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Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. Despite extensive research and multiple clinical trials, there are still no FDA-approved therapies to treat the most severe forms of NAFLD. This is largely due to its complicated etiology and pathogenesis, which involves visceral obesity, insulin resistance, gut dysbiosis, etc. Although inflammation is generally believed to be one of the critical factors that drive the progression of simple steatosis to nonalcoholic steatohepatitis (NASH), the exact type of inflammation and how it contributes to NASH pathogenesis remain largely unknown. Liver inflammation is accompanied by the elevation of inflammatory mediators, including cytokines and chemokines and consequently intrahepatic infiltration of multiple types of immune cells. Recent studies revealed that extracellular vesicles (EVs) derived from inflammatory cells and hepatocytes play an important role in controlling liver inflammation during NASH. In this review, we highlight the roles of innate and adaptive immune cells and their microRNA-enriched EVs during NAFLD development and discuss potential drugs that target inflammatory pathways for the treatment of NAFLD.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and Aspergillus conidiae than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on β1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.

Coronavirus disease 2019 (COVID-19) is associated with an increased risk of thrombotic events. Ischemic stroke in COVID-19 patients entails high severity and mortality rates. Here we aimed to analyze cerebral thrombi of COVID-19 patients with large vessel occlusion (LVO) acute ischemic stroke to expose molecular evidence for SARS-CoV-2 in the thrombus and to unravel any peculiar immune-thrombotic features. We conducted a systematic pathological analysis of cerebral thrombi retrieved by endovascular thrombectomy in patients with LVO stroke infected with COVID-19 (n = 7 patients) and non-covid LVO controls (n = 23). In thrombi of COVID-19 patients, the SARS-CoV-2 docking receptor ACE2 was mainly expressed in monocytes/macrophages and showed higher expression levels compared to controls. Using polymerase chain reaction and sequencing, we detected SARS-CoV-2 Clade20A, in the thrombus of one COVID-19 patient. Comparing thrombus composition of COVID-19 and control patients, we noted no overt differences in terms of red blood cells, fibrin, neutrophil extracellular traps (NETs), von Willebrand Factor (vWF), platelets and complement complex C5b-9. However, thrombi of COVID-19 patients showed increased neutrophil density (MPO+ cells) and a three-fold higher Neutrophil-to-Lymphocyte Ratio (tNLR). In the ROC analysis both neutrophils and tNLR had a good discriminative ability to differentiate thrombi of COVID-19 patients from controls. In summary, cerebral thrombi of COVID-19 patients can harbor SARS-CoV2 and are characterized by an increased neutrophil number and tNLR and higher ACE2 expression. These findings suggest neutrophils as the possible culprit in COVID-19-related thrombosis. Graphical Abstract

Neutrophils are vital in defence against pathogens, but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome. COVID-19 is associated with systemic expansion of immature neutrophils, but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10−subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable 12 wk post-hospitalisation, indicating long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients down-regulate the chemokine receptor CXCR2, whereas neutrophils from severely ill individuals fail to do so, suggesting an altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10−and CXCR2hineutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for the identification of individuals at high risk of progressing to severe COVID-19.

© Springer-Verlag GmbH Germany, part of Springer Nature 2018, Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells., This work was supported by COST (European Cooperation in Science and Technology) and the COST Action BM1404 Mye-EUNITER (http://www.mye-eunit er.eu). COST is part of the EU Framework Programme Horizon 2020. This work was also supported by Grants from the Cooperation between German Cancer Research Center (DKFZ) and Ministry of Science, Technology and Space of Israel (MOST) in Cancer Research (CA181 to V. Umansky and Z.G. Fridlender).

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models., Anti-Ly6G or ant-Gr1 antibodies are commonly used to deplete neutrophils in vivo. Here the authors provide mechanistic insight into why these approaches may not specifically or durably reduce the number of neutrophils in mice, and also present a new method that overcomes these limitations to have potentially wide applicability in experimental studies.

Tumor-infiltrating neutrophils have been implicated in malignant development and progression, but mechanisms are ill defined. Neutrophils produce a proliferation-inducing ligand APRIL/TNFSF13, a factor that promotes development of tumors from diverse origins, including diffuse large B-cell lymphoma (DLBCL). High APRIL expression in DLBCL correlates with reduced patient survival, but the pathway(s) dictating APRIL expression are not known. Here, we show that all blood neutrophils constitutively secrete APRIL, and inflammation-associated stimuli, such as TNF, further upregulate APRIL. In a significant fraction of DLBCL patients, tumor cells constitutively produced the ELC-CXC chemokine CXCL-8 (IL8), enabling them to recruit APRIL-producing blood neutrophils. CXCL-8 production in DLBCL was unrelated to the cell of origin, as APRIL-producing neutrophils infiltrated CXCL-8+ DLBCL from both germinal center (GC) and non-GC subtypes. Rather, CXCL-8 production implied events affecting DNA methylation and acetylation. Overall, our results showed that chemokine-mediated recruitment of neutrophils secreting the tumor-promoting factor APRIL mediates DLBCL progression. Cancer Res; 77(5); 1097–107. ©2016 AACR.

