important to assess efficacies of pharmaceuticals predicted to block recruitment of neutrophils or attenuate their ROS production. Identified antagonists to neutrophil functions may help to reduce tissue damage following inflammation. Described are detailed assays to test these functions, along with steps to generate neutrophils from ex vivo-cultured murine bone marrow that produce robust responses in either assay. The first function protocol details a quantitative assay for chemotaxis that involves culture plates with dual chamber wells that separate cells from a chemokine with small pore-sized membranes. Quantitative measurements of cell numbers in the chemokine-containing chamber are performed with either fluorescence or luminescence detection reagents, which provide signals directly proportional to the numbers of migrated cells. Multiwell plates are used for rapidly testing a variety of conditions and/or chemoattractants. Described in the second function protocol is an assay to measure ROS produced by stimulated neutrophils, again using a multiwell platform for rapid, quantitative measurements of several conditions simultaneously.

(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC 50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC 50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future., Competing Interests: The authors declare no conflict of interest.

Neutrophils contribute to low oxygen availability at inflammatory sites through the generation of reactive oxidants. They are also functionally affected by hypoxia, which delays neutrophil apoptosis. However, the eventual fate of neutrophils in hypoxic conditions is unknown and this is important for their effective clearance and the resolution of inflammation. We have monitored the survival and function of normal human neutrophils exposed to hypoxia over a 48 h period. Apoptosis was delayed, and the cells remained intact even at 48 h. However, hypoxia promoted significant changes in neutrophil morphology with the appearance of many new cytoplasmic vesicles, often containing cell material, within 5 hours of exposure to low O 2 . This coincided with an increase in LC3B-II expression, indicative of autophagosome formation and an autophagy-like process. In hypoxic conditions, neutrophils preferentially lost myeloperoxidase, a marker of azurophil granules. Short-term (2 h) hypoxic exposure resulted in sustained potential to generate superoxide when O 2 was restored, but the capacity for oxidant production was lost with longer periods of hypoxia. Phagocytic ability was unchanged by hypoxia, and bacterial killing by neutrophils in both normoxic and hypoxic conditions was substantially diminished after 24 hours. However, pre-exposure to hypoxia resulted in an enhanced ability to kill bacteria by oxidant-independent mechanisms. Our data provide the first evidence for hypoxia as a driver of neutrophil autophagy that can influence the function and ultimate fate of these cells, including their eventual clearance and the resolution of inflammation., (©2019 Society for Leukocyte Biology.)

Neutrophils are the most abundant innate immune cells. The pathogenic roles of neutrophils are related to chronic inflammation and autoimmune diseases. Psoriasis is a chronic systemic inflammatory disease affecting ~2-3% of the world population. The abundant presence of neutrophils in the psoriatic skin lesions serves as a typical histopathologic hallmark of psoriasis. Recent reports indicated that oxidative stress, granular components, and neutrophil extracellular traps from psoriatic neutrophils are related to the initial and maintenance phases of psoriasis. This review provides an overview on the recent (up to 2019) advances in understanding the role of neutrophils in the pathophysiology of psoriasis, including the effects of respiratory burst, degranulation, and neutrophil extracellular trap formation on psoriatic immunity and the clinical relationships., (Copyright © 2019 Chiang, Cheng, Korinek, Lin and Hwang.)