N-3 docosapentaenoic acid-derived resolvin D5 (RvD5n-3 DPA) is diurnally regulated in peripheral blood and exerts tissue-protective actions during inflammatory arthritis. Here, using an orphan GPCR screening approach coupled with functional readouts, we investigated the receptor(s) involved in mediating the leukocyte-directed actions of RvD5n-3 DPA and identified GPR101 as the top candidate. RvD5n-3 DPA bound to GPR101 with high selectivity and stereospecificity, as demonstrated by a calculated KD of approximately 6.9 nM. In macrophages, GPR101 knockdown limited the ability of RvD5n-3 DPA to upregulate cyclic adenosine monophosphate, phagocytosis of bacteria, and efferocytosis. Inhibition of this receptor in mouse and human leukocytes abrogated the pro-resolving actions of RvD5n-3 DPA, including the regulation of bacterial phagocytosis in neutrophils. Knockdown of the receptor in vivo reversed the protective actions of RvD5n-3 DPA in limiting joint and gut inflammation during inflammatory arthritis. Administration of RvD5n-3 DPA during E. coli-initiated inflammation regulated neutrophil trafficking to the site of inflammation, increased bacterial phagocytosis by neutrophils and macrophages, and accelerated the resolution of infectious inflammation. These in vivo protective actions of RvD5n-3 DPA were limited when Gpr101 was knocked down. Together, our findings demonstrate a fundamental role for GPR101 in mediating the leukocyte-directed actions of RvD5n-3 DPA.

Item does not contain fulltext BACKGROUND: Eosinophils are a prominent cell type in the host response to helminths, and some evidence suggests that neutrophils might also play a role. However, little is known about the activation status of these granulocytes during helminth infection. METHODS: We analyzed the expression of eosinophil and neutrophil activation markers in peripheral blood by flow cytometry and measured serum levels of eosinophil granule proteins in 300 subjects residing in an area endemic for soil-transmitted helminths (STH). The data generated are on samples before and after 1 year of 3-monthly albendazole treatment. RESULTS: Anthelmintic treatment significantly reduced the prevalence of STH. While eosinophil numbers were significantly higher in STH-infected compared to uninfected subjects and significantly decreased following albendazole treatment, there was no effect exerted by the helminths on either eosinophil nor neutrophil activation. Although at baseline eosinophil granule protein levels were not different between STH-infected and uninfected subjects, treatment significantly reduced the levels of eosinophil-derived neurotoxin (EDN) in those infected at baseline. CONCLUSIONS: These results show that besides decreasing eosinophil numbers, anthelmintic treatment does not significantly change the activation status of eosinophils, nor of neutrophils, and the only effect seen was a reduction in circulating levels of EDN. CLINICAL TRIALS REGISTRATION: http://www.isrctn.com/ISRCTN75636394.

Summary Tumor-infiltrating myeloid cells (TIMs) comprise monocytes, macrophages, dendritic cells, and neutrophils, and have emerged as key regulators of cancer growth. These cells can diversify into a spectrum of states, which might promote or limit tumor outgrowth but remain poorly understood. Here, we used single-cell RNA sequencing (scRNA-seq) to map TIMs in non-small-cell lung cancer patients. We uncovered 25 TIM states, most of which were reproducibly found across patients. To facilitate translational research of these populations, we also profiled TIMs in mice. In comparing TIMs across species, we identified a near-complete congruence of population structures among dendritic cells and monocytes; conserved neutrophil subsets; and species differences among macrophages. By contrast, myeloid cell population structures in patients' blood showed limited overlap with those of TIMs. This study determines the lung TIM landscape and sets the stage for future investigations into the potential of TIMs as immunotherapy targets.

Acute inflammation recruits neutrophils with a band-shaped nucleus to the circulation. This neutrophil population was recently shown to have superior antibacterial capacity. Early recruitment of banded neutrophils to an infection site will likely improve the outcome of the immune response, yet it critically depends on efficient migration. However, the current dogma states that the segmentation of the mature neutrophil nucleus has evolved to favor migration through narrow pores as found between endothelial cells and in the interstitium. Therefore, we hypothesized that banded neutrophils migrate less efficiently than neutrophils with segmented nuclei, whereas recently described neutrophils with hypersegmented nuclei would in turn migrate more efficiently. Acute inflammation was evoked in a human model of experimental endotoxemia to recruit neutrophil subsets with different nuclear segmentation to the circulation. To simulate migration toward an infection site, migration of the subsets was studied in in vitro models of transendothelial migration or interstitial chemokinesis and chemotaxis. In both models, nuclear segmentation did not increase migration speed. In dense collagen matrices, the speed of the hypersegmented neutrophils was even reduced compared with the banded neutrophils. Fluorescence microscopy suggested that the hypersegmented neutrophils displayed reduced rear release and deposited more membrane vesicles. Vice versa, migration through narrow pores did not induce nuclear segmentation in the neutrophils. In conclusion, like neutrophils with a segmented nucleus, the banded subset exhibited efficient migration through narrow pores. These findings suggest that the nucleus does not preclude the banded subset from reaching an infection site.