Aim: To study the role of complement receptor 1 (CR1) for binding of Escherichia coli (E. coli) to erythrocytes, for leukocyte phagocytosis, oxidative burst and complement activation in human whole blood from a CR1 deficient (CR1D) patient and healthy controls with low, medium and high CR1 numbers., Methods: Alexa-labelled bacteria were used to quantify erythrocyte-bound bacteria, free bacteria in plasma and phagocytosis using flow cytometry. Complement activation in plasma was measured by enzyme-linked immunosorbent assay. The CR1 numbers as well as C3bc and C4bc deposition on erythrocytes were measured by flow cytometry. Cytokines were measured using multiplex technology, and bacterial growth was measured by colony forming units. CR1 was blocked using the anti-CR1 blocking mAb 3D9., Results: Approximately 85% of E. coli bound to erythrocytes after 15 min incubation in donor blood with high and medium CR1 numbers, 50% in the person with low CR1 numbers and virtually no detectable binding in the CR1D (r 2  = 0.87, P < 0.0007). The number of free bacteria in plasma was inversely related to erythrocyte CR1 numbers (r 2  = 0.98, P < 0.0001). E. coli-induced phagocytosis and oxidative burst were significantly enhanced by the anti-CR1 mAb 3D9 and in the CR1D and the donor with low CR1 numbers. E. coli-induced complement activation in plasma, C3bc and C4bc deposition on erythrocytes, and bacterial growth were similar in all four cases., Conclusions: CR1D and low CR1 numbers prevented E. coli binding to erythrocytes, increased free bacteria in plasma, phagocytosis and oxidative burst, but did not affect plasma or surface complement activation and bacterial growth., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Innovative nanotechnology aims to develop particles that are small, monodisperse, smart, and do not cause unintentional side effects. Uniform magnetic Fe 3 O 4 nanoparticles (12 nm in size) were prepared by thermal decomposition of iron(III) oleate. To make them colloidally stable and dispersible in water and cell culture medium, they were modified with phosphonic acid- (PA) and hydroxamic acid (HA)-terminated poly(ethylene glycol) yielding PA-PEG@Fe 3 O 4 and HA-PEG@Fe 3 O 4 nanoparticles; conventional γ-Fe 2 O 3 particles were prepared as a control. Advanced techniques were used to evaluate the properties and safety of the particles. Completeness of the nanoparticle coating was tested by real-time polymerase chain reaction. Interaction of the particles with primary human peripheral blood cells, cellular uptake, cytotoxicity, and immunotoxicity were also investigated. Amount of internalized iron in peripheral blood mononuclear cells was 72, 38, and 25 pg Fe/cell for HA-PEG@Fe 3 O 4 , γ-Fe 2 O 3 , and PA-PEG@Fe 3 O 4 , respectively. Nanoparticles were localized within the cytoplasm and in the extracellular space. No cytotoxic effect of both PEGylated nanoparticles was observed (0.12-75 μg/cm 2 ) after 24 and 72-h incubation. Moreover, no suppressive effect was found on the proliferative activity of T-lymphocytes and T-dependent B-cell response, phagocytic activity of monocytes and granulocytes, and respiratory burst of phagocytes. Similarly, no cytotoxic effect of γ-Fe 2 O 3 particles was observed. However, they suppressed the proliferative activity of T-lymphocytes (75 μg/cm 2 , 72 h) and also decreased the phagocytic activity of monocytes (15 μg/cm 2 , 24 h; 3-75 μg/cm 2 , 72 h). We thus show that newly developed particles have great potential especially in cancer diagnostics and therapy.

The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

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., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Neutrophils are recruited to the airways of patients with acute respiratory distress syndrome (ARDS) where they acquire an activated pro-survival phenotype with an enhanced respiratory burst thought to contribute to ARDS pathophysiology. Our in vitro model enables blood neutrophil transepithelial migration into cell-free tracheal aspirate fluid from patients to recapitulate the primary airway neutrophil phenotype observed in vivo. Neutrophils transmigrated through our model toward airway fluid from children with lower respiratory viral infections coinfected with bacteria had elevated levels of neutrophil activation markers but paradoxically exhibited an inability to kill bacteria and a defective respiratory burst compared with children without bacterial coinfection. The airway fluid from children with bacterial coinfections had higher levels of neutrophil elastase activity, as well as myeloperoxidase levels compared to children without bacterial coinfection. Neutrophils transmigrated into the aspirate fluid from children with bacterial coinfection showed decreased respiratory burst and killing activity against H. influenzae and S. aureus compared to those transmigrated into the aspirate fluid from children without bacterial coinfection. Use of a novel transmigration model recapitulates this pathological phenotype in vitro that would otherwise be impossible in a patient, opening avenues for future mechanistic and therapeutic research.

The sea turtles maintaining in rescue centres can cause physiological stress to the animals with subsequent effects, such as the imbalance of immune system components. It is therefore crucial to investigate how innate immune responses are influenced by stress within recovery centers with the aim to use them as primary tools for the evaluation of the rescued specimens' health status and for developing more effective conservative strategies. In this study we monitored for the first time different immune responses in hospitalized Mediterranean loggerhead sea turtles (Caretta caretta) (n = 88), comparing them with free-ranging animals (n = 11). The final scope was to identify sensitive tools based on immune responses parameters that rescue centers operators could use to verify the stress condition of hospitalized specimens. Blood samples were obtained from loggerhead sea turtles hospidalized for different periods (≤2 months; >2 months and ≤1 year; >1 year) in various Italian rescue centers. Free-ranging turtles were captured in the South of Spain during a shipboard survey with a minimal invasive technique. Samples were analyzed for different stress-associated parameters (white cells count, heterophils:lymphocytes (H:L) ratio, respiratory burst, total antioxidant status (TAS), lysozyme). Free-ranging specimens showed lower values for most of the measured parameters. The highest values of TAS, lysozyme activity, respiratory burst and leukocytes profile were detected during the first 2 months of hospitalization, that resulted as the most critical period for the rehabilitation of turtles. After more than 1 year, immune values were similar to the values of free-ranging, indicating an acclimatization to captivity and health conditions amelioration. Moreover, monocytes low levels and eosinophils significant decrease in hospitalized animals indicated the absence of chronic inflammations and reduction of parasitic load during rehabilitation. Statistical analysis pointed out that lysozyme activity and eosinophyls count represent valid methods to diagnose physiological stress and inflammation in hospitalized loggerhead sea turtles., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The phagocyte NADPH oxidase NOX2 was the first NOX family member to be discovered. It is responsible for the production of reactive oxygen species that are required for bacterial killing and host defense. Activated NOX2 is an enzymatic complex composed of two membrane proteins, p22 phox and gp91 phox (renamed NOX2), which form the cytochrome b 558 , and four cytosolic proteins, p47 phox , p67 phox , p40 phox , and the small GTPase Rac2. Except for Rac2, all proteins from the complex become phosphorylated during neutrophil activation, suggesting the importance of this process in NOX2 regulation. The phosphorylation of the cytosolic components, and in particular p47phox, has been extensively studied; however, the phosphorylation of the membrane proteins was less studied, in part due to the lack of good antibodies and accurate membrane solubilization techniques. In this chapter, we describe a method we have used to study NOX2 phosphorylation, which is based on the labeling of the intracellular ATP pool with 32 P prior to applying a stimulus inducing protein phosphorylation. We also describe the solubilization of membrane-bound gp91 phox /NOX2 and analysis by immunoprecipitation, polyacrylamide gel electrophoresis, electrophoretic transfer, phosphoamino acid analysis, and autoradiography. This protocol can also be used to study the possible phosphorylation of other NOX family members.

Although it is well-recognized that inflammation enhances leukocyte bactericidal activity, the underlying mechanisms are not clear. Here we report that PRL2 is sensitive to oxidative stress at inflamed sites. Reduced PRL2 in phagocytes causes increased respiratory burst activity and enhances phagocyte bactericidal activity. PRL2 (Phosphatase Regenerating Liver 2) is highly expressed in resting immune cells, but is markedly downregulated by inflammation. in vitro experiments showed that PRL2 was sensitive to hydrogen peroxide (H 2 O 2 ), a common damage signal at inflamed sites. In response to infection, PRL2 knockout (KO) phagocytes were hyper activated, produced more reactive oxygen species (ROS) and exhibited enhanced bactericidal activity. Mice with PRL2 deficiency in the myeloid cell compartment were resistant to lethal listeria infection and cleared the bacteria more rapidly and effectively. Moreover, in vitro experiments demonstrated that PRL2 binds to GTPase Rac and regulates ROS production. Rac GTPases were more active in PRL2 (KO) phagocytes than in wild type cells after bacterium infection. Our findings indicate that PRL2 senses ROS at inflamed sites and regulates ROS production in phagocytes. This positive feedback mechanism promotes bactericidal activity of phagocytes and may play an important role in innate anti-bacterial immunity.

Background: Haemophilus influenzae is found in the nasopharynx of 80% of the human population. While colonisation with non-typeable Haemophilus influenzae (NTHi) is usually asymptomatic, it is capable of causing acute and chronic otitis media (OM) in infants, invasive disease in susceptible groups and is the leading cause of exacerbations of patients with chronic obstructive pulmonary disease (COPD). Current methods for assessing functional antibody immunity to NTHi are limited and labour intensive. Flow cytometric assays could provide an attractive alternative to evaluate immune responses to candidate vaccines in clinical trials., Results: We have developed a duplexed flow-cytometric uptake and oxidative burst opsonophagocytosis assay (fOPA). We have also developed a duplexed antibody-mediated complement C3b/iC3b and C5b-9 deposition assay (CDA). Antibody-mediated C3b/iC3b deposition correlated with opsonophagocytic uptake (r = 0.65) and with opsonophagocytic oxidative burst (r = 0.69). Both fOPA and CDA were reproducible, with the majority of samples giving a coefficient of variation (CV) of < 20% and overall assay CVs of 14% and 16% respectively., Conclusions: The high-throughput flow cytometric assays developed here were successfully optimised for use with NTHi. Assays proved to be sensitive and highly reproducible for the measurement of bacterial uptake and oxidative burst opsonophagocytosis and antibody-mediated deposition of C3b/iC3b and C5b-9. These assays are useful tools for use in large scale epidemiological studies and to assist in the assessment of functional antibody induced by NTHi candidate vaccines.

Molecular oxygen is a necessary compound for all aerobic organisms, although oxygen is a potent oxidant, which can cause oxidative stress (OS). OS occurs when there is an imbalance between the production of oxidant and antioxidants components, are result of normal cell metabolism, and many of these compounds play a fundamental role in several metabolic pathways. The organism produces several reactive oxygen species (ROS), but they are balanced by an antioxidant defense system that maintains the levels of these oxidizing compounds at an acceptable level. Many of these components are essential in the organism defense and their byproducts are considered potent bactericides that actively act in the destruction of invading pathogens. Fish immune system is composed of innate and acquired mechanisms of defense. Phagocytosis is an innate process of defense, which interconnects these two systems, since the pathogens processing by professional phagocytes is a fundamental stage for antibodies production. During phagocytosis there is production of ROS and consequent production of free radicals (FR), these compounds lead to the formation of potent bactericides to combat microorganisms. However, it is known that OS limits the immune response, with an impairment in defense compounds in an attempt to decrease the ROS production. Studies of fish FR production are preliminary and should be executed to evaluate the effects of ROS on fish, including their beneficial action against pathogens and its deleterious action on the oxidation of cellular components.