Background. Chronic lung allograft dysfunction is a heterogeneous entity limiting long-term survival after lung transplantation. Different clinical phenotypes (bronchiolitis obliterans syndrome [BOS]-neutrophilic BOS-restrictive allograft syndrome [RAS]) have been identified but the mechanisms remain elusive. Methods. In this study, we measured 34 different cytokines, chemokines, and growth factors in bronchoalveolar lavage fluid of 20 stable patients, 20 patients suffering from non-neutrophilic BOS, 17 from neutrophilic BOS, and 20 from RAS using classic enzyme-linked immunosorbent assay and multiplex technology. Results. Total cell count and % neutrophils were elevated in neutrophilic BOS and RAS compared to stable and non-neutrophilic BOS patients, whereas also the % eosinophils was elevated at diagnosis of RAS. Levels of interleukin (IL)-1 beta (P

An animal model involving the subcutaneous implantation of tissue cages into guinea pigs and subsequent infection with Staphylococcus aureus was used for study factors pertinent to foreign body infection. Whereas 10(8) colony-forming units (cfu) of S. aureus strain Wood 46 did not produce any abscesses in the absence of foreign material, 10(2) cfu was sufficient to infect 95% of the tissue cages despite the presence of polymorphonuclear leukocytes (PMNLs) in sterile tissue cage fluid. Opsonization of S. aureus by tissue cage fluid was adequate during the first hour of infection, but opsonic coating of the organisms decreased at 20 hr after the induction of infection. PMNLs from sterile tissue cage fluid showed decreased phagocytic and bactericidal activities when compared with PMNLs from either blood or peritoneal exudate obtained after short- or long-term stimulation (P less than 0.001).

Background Immunometabolism plays a central role in many cardiometabolic diseases. However, a robust map of immune-related gene networks in circulating human cells, their interactions with metabolites, and their genetic control is still lacking. Here, we integrate blood transcriptomic, metabolomic, and genomic profiles from two population-based cohorts (total N = 2168), including a subset of individuals with matched multi-omic data at 7-year follow-up. Results We identify topologically replicable gene networks enriched for diverse immune functions including cytotoxicity, viral response, B cell, platelet, neutrophil, and mast cell/basophil activity. These immune gene modules show complex patterns of association with 158 circulating metabolites, including lipoprotein subclasses, lipids, fatty acids, amino acids, small molecules, and CRP. Genome-wide scans for module expression quantitative trait loci (mQTLs) reveal five modules with mQTLs that have both cis and trans effects. The strongest mQTL is in ARHGEF3 (rs1354034) and affects a module enriched for platelet function, independent of platelet counts. Modules of mast cell/basophil and neutrophil function show temporally stable metabolite associations over 7-year follow-up, providing evidence that these modules and their constituent gene products may play central roles in metabolic inflammation. Furthermore, the strongest mQTL in ARHGEF3 also displays clear temporal stability, supporting widespread trans effects at this locus. Conclusions This study provides a detailed map of natural variation at the blood immunometabolic interface and its genetic basis, and may facilitate subsequent studies to explain inter-individual variation in cardiometabolic disease., published version, peerReviewed