The current study was conducted to investigate the effect of ExcelMOS® in enhancing the immune system of Sparus aurata broodstock and their impact on offspring health through displaying the maternal transfer of immunity. Broodstock were divided into two groups: one was injected intraperitoneally with ExcelMOS® 1 month before spawning, while the other group was used as a control (without injection). Comprehensive increase in survival rate was observed for larvae hatched from ExcelMOS®-injected broodstock than those of the control (P ≤ 0.05). Hematological analysis showed increases in leukocyte count and hematocrit percentage (P ≤ 0.05) and significant enhancement in immune assays as phagocytic, respiratory burst, lysozyme activities in ExcelMOS®-injected broodstock (P ≤ 0.05). Additionally, total immunoglobulin levels in the serum, eggs, and larvae resulted from ExcelMOS®-injected broodstock were highly significant (P ≤ 0.05) than those in the control ones. Transmission electron microscopy and semi-thin sections in posterior intestine of ExcelMOS®-injected broodstock revealed reinforcement of the epithelial barrier structure, intestinal integrity, and functionality in combination with the stimulation of innate immune system. In conclusion, immunostimulation of Sparus aurata broodstock using ExcelMOS® has improved survival of larvae and enhanced both innate and adaptive immune defense mechanisms. Further investigations are required to show the effect of ExcelMOS® on fish cultured in intensive culture systems.

Background and Aim: Due to a dysregulated immune response, patients with acute-on-chronic liver failure (ACLF) have increased risk of infection and multi organ failure in comparison to compensated cirrhosis. The comparative data on the presence of 'immune paresis' in patients with ACLF and decompensated cirrhosis without ACLF is not available. Aim of the present study was to compare the immunological parameters in patients with decompensated cirrhosis with and without ACLF., Methodology: In a prospective study, 76 patients with decompensated cirrhosis with (n = 38) and without (n = 38) ACLF and 10 healthy controls (HC) were evaluated for monocytic human leukocyte antigen-antigen D Related (HLA-DR) expression, mean density of HLA-DR expressed on the surface of these cells, neutrophil oxidative burst (NOB) capacity and serum levels of cytokines (IL-1, IL-6, IL-8, IL10, IL-12, and TNF-α)., Results: Patients of decompensated cirrhosis with and without ACLF demonstrated significantly lower mean percentage of monocytes expressing HLA-DR and quantitative increase in the NOB after stimulation with PMA when compared to HC. However there was no difference in mean percentage of monocytes with HLA-DR expression (43.61±26.56% vs. 43.10±20.98%) (p = 0.91), mean density of HLA-DR expression on the surface (30.34±29.32 vs. 41.71±52.13) (p = 0.42) and quantitative increase in NOB after stimulation with PMA (16.55±11.91 vs. 17.24±16.18) (p = 0.47) amongst patients with decompensated cirrhosis with and without ACLF. Patients with ACLF had significantly higher pro-inflammatory and anti-inflammatory cytokines in comparison to patients with decompensated cirrhosis without ACLF., Conclusion: Patients with decompensated cirrhosis demonstrate a component of immune-paresis, however there is similar impairment in HLA-DR expression and NOB capacity in patients with and without ACLF. Both inflammatory and anti-inflammatory cytokines are increased in patients with ACLF in comparison to patients with decompensated cirrhosis without ACLF., Competing Interests: The authors have declared that no competing interests exist.

Recognition of the fungal cell wall carbohydrate β-glucan by the host receptor Dectin-1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal β-glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat-killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin-dependent phagocytosis of particulate (1-3,1-6)-β-glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal β-glucan particles upon actin cytoskeleton disruption, relies on Dectin-1 and results in the classical caspase-1 inflammasome formation through NLRP3, generation of an oxidative burst, NF-κB activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose of Review: Hemolytic anemias caused by premature destruction of red blood cells occur in many disorders including hemoglobinopathies, autoimmune conditions, during infection or following reaction to drugs or transfusions. Recent studies which will be reviewed here have uncovered several novel mechanisms by which hemolysis can alter immunological functions and increase the risk of severe complications in hemolytic disorders., Recent Findings: Plasma-free heme can induce the formation of neutrophil extracellular traps (NETs) through reactive oxygen species signaling. Although NETs protect the host against infections, in patients with sickle disease, they are associated with vaso-occlusive crises. Heme may increase host susceptibility to infections by inducing heme oxygenase 1 (HO-1) in immature neutrophils, thereby inhibiting oxidative burst required for clearance of engulfed bacteria. In addition, heme impairs macrophage phagocytosis and microbial clearance through inhibition of cytoskeletal remodeling. Hemolysis can also favor anti-inflammatory immune cell polarization by inhibiting dendritic cell maturation necessary for effector T-cell responses, inducing differentiation of monocytes into red pulp macrophages, important for iron recycling from senescent erythrocytes, and driving regulatory T-cell expansion through modulation of HO-1 expression in nonclassical monocytes., Summary: Hemolysis breakdown products show remarkable effects on the regulation of immune cell differentiation and function.

Inbred mouse strains differ in their susceptibility to infection with the facultative intracellular bacterium Listeria monocytogenes , largely due to delayed or deficient innate immune responses. Previous antibody depletion studies suggested that neutrophils (polymorphonuclear leukocytes [PMN]) were particularly important for clearance in the liver, but the ability of PMN from susceptible and resistant mice to directly kill L. monocytogenes has not been examined. In this study, we showed that PMN infiltrated the livers of BALB/c/By/J (BALB/c) and C57BL/6 (B6) mice in similar numbers and that both cell types readily migrated toward leukotriene B4 in an in vitro chemotaxis assay. However, CFU burdens in the liver were significantly higher in BALB/c mice than in other strains, suggesting that PMN in the BALB/c liver might not be able to clear L. monocytogenes as efficiently as B6 PMN. Unprimed PMN harvested from either BALB/c or B6 bone marrow killed L. monocytogenes directly ex vivo , and pretreatment with autologous serum significantly enhanced killing efficiency for both. L. monocytogenes were internalized within 10 min and rapidly triggered intracellular production of reactive oxygen species in a dose-dependent manner. However, PMN from gp91 phox -deficient mice also readily killed L. monocytogenes , which suggested that nonoxidative killing mechanisms may be sufficient for bacterial clearance. Together, these results indicate that there is not an intrinsic defect in the ability of PMN from susceptible BALB/c mice to kill L. monocytogenes and further suggest that if PMN function is impaired in BALB/c mice, it is likely due to locally produced modulating factors present in the liver during infection., (Copyright © 2018 American Society for Microbiology.)

A prolonged stress burden is known to hamper the efficiency of both the innate and the adaptive immune systems and to attenuate the stress responses by the catecholaminergic and endocannabinoid (EC) systems. Key mechanisms of innate immunity are the eradication of pathogens through phagocytosis and the respiratory burst. We tested the concentration-dependent, spontaneous and stimulated (via TNFα and N-formylmethionine-leucyl-phenylalanine) release of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMNs) in vitro in response to norepinephrine (NE) and AM1241, a pharmacological ligand for the EC receptor CB2. We evaluated phagocytosis of Dectin-1 ligating zymosan particles and tested the cytokine response against Candida antigen in an in vitro cytokine release assay. Increasing concentrations of NE did not affect phagocytosis, yet stimulated ROS release was attenuated gradually reaching maximum suppression at 500 nM. Adrenergic receptor (AR) mechanisms using non-AR-selective (labetalol) as well as specific α-(prazosin) and β-(propranolol) receptor antagonists were tested. Results show that only labetalol and propranolol were able to recuperate cytotoxicity in the presence of NE, evidencing a β-receptor-mediated effect. The CB2 agonist, AM1241, inhibited phagocytosis at 10 µM and spontaneous peroxide release by PMNs. Use of the inverse CB2 receptor agonist SR144528 led to partial recuperation of ROS production, confirming the functional role of CB2. Additionally, AM1241 delayed early activation of monocytes and induced suppression of IL-2 and IL-6 levels in response to Candida via lower activity of mammalian target of rapamycin (mTOR). These findings provide new insights into key mechanisms of innate immunity under stressful conditions where ligands to the sympatho-adrenergic and EC system are released.

Despite advances, identification and formulation of safe and effective vaccine for long-lasting protection against leishmaniasis is still inadequate. In this study, we have identified a novel antigen, leishmanial elongation factor-1α (EF1-α), as an immunodominant component of solubilized leishmanial membrane antigens that reacts with visceral leishmaniasis (VL) sera and induces cellular proliferative and cytokine response in PBMCs of cured VL subjects. Leishmanial EF1-α is a 50 kDa antigen that plays a crucial role in pathogen survival by regulating oxidative burst in the host phagocytes. Previously, immunodominant truncated forms of EF1-α from different species of Leishmania have been reported. Formulation of the L. donovani 36 kDa truncated as well as the cloned recombinant EF1-α in cationic liposomes induce strong resistance to parasitic burden in liver and spleen of BALB/c mice through induction of DTH and a IL-10 and TGF-β suppressed mixed Th1/Th2 cytokine responses. Multiparametric analysis of splenocytes for generation of antigen-specific IFN-γ, IL2, and TNF-α producing lymphocytes indicate that cationic liposome facilitates expansion of both CD4 + as well as CD8 + memory and effector T cells. Liposomal EF1-α is a novel and potent vaccine formulation against VL that imparts long-term protective responses. Moreover, the flexibility of this formulation opens up the scope to combine additional adjuvants and epitope selected antigens for use in other disease forms also.