INTRODUCTION: Life expectancy evaluation is crucial when selecting patients who may benefit from phase I studies. The Royal Marsden Hospital (RMH) prognostic score, based on three objective variables (number of metastatic sites, lactate dehydrogenase (LDH) and serum albumin) was validated in patients treated with cytotoxics and targeted therapies. We aimed to determine if those factors were applicable to immune-checkpoint therapies (ICTs) in phase I trials and to evaluate new variables that may preclude a better prognosis in patients receiving ICT. PATIENTS AND METHODS: We conducted a retrospective analysis of survival risk factors in a discovery cohort of 155 patients enrolled into ICT phase I trials at our institution. We computed univariate analysis and multivariate analysis (MVA) of demographics, clinical and biological data to assess their prognostic value for overall survival (OS). MVA results were used to build a prognostic score for OS. A validation cohort of 113 patients enrolled in phase I ICT trials was used to prospectively validate this score. RESULTS: A total of 155 patients (M/F: 83/72; median age 59) receiving an experimental ICT between March 2012 and January 2016 were included in the discovery cohort. An MVA assessing the RMH score variables showed that low albumin (hazard ratio [HR] 1.73, 95% confidence interval [CI] 1.05-2.86) and LDH > upper limit normal (ULN) (HR 1.88, 95% CI 1.12-3.15) were independent negative prognostic factors for OS. Interestingly, neutrophil-to-lymphocyte ratio (NLR) > 6 (HR 1.75, 95% CI 1.04-2.95) was associated with a decrease in OS. The number of metastases was not associated with a poorer outcome for this ICT cohort (HR 0.83, 95% CI 0.51-1.35). A risk score based on the results of the MVA (NLR > 6 = 1; LDH > ULN = 1; albumin < 35 g/l = 1) showed that patients presenting a high score (>1) had a significantly shorter OS (20.4 weeks; 95% CI 5.7-35.2) compared to those with a low score (0 or 1) (68.9 weeks; 95% CI 50-83.7) (HR 2.9, 95% CI 1.87-4.64). In the validation cohort of 113 patients, again the patients presenting a high score showed an inferior OS (HR 6.3, 95% CI 2.7-14.8). CONCLUSION: In ICT phase I trials, traditional prognostic variables included in the RMH score may be suboptimal to determine patient's prognosis. In this context, the NLR is a significant prognostic variable. The Gustave Roussy Immune Score, based on albumin, LDH and NLR, allows a better selection of patients for ICT phase I trials.

Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked., Author Summary Pathogenic microbes have evolved elaborate strategies to manipulate host defenses to establish and spread in the host population. One such mechanism involves disruption of the immune signaling cascade orchestrated by the Toll-like receptors (TLRs), which sense microbial attack. TLR signaling elicits a proinflammatory response that controls immune cell recruitment to infected tissues. Here, we show that Staphylococcus aureus, an opportunistic human pathogen, expresses a host defense–like protein, TirS, that actively perturbs the initial TLR activation stage. Results with isolated human cells and mouse models show that TirS is a broad innate immune inhibitor of TLR-dependent signaling and modulates bacterial virulence, attenuating local inflammation. Moreover, the tirS gene lies near antimicrobial resistance genes for an antibiotic that enhances TirS production, shifting the balance to favor the pathogen and promote disease. Understanding mechanisms by which S. aureus modulates the immune response may lead to novel approaches for preventing and treating infection.

In clinical conditions trauma is associated with high mortality and morbidity. Neutrophils play a key role in the development of multiple organ failure after trauma. To have a detailed understanding of the neutrophil activation at primary stages after trauma, neutrophils were isolated from control and surgical trauma rats in this study. Extracted proteins were analyzed using nano liquid chromatography coupled to tandem mass spectrometry. A total of 2924 rat neutrophil proteins were identified in our analysis, of which 393 were found differentially regulated between control and trauma groups. By using functional pathways analysis of the 190 proteins up-regulated in surgical trauma we found proteins related to transcription initiation and protein biosynthesis. On the other hand, among the 203 proteins down-regulated in surgical trauma we found enrichment for proteins of the immune response, proteasome degradation and actin cytoskeleton. Overall, enzyme prediction analysis revealed that regulated enzymes are directly involved in neutrophil apoptosis, directional migration and chemotaxis. Our observations were then confirmed by in silico protein-protein interaction analysis. Collectively, our results reveal that neutrophils drastically regulate their biochemical pathways after the early stages of surgical trauma, showing lower activity. This implies higher susceptibility of the trauma patients to infection and bystander tissues damage. This article is protected by copyright. All rights reserved.

The acute symptoms of gout are triggered by the inflammatory response to monosodium urate crystals, mediated principally by macrophages and neutrophils. Innate immune pathways are of key importance in the pathogenesis of gout, in particular the activation of the NLRP3 inflammasome, which leads to the release of IL-1β and other pro-inflammatory cytokines. The orchestration of this pro-inflammatory cascade involves multiple intracellular and extracellular receptors and enzymes interacting with environmental influences that modulate the inflammatory state. Furthermore, the resolution of inflammation in gout is becoming better understood. This Review highlights recent advances in our understanding of both positive and negative regulatory pathways, as well as the genetic and environmental factors that modulate the inflammatory response. Some of these pathways can be manipulated and present novel therapeutic opportunities for the treatment of acute gout attacks.