Background: People with critical illness (CI) commonly develop various forms of immune dysfunction, however, there is limited information concerning immune dysfunction in dogs with CI., Hypothesis: The immune response in CI dogs differs from that of healthy dogs., Animals: Immunologic variables were compared between 14 dogs with CI, defined as APPLE fast score of >20 points, admitted to the University of Missouri Veterinary Health Center Small Animal Clinic Intensive Care Unit and healthy controls (n = 15)., Methods: Cohort study evaluating constitutive and lipopolysaccharide (LPS)-stimulated TNF-α, IL-6, and IL-10 production, phagocytosis of opsonized E. coli and respiratory burst capacity after opsonized E. coli or phorbol 12-myristate 13-acetate (PMA) stimulation, peripheral blood lymphocyte phenotype, and monocyte expressions of HLA-DR and TLR-4., Results: Lipopolysaccharide-stimulated leukocyte TNF-α (median, Q1, Q3; CI, 49, 49, 120; control, 655, 446, 1174 pg/mL; P = < 0.001), IL-6 (median, Q1, Q3; CI, 49, 49, 64; control, 100, 49, 166 pg/mL; P = 0.029), and IL-10 (CI, 49, 49, 56; control, 96, 49, 203 pg/mL; P = 0.014) production and both E. coli (median, Q1, Q3; CI, 60.5, 43, 88.5; control, 86.6, 81, 89.2%; P = 0.047) and PMA (CI, 40, 11.7, 70; control, 93, 83, 97.6%; P = < 0.001)-stimulated respiratory burst capacity significantly decreased in CI dogs. Percentage of monocytes expressing TLR-4 greater in the CI dogs (median, Q1, Q3; CI, 46.9, 24.3, 64.2; control, 16.4, 9.4, 26.2%; P = 0.005)., Conclusion: These findings suggest dogs with CI develop immune system alterations that result in reduced respiratory burst function and cytokine production despite upregulation of TLR-4., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

The importance of metabolism in macrophage function has been reported, but the in vivo relevance of the in vitro observations is still unclear. Here we show that macrophage metabolites are defined in a specific tissue context, and these metabolites are crucially linked to tissue-resident macrophage functions. We find the peritoneum to be rich in glutamate, a glutaminolysis-fuel that is exploited by peritoneal-resident macrophages to maintain respiratory burst during phagocytosis via enhancing mitochondrial complex-II metabolism. This niche-supported, inducible mitochondrial function is dependent on protein kinase C activity, and is required to fine-tune the cytokine responses that control inflammation. In addition, we find that peritoneal-resident macrophage mitochondria are recruited to phagosomes and produce mitochondrially derived reactive oxygen species, which are necessary for microbial killing. We propose that tissue-resident macrophages are metabolically poised in situ to protect and exploit their tissue-niche by utilising locally available fuels to implement specific metabolic programmes upon microbial sensing.

Identification of parasite antigens targeted by immune effector mechanisms that confer protection against malaria is important for the design of a multi-component malaria vaccine. Here, the association of antibodies reacting with the Plasmodium falciparum merozoite surface protein-4 (MSP4) with protection against clinical malaria was investigated in a Senegalese community living in an area of moderate, seasonal malaria transmission. Blood samples were collected at the end of an 8-month long dry season without any recorded parasite transmission from 206 residents enrolled in a prospective follow-up study. Active daily clinical monitoring was implemented during the subsequent five months. Entomologic monitoring documented parasite transmission during the first three months of follow-up. Serum IgG levels were determined by ELISA against three MSP4 baculovirus-encoded recombinant protein constructs, namely the full-length MSP4p40, MSP4p30 devoid of a highly polymorphic sequence stretch and the conserved C-terminal EGF-containing MSP4p20, as well as against a merozoite crude extract. Community seroprevalence against all three constructs was quite high, the lowest being against MSP4p30. Seroprevalence and antibody levels against the three MSP4 constructs were age-dependent. IgG1 dominated the anti-MSP4p20 responses, while both IgG1 and IgG3 were observed against MSP4p40. Anti-MSP4 antibodies were associated with the antibody-dependent respiratory burst (ADRB) activity in a functional assay of merozoite phagocytosis by polymorphonuclear cells. Importantly, high antibody levels against each of the three MSP4 constructs at the end of the dry season were associated with reduced morbidity during the subsequent transmission season in an age-adjusted Poisson regression model (IRR = 0.65 [0.50-0.83], P<0.001 for responses over the median values). These data are consistent with a protective role for the naturally acquired anti-MSP4 antibodies and support further development of MSP4 as a candidate component of malaria vaccine., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Microorganisms are known to devise various strategies to thwart protective responses by the host. One such strategy is to incorporate sequences and domains in their genes/proteins that have similarity to various domains of the host proteins. In this study, we report that Mycobacterium tuberculosis protein Rv3529c exhibits significant similarity to the death domain of the TLR pathway adaptor protein MyD88. Incubation of macrophages with Rv3529c specifically inhibited TLR2-mediated proinflammatory responses. This included attenuated oxidative burst, reduced phosphorylation of MAPK-ERK, reduced activation of transcription factor NF-κB and reduced secretion of proinflammatory cytokines IFN-γ, IL-6, and IL-17A with a concomitant increased secretion of suppressor cytokines IL-10 and TGF-β. Importantly, Rv3529c significantly inhibited TLR2-induced association of MyD88 with IRAK1 by competitively binding with IRAK1. Further, Rv3529c mediated inhibition of apoptosis and phagosome-lysosome fusion. Lastly, incubation of macrophages with Rv3529c increased bacterial burden inside macrophages. The data presented show another strategy evolved by M. tuberculosis toward immune evasion that centers on incorporating sequences in proteins that are similar to crucial proteins in the innate immune system of the host., (© Society for Leukocyte Biology.)