CXCL8 or interleukin (IL)-8 directs neutrophil migration and activation through interaction with CXCR1 and CXCR2 that belong to the family of G protein-coupled receptors (GPCRs). Naturally occurring posttranslational modifications of the NH2-terminal region of CXCL8 affect its biological activities, but the underlying molecular mechanisms are only partially understood. Here, we studied the implications of site-specific citrullination and truncation for the signaling potency of CXCL8. Native CXCL8(1-77), citrullinated [Cit5]CXCL8(1-77) and the major natural isoform CXCL8(6-77) were chemically synthesized and tested in internalization assays using human neutrophils. Citrullinated and truncated isoforms showed a moderately enhanced capacity to induce internalization of CXCR1 and CXCR2. Moreover, CXCL8-mediated activation of G&alpha, i-dependent signaling through CXCR1 and CXCR2 was increased upon modification to [Cit5]CXCL8(1-77) or CXCL8(6-77). All CXCL8 variants promoted recruitment of &beta, arrestins 1 and 2 to CXCR1 and CXCR2. Compared to CXCL8(1-77), CXCL8(6-77) showed an enhanced potency to recruit &beta, arrestin 2 to both receptors, while for [Cit5]CXCL8(1-77) only the capacity to induce &beta, arrestin 2 recruitment to CXCR2 was increased. Both modifications had no biasing effect, i.e., did not alter the preference of CXCL8 to activate either G&alpha, i-protein or &beta, arrestin-dependent signaling through its receptors. Our results support the concept that specific chemokine activities are fine-tuned by posttranslational modifications.

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.

The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo. We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Summary: Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions. : Matlung et al. identify trogoptosis as an immune cell-mediated mechanism of cytotoxicity, demonstrating that neutrophil-mediated destruction of antibody-opsonized cancer cells occurs through a specific process that is distinct from that used by other immune cells. Keywords: antibody-dependent cellular cytotoxicity, neutrophils, CD47-SIRPα interaction, trogocytosis, trogoptosis

The methyltransferase Ezh2, an epigenetic regulator associated with tumor-cell metastasis, also methlyates the cytoplasmic integrin adaptor talin. This modification inhibits the binding of talin to F-actin, which enhances the migration and invasion of dendritic cells and neutrophils.

Psoriasis is characterized by an apoptosis-resistant and metabolic active epidermis, while a hallmark for allergic contact dermatitis (ACD) is T cell-induced keratinocyte apoptosis. Here, we induced ACD reactions in psoriasis patients sensitized to nickel (n = 14) to investigate underlying mechanisms of psoriasis and ACD simultaneously. All patients developed a clinically and histologically typical dermatitis upon nickel challenge even in close proximity to pre-existing psoriasis plaques. However, the ACD reaction was delayed as compared to non-psoriatic patients, with a maximum intensity after 7 days. Whole genome expression analysis revealed alterations in numerous pathways related to metabolism and proliferation in non-involved skin of psoriasis patients as compared to non-psoriatic individuals, indicating that even in clinically non-involved skin of psoriasis patients molecular events opposing contact dermatitis may occur. Immunohistochemical comparison of ACD reactions as well as in vitro secretion analysis of lesional T cells showed a higher Th17 and neutrophilic migration as well as epidermal proliferation in psoriasis, while ACD reactions were dominated by cytotoxic CD8+ T cells and a Th2 signature. Based on these findings, we hypothesized an ACD reaction directly on top of a pre-existing psoriasis plaque might influence the clinical course of psoriasis. We observed a strong clinical inflammation with a mixed psoriasis and eczema phenotype in histology. Surprisingly, the initial psoriasis plaque was unaltered after self-limitation of the ACD reaction. We conclude that sensitized psoriasis patients develop a typical, but delayed ACD reaction which might be relevant for patch test evaluation in clinical practice. Psoriasis and ACD are driven by distinct and independent immune mechanisms. peerReviewed

Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule in the cytosol of myeloid cells, required for induction of T-helper cells producing interleukin-17 (Th17 cells) and important in antifungal immunity. In a patient suffering from Candida dubliniensis meningoencephalitis, mutations in the CARD9 gene were found to result in the loss of protein expression. Apart from the reduced numbers of CD4(+) Th17 lymphocytes, we identified a lack of monocyte-derived cytokines in response to Candida strains. Importantly, CARD9-deficient neutrophils showed a selective Candida albicans killing defect with abnormal ultrastructural phagolysosomes and outgrowth of hyphae. The neutrophil killing defect was independent of the generation of reactive oxygen species by the reduced NAD phosphate oxidase system. Taken together, this demonstrates that human CARD9 deficiency results in selective defect in the host defense against invasive fungal infection, caused by an impaired phagocyte killing.