Objective: Trauma induces a complex immune response that requires a systems biology research approach. Here, we used a novel technology, mass cytometry by time-of-flight, to comprehensively characterize the multicellular response to trauma., Design: Peripheral blood mononuclear cells samples were stained with a 38-marker immunophenotyping cytometry by time-of-flight panel. Separately, matched peripheral blood mononuclear cells were stimulated in vitro with heat-killed Streptococcus pneumoniae or CD3/CD28 antibodies and stained with a 38-marker cytokine panel. Monocytes were studied for phagocytosis and oxidative burst., Setting: Single-institution level 1 trauma center., Patients or Subjects: Trauma patients with injury severity scores greater than 20 (n = 10) at days 1, 3, and 5 after injury, and age- and gender-matched controls., Interventions: None., Measurements and Main Results: Trauma-induced expansion of Th17-type CD4 T cells was seen with increased expression of interleukin-17 and interleukin-22 by day 5 after injury. Natural killer cells showed reduced T-bet expression at day 1 with an associated decrease in tumor necrosis factor-β, interferon-γ, and monocyte chemoattractant protein-1. Monocytes showed robust expansion following trauma but displayed decreased stimulated proinflammatory cytokine production and significantly reduced human leukocyte antigen - antigen D related expression. Further analysis of trauma-induced monocytes indicated that phagocytosis was no different from controls. However, monocyte oxidative burst after stimulation increased significantly after injury., Conclusions: Using cytometry by time-of-flight, we were able to identify several major time-dependent phenotypic changes in blood immune cell subsets that occur following trauma, including induction of Th17-type CD4 T cells, reduced T-bet expression by natural killer cells, and expansion of blood monocytes with less proinflammatory cytokine response to bacterial stimulation and less human leukocyte antigen - antigen D related. We hypothesized that monocyte function might be suppressed after injury. However, monocyte phagocytosis was normal and oxidative burst was augmented, suggesting that their innate antimicrobial functions were preserved. Future studies will better characterize the cell subsets identified as being significantly altered by trauma using cytometry by time-of-flight, RNAseq technology, and functional studies.

Reactive oxygen species (ROS) generated by NADPH oxidase play an important role in antimicrobial host defense and inflammation. Their deficiency in humans results in recurrent and severe bacterial infections, while their unregulated release leads to pathology from excessive inflammation. The release of high concentrations of ROS aids in clearance of invading bacteria. Localization of ROS release to phagosomes containing pathogens limits tissue damage. Host immune cells, like neutrophils, also known as PMNs, will release large amounts of ROS at the site of infection following the activation of surface receptors. The binding of ligands to G-protein-coupled receptors (GPCRs), toll-like receptors, and cytokine receptors can prime PMNs for a more robust response if additional signals are encountered. Meanwhile, activation of Fc and integrin directly induces high levels of ROS production. Additionally, GPCRs that bind to the bacterial-peptide analog fMLP, a neutrophil chemoattractant, can both prime cells and trigger low levels of ROS production. Engagement of these receptors initiates intracellular signaling pathways, resulting in activation of downstream effector proteins, assembly of the NADPH oxidase complex, and ultimately, the production of ROS by this complex. Within PMNs, ROS released by the NADPH oxidase complex can activate granular proteases and induce the formation of neutrophil extracellular traps (NETs). Additionally, ROS can cross the membranes of bacterial pathogens and damage their nucleic acids, proteins, and cell membranes. Consequently, in order to establish infections, bacterial pathogens employ various strategies to prevent restriction by PMN-derived ROS or downstream consequences of ROS production. Some pathogens are able to directly prevent the oxidative burst of phagocytes using secreted effector proteins or toxins that interfere with translocation of the NADPH oxidase complex or signaling pathways needed for its activation. Nonetheless, these pathogens often rely on repair and detoxifying proteins in addition to these secreted effectors and toxins in order to resist mammalian sources of ROS. This suggests that pathogens have both intrinsic and extrinsic mechanisms to avoid restriction by PMN-derived ROS. Here, we review mechanisms of oxidative burst in PMNs in response to bacterial infections, as well as the mechanisms by which bacterial pathogens thwart restriction by ROS to survive under conditions of oxidative stress.