Rationale: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this hypothesis is lacking. Objective: To assess the role of macrophage cholesterol efflux pathways in atherogenesis. Methods and Results: We developed mice with efficient deletion of the ATP-binding cassette transporters A1 and G1 (ABCA1 and ABCG1) in macrophages (MAC-ABC DKO mice) but not in hematopoietic stem or progenitor populations. MAC-ABC DKO bone marrow (BM) was transplanted into Ldlr −/− recipients. On the chow diet, these mice had similar plasma cholesterol and blood monocyte levels but increased atherosclerosis compared with controls. On the Western-type diet, MAC-ABC DKO BM–transplanted Ldlr −/− mice had disproportionate atherosclerosis, considering they also had lower very low-density lipoprotein/low-density lipoprotein cholesterol levels than controls. ABCA1/G1-deficient macrophages in lesions showed increased inflammatory gene expression. Unexpectedly, Western-type diet–fed MAC-ABC DKO BM–transplanted Ldlr −/− mice displayed monocytosis and neutrophilia in the absence of hematopoietic stem and multipotential progenitor cells proliferation. Mechanistic studies revealed increased expressions of machrophage colony stimulating factor and granulocyte colony stimulating factor in splenic macrophage foam cells, driving BM monocyte and neutrophil production. Conclusions: These studies show that macrophage deficiency of ABCA1/G1 is proatherogenic likely by promoting plaque inflammation and uncover a novel positive feedback loop in which cholesterol-laden splenic macrophages signal BM progenitors to produce monocytes, with suppression by macrophage cholesterol efflux pathways.

Perinuclear antineutrophil cytoplasmic antibodies have recently been demonstrated in the sera of patients with inflammatory bowel disease. Three hundred and sixty six sera obtained from 120 patients with ulcerative colitis, 105 patients suffering from Crohn's disease and 49 non-inflammatory bowel disease controls were tested in two laboratories, using an indirect immunofluorescence assay. In addition, a fixed-neutrophil enzyme linked immunoadsorbent assay (ELISA) was evaluated in one of the two laboratories. The results in the immunofluorescence test showed a high degree of correlation between the two laboratories (Kappa coefficient = 0.8). Ninety five of the 120 (79%) ulcerative colitis patients had a positive test whereas only 14 of the 105 (13%) patients with Crohn's disease were positive. Sera from four patients suffering from primary sclerosing cholangitis were positive as well as four of the 45 control sera (9%). The sensitivity of the perinuclear antineutrophil cytoplasmic antibody immunofluorescence test for the diagnosis of ulcerative colitis was 0.75 with a specificity of 0.88 and a positive predictive value of 0.88 (all sera). In the ELISA technique 37 of 94 ulcerative colitis sera and one of the 68 Crohn's disease sera were positive. In the control group only one of the patients suffering from primary sclerosing cholangitis reacted positively (32 non-inflammatory bowel disease sera tested). The ELISA technique had a high specificity (0.97), but a low sensitivity (0.39). There was no relation of perinuclear antineutrophil cytoplasmic antibodies in ulcerative colitis patients or in Crohn's disease patients with disease activity, duration of illness, localisation, extent of disease, previous bowel operations or medical treatment. The clinical significance of perinuclear antineutrophil cytoplasmic antibody positive and negative subsets in both groups of patients thus remains unexplained. Our study confirms that determination of serum antineutrophil cytoplasmatic antibodies in patients with inflammatory bowel disease may differentiate ulcerative colitis from Crohn's disease. Further immunological studies are needed to explain the absence of these antibodies in a subset of ulcerative colitis patients and their role in the pathogenesis of the disease.

Five patients with subglottic stenosis, occurring either as a presenting symptom or as a manifestation in the course of a systemic disease, are described. Indirect immunofluorescence revealed the presence of circulating autoantibodies against both cytoplasmic and perinuclear constituents of neutrophils in all five. Antibodies directed against a 29 kDa antigen of the azurophilic granules (two patients), against myeloperoxidase (one patient), and against both the 29 kDa antigen and myeloperoxidase (one patient) were found by enzyme-linked immunosorbent assay. These autoantibodies have previously been found in patients with Wegener's granulomatosis, microscopic polyarteritis, (idiopathic) glomerulonephritis and Churg-Strauss syndrome. However, only one of these five patients fulfilled the criteria for these conditions. Since these autoantibodies are seldom observed in other conditions, and other diseases had been excluded by careful evaluation, we suggest that their presence places subglottic stenosis within the spectrum of necrotizing (granulomatous) vasculitis. Whether immunosuppressive therapy is always warranted in patients with subglottic stenosis and circulating anti-neutrophil cytoplasmic antibodies is a matter of debate.

Neutrophils are key components of the inflammatory response and as such contribute to the killing of microorganisms. In addition, recent evidence suggests their involvement in the development of the immune response. The role of neutrophils during the first weeks post-infection withLeishmania donovaniwas investigated in this study. WhenL. donovani-infected mice were selectively depleted of neutrophils with the NIMP-R14 monoclonal antibody, a significant increase in parasite numbers was observed in the spleen and bone marrow and to a lesser extent in the liver. Increased susceptibility was associated with enhanced splenomegally, a delay in the maturation of hepatic granulomas, and a decrease in inducible nitric oxide synthase expression within granulomas. In the spleen, neutrophil depletion was associated with a significant increase in interleukin 4 (IL-4) and IL-10 levels and reduced gamma interferon secretion by CD4+and CD8+T cells. Increased production of serum IL-4 and IL-10 and higher levels ofLeishmania-specific immunoglobulin G1 (IgG1) versus IgG2a revealed the preferential induction of Th2 responses in neutrophil-depleted mice. Altogether, these data suggest a critical role for neutrophils in the early protective response againstL. donovani, both as effector cells involved in the killing of the parasites and as significant players influencing the development of a protective Th1 immune response.

Jam-C(-/-) mice exhibit growth retardation and multilobular pneumonia concomitant with poor survival of the mice under conventional housing conditions. The deficient mice present a mega-oesophagus and have altered airway responsiveness. In addition, the number of circulating granulocytes is increased in Jam-C(-/-) mice as compared to control animals. These phenotypes probably reflect the different functions of JAM-C expressed by endothelial and mesenchymal cells. Indeed, the deregulation in the number of circulating granulocytes is caused by the lack of JAM-C expression on endothelial cells since rescuing endothelial expression of the protein in the Jam-C(-/-) mice is sufficient to restore homeostasis. More importantly, the rescue of vascular JAM-C expression is accompanied by better survival of deficient mice, suggesting that endothelial expression of JAM-C is mandatory for animal survival from opportunistic infections and fatal pneumonia.

To determine whether neutrophil depletion and Kupffer cell inhibition might combine their protective effects to decrease the severity of acute pancreatitis.Mice had cerulein administration to induce acute pancreatitis and were pretreated with either anti-mouse neutrophil serum or gadolinium chloride (GdCl3) to prevent Kupffer cell activation, or both treatments. Injury was assessed in pancreas and lungs. Myeloperoxidases (MPO) assessed neutrophil infiltration. Interleukin-6 (IL-6) and IL-10 were measured in serum, pancreas, lungs and liver.In mice with acute pancreatitis, neutrophil depletion reduced the severity of pancreatitis and pancreatitis-associated lung injury. Kupffer cell inactivation by GdCl3 had less protective effect, although IL-6 and IL-10 concentrations were significantly decreased. The protective treatment brought by neutrophil depletion was not enhanced by Kupffer cell inactivation and both treatments did not combine their protective effects.Our results confirm the role of activated neutrophils in aggravating organ injury in acute pancreatitis while the role of Kupffer cell activation is less obvious.

The main regulators of leukocyte trafficking during inflammatory responses are chemokines. However, another class of recently identified chemotactic agents is extracellular cyclophilins, the proteins mostly known as receptors for the immunosuppressive drug, cyclosporine A. Cyclophilins can induce leukocyte chemotaxis in vitro and have been detected at elevated levels in inflamed tissues, suggesting that they might contribute to inflammatory responses. We recently identified CD147 as the main signaling receptor for cyclophilin A. In the current study we examined the contribution of cyclophilin-CD147 interactions to inflammatory responses in vivo using a mouse model of acute lung injury. Blocking cyclophilin-CD147 interactions by targeting CD147 (using anti-CD147 Ab) or cyclophilin (using nonimmunosuppressive cyclosporine A analog) reduced tissue neutrophilia by up to 50%, with a concurrent decrease in tissue pathology. These findings are the first to demonstrate the significant contribution of cyclophilins to inflammatory responses and provide a potentially novel approach for reducing inflammation-mediated diseases.

Heterophils, the principal avian polymorphonuclear leukocytes (PMNs) equivalent to the mammalian neutrophil, function as professional phagocytes against bacterial infections, mediate acute inflammation, and respond to cytokine stimulation to aid in regulation of innate host defenses. Interleukin-2 (IL-2) has been found to exercise an array of biological effects on other cell types besides T lymphocytes, including NK cells, B cells, monocytes, and neutrophils. In the present experiments, using real-time quantitative RT-PCR, we evaluated the role of rChIL-2 as a priming mediator controlling heterophil responses at the level of gene transcription by examining the expression of mRNA for pro-inflammatory (IL-1, IL-6, IL-8) and Th1 (IL-18 and IFN-) cytokine genes following stimulation with phagocytosis agonists; i.e., opsonized and nonopsonized Salmonella enteritidis. Peripheral blood heterophils were isolated and incubated with rChIL-2 from transfected COS cells. rChIL-2 selectively primed the heterophils for an increase in transcription of the pro-inflammatory cytokine IL-8 and of theTh1 cytokine IL-18 induced by all three phagocytic agonists. Although rChIL-2 priming modulated the expression of specific cytokine mRNA in heterophils stimulated by different phagocytic agonists, the rChIL-2 by itself did not directly induce gene expression of either the pro-inflammatory or Th1 cytokines. We propose that rChIL-2 could be priming heterophils solely to function as more efficient innate effector cells to limit bacterial growth through the selective increase of IL-8 and IL-18 gene expression. Published by Elsevier Ltd.