Parasites can be a major cause of natural selection on hosts, which consequently evolve a variety of strategies to avoid, eliminate, or tolerate infection. When ecologically similar host populations present disparate infection loads, this natural variation can reveal immunological strategies underlying adaptation to infection and population divergence. For instance, the tapeworm Schistocephalus solidus persistently infects 0-80% of threespine stickleback ( Gasterosteus aculeatus ) in lakes on Vancouver Island. To test whether these heterogeneous infection rates result from evolved differences in immunity, we experimentally exposed laboratory-reared fish from ecologically similar high-infection and no-infection populations to controlled doses of Schistocephalus We observed heritable between-population differences in several immune traits: Fish from the naturally uninfected population initiated a stronger granulocyte response to Schistocephalus infection, and their granulocytes constitutively generate threefold more reactive oxygen species in cell culture. Despite these immunological differences, Schistocephalus was equally successful at establishing initial infections in both host populations. However, the no-infection fish dramatically suppressed tapeworm growth relative to high-infection fish, and parasite size was intermediate in F1 hybrid hosts. Our results show that stickleback recently evolved heritable variation in their capacity to suppress helminth growth by two orders of magnitude. Data from many natural populations indicate that growth suppression is widespread but not universal and, when present, is associated with reduced infection prevalence. Host suppression of helminth somatic growth may be an important immune strategy that aids in parasite clearance or in mitigating the fitness costs of persistent infection., Competing Interests: The authors declare no conflict of interest.

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that promotes inflammatory cytokine production by activating the MEK/ERK pathway. Tpl2 has been shown to be important for eliciting the inflammatory properties of macrophages; however, there is relatively little known about the contribution of Tpl2 to neutrophil effector functions. This is an important consideration, as neutrophils provide the first line of defense against infection in the innate immune system. We found that Tpl2 is expressed in both human and murine neutrophils, suggesting a potential function for Tpl2 in this lineage. Despite significantly higher proportions of bone marrow (BM) neutrophils in Tpl2-deficient ( Tpl2 -/- ) mice compared with wild-type (WT) mice, Tpl2 -/- mice have significantly reduced proportions of circulating neutrophils. Tpl2 -/- neutrophils show impaired recruitment to thioglycollate, which was primarily a result of neutrophil-extrinsic factors in the host. In response to infection, neutrophils secrete inflammatory cytokines and produce reactive oxygen species (ROS), which promote bacterial killing. Tpl2 ablation impaired neutrophil TNF secretion in response to LPS stimulation, superoxide generation in response to the chemotactic peptide fMLP, and killing of the extracellular bacterium, Citrobacter rodentium , despite normal bacterial phagocytosis. These results implicate Tpl2 in the regulation of multiple neutrophil antimicrobial pathways, including inflammatory cytokine secretion and oxidative burst. Furthermore, they indicate that Tpl2 functions early during infection to bolster neutrophil-mediated innate immunity against extracellular bacteria., (© Society for Leukocyte Biology.)

Exposure to pro-inflammatory cytokines, chemokines, mitochondrial contents, and bacterial and viral products induces neutrophils to transition from a basal state into a primed one, which is currently defined as an enhanced response to activating stimuli. Although, typically associated with enhanced generation of reactive oxygen species (ROS) by the NADPH oxidase, primed neutrophils show enhanced responsiveness of exocytosis, NET formation, and chemotaxis. Phenotypic changes associated with priming also include activation of a subset of functions, including adhesion, transcription, metabolism, and rate of apoptosis. This review summarizes the breadth of phenotypic changes associated with priming and reviews current knowledge of the molecular mechanisms behind those changes. We conclude that the current definition of priming is too restrictive. Priming represents a combination of enhanced responsiveness and activated functions that regulate both adaptive and innate immune responses.

Aim: Irisin, the adipomyokine, released mainly by exercised muscles, participate in the browning of adipose tissue and contribute to the restriction of insulin resistance and diabetes mellitus 2 development. Because of the limited reports describing the effect of irisin on inflammation and immunocompetent cells activation, the present study attempted to assess the influence of various irisin concentrations on basic macrophage activity., Main Methods: Studies were carried out on murine RAW 264.7 macrophages cultured in medium enriched with irisin (0nM, 10nM, 50nM, or 100nM). General cell activity, viability, and proliferation were assessed along with phagocytosis process, and respiratory burst generation., Key Finding: Irisin level positively correlates with general cell activity and cell cycle progression as well as with phagocytosis intensity, but negatively correlates with the intensity of respiratory burst generation. No influence of irisin on quiescent cell viability, including apoptosis or necrosis, was observed., Significance: This research is the first to show that irisin modulates macrophage activity by reducing reactive oxygen species (ROS) overproduction, which could suggest its potential anti-inflammatory properties. Therefore, further studies are needed for the evaluation of influence of irisin on immunocompetent cell function., (Copyright © 2017 Elsevier Inc. All rights reserved.)