Phagocytosis and superoxide production of neutrophils stimulated with Pneumocystis carinii were studied using P. carinii obtained from rats and neutrophils and serum from healthy blood donors. Superoxide generation increased in a dose-related way to the number of pneumocysts added and was significantly higher when the parasites were opsonized. The use of heat-inactivated serum for opsonization reduced superoxide production somewhat, but not significantly. Preincubation of neutrophils with recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) increased superoxide production in neutrophils stimulated with pneumocysts from 7.0 to 9.6 fmol/cell/20 min; however, the production in unstimulated cells increased by the same amount. Activation of the respiratory burst was paralleled by phagocytosis, and very few phagocytic vacuoles were found in neutrophils stimulated with unopsonized pneumocysts. By electron microscopy, various stages of cyst degradation were seen.

We investigated the clinical features and outcome of 60 patients with antineutrophil cytoplasmic autoantibodies with cytoplasmic staining (C-ANCA)- and with (peri)nuclear staining (P-ANCA)-associated glomerulonephritis. Virtually all patients with C-ANCA had antibodies against proteinase 3, which corresponded with a clinical and/or histological diagnosis of Wegener's granulomatosis (WG). P-ANCA were associated with antibodies against myeloperoxidase. Although more often associated with renal-limited disease, a significant number of patients with P-ANCA had also symptoms of organs known to be preferentially affected with WG. The majority of patients presented with rapidly progressive glomerulonephritis which responded favorably to immunosuppressive therapy, albeit at the expense of a considerable number of fatal infections. Furthermore, a more protracted form of glomerulonephritis was recognized. A kidney biopsy scoring system appeared to be of value in predicting which patients would benefit most from treatment. C-ANCA and P-ANCA appear to identify a subset of patients with vasculitis that share common clinical features and a favorable response to immunosuppressive therapy.

We have evaluated the therapeutic activity of recombinant erythropoietin (rEpo), in comparison with recombinant interleukin-3 (rIL-3) and granulocyte-macrophage colony-stimulating factor (rGM-CSF), on a lethal form of acute anemia resulting from Fc gamma receptor-mediated erythrophagocytosis after a single injection (500 micrograms) of a monoclonal anti-mouse red blood cell (MRBC) autoantibody. Continuous perfusion of rEpo before the administration of anti-MRBC monoclonal antibody completely protected animals from death due to anemia with a rapid recovery, while no protection was obtained by rIL-3 perfusion. In contrast, rGM-CSF perfusion markedly accelerated the progression of anemia and the mortality rate. This was found to result from an enhancement of erythrophagocytosis by Kupffer cells and by polymorphonuclear leukocytes that massively infiltrated the livers. Even after the injection of a sublethal dose (100 micrograms) of anti-MRBC monoclonal antibody, rGM-CSF-perfused mice died of a severe form of acute anemia. Furthermore, we have shown that rEpo was able to treat efficiently a spontaneous form of autoimmune hemolytic anemia in a majority of anemic NZB mice, whereas rGM-CSF markedly aggravated anemia. This may be of clinical importance, because GM-CSF administration could exhibit an adverse effect in some autoimmune diseases that involve autoimmune anemia.

Summary Breaching endothelial cells (ECs) is a decisive step in the migration of leukocytes from the vascular lumen to the extravascular tissue, but fundamental aspects of this response remain largely unknown. We have previously shown that neutrophils can exhibit abluminal-to-luminal migration through EC junctions within mouse cremasteric venules and that this response is elicited following reduced expression and/or functionality of the EC junctional adhesion molecule-C (JAM-C). Here we demonstrate that the lipid chemoattractant leukotriene B4 (LTB4) was efficacious at causing loss of venular JAM-C and promoting neutrophil reverse transendothelial cell migration (rTEM) in vivo. Local proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) drove this cascade of events as supported by presentation of NE to JAM-C via the neutrophil adhesion molecule Mac-1. The results identify local LTB4-NE axis as a promoter of neutrophil rTEM and provide evidence that this pathway can propagate a local sterile inflammatory response to become systemic., Graphical Abstract, Highlights • Endogenous LTB4 mediates reduced expression of endothelial cell (EC) JAM-C in I-R • LTB4 can stimulate proteolytic cleavage of EC JAM-C by neutrophil elastase (NE) • Activation of a local LTB4-NE axis induces neutrophil reverse TEM in vivo • Activation of a local LTB4-NE axis can promote distant organ damage, The mechanisms and implications of aberrant modes of neutrophil transendothelial cell migration remain largely unknown. Here, Nourshargh and colleagues demonstrate that local leukotriene B4-induced proteolytic cleavage of endothelial JAM-C by neutrophil elastase promotes neutrophil reverse transendothelial cell migration back into the circulation and is decisive in dissemination of systemic inflammation.