Migration Of Polymorphonuclear Neutrophils Research Articles

Mesenchymal Stromal/Stem Cells Know Best: The Remarkable Complexities of Its Interactions With Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs), the predominant immune cell type in humans, have long been known as first-line effector cells against bacterial infections mainly through phagocytosis and production of reactive oxygen species (ROS). However, recent research has unveiled novel and pivotal roles of these abundant but short-lived granulocytes in health and disease. Human mesenchymal stromal/stem cells (MSCs), renowned for their regenerative properties and modulation of T lymphocytes from effector to regulatory phenotypes, exhibit complex and context-dependent interactions with PMNs. Regardless of species or source, MSCs strongly abrogate PMN apoptosis, a critical determinant of PMN function, except if PMNs are highly stimulated. MSCs also have the capacity to fine-tune PMN activation, particularly in terms of CD11b expression and phagocytosis. Moreover, MSCs can modulate numerous other PMN functions, spanning migration, ROS production, and neutrophil extracellular trap (NET) formation/NETosis, but directionality is remarkably dependent on the underlying context: in normal nondiseased conditions, MSCs enhance PMN migration and ROS production, whereas in inflammatory conditions, MSCs reduce both these functions and NETosis. Furthermore, the state of the MSCs themselves, whether isolated from diseased or healthy donors, and the specific secreted products and molecules, can impact interactions with PMNs; while healthy MSCs prevent PMN infiltration and NETosis, MSCs isolated from patients with cancer promote these functions. This comprehensive analysis highlights the intricate interplay between PMNs and MSCs and its profound relevance in healthy and pathological conditions, shedding light on how to best strategize the use of MSCs in the expanding list of diseases with PMN involvement.

Fasciola hepatica soluble antigens (FhAg) induce ovine PMN innate immune reactions and NET formation in vitro and in vivo

Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.

Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

BackgroundExcess polymorphonuclear neutrophil (PMN) recruitment or excessive neutrophil extracellular trap (NET) formation can lead to the development of multiple organ dysfunction during sepsis. M2 macrophage-derived exosomes (M2-Exos) have exhibited anti-inflammatory activities in some inflammatory diseases to mediate organ functional protection, but their role in treating sepsis-related acute lung injury (ALI) remains unclear. In this study, we sought to investigate whether M2-Exos could prevent potentially deleterious inflammatory effects during sepsis-related ALI by modulating abnormal PMN behaviours.MethodsC57BL/6 wild-type mice were subjected to a caecal ligation and puncture (CLP) mouse model to mimic sepsis in vivo, and M2-Exos were administered intraperitoneally 1 h after CLP. H&E staining, immunofluorescence and immunohistochemistry were conducted to investigate lung tissue injury, PMN infiltration and NET formation in the lung. We further demonstrated the role of M2-Exos on PMN function and explored the potential mechanisms through an in vitro coculture experiment using PMNs isolated from both healthy volunteers and septic patients.ResultsHere, we report that M2-Exos inhibited PMN migration and NET formation, alleviated lung injury and reduced mortality in a sepsis mouse model. In vitro, M2-Exos significantly decreased PMN migration and NET formation capacity, leading to lipid mediator class switching from proinflammatory leukotriene B4 (LTB4) to anti-inflammatory lipoxin A4 (LXA4) by upregulating 15-lipoxygenase (15-LO) expression in PMNs. Treatment with LXA4 receptor antagonist attenuated the effect of M2-Exos on PMNs and lung injury. Mechanistically, prostaglandin E2 (PGE2) enriched in M2-Exos was necessary to increase 15-LO expression in PMNs by functioning on the EP4 receptor, upregulate LXA4 production to downregulate chemokine (C-X-C motif) receptor 2 (CXCR2) and reactive oxygen species (ROS) expressions, and finally inhibit PMN function.ConclusionsOur findings reveal a previously unknown role of M2-Exos in regulating PMN migration and NET formation through lipid mediator class switching, thus highlighting the potential application of M2-Exos in controlling PMN-mediated tissue injury in patients with sepsis.

The Effect of Local Anesthetics on Neutrophils in the Context of Different Isolation Techniques.

Various functions of polymorphonuclear neutrophils (PMNs) are related to diseases and postoperative plasma changes. The influence of some local anesthetics (LAs) on PMNs obtained by conventional isolation methods and their functions has already been demonstrated. This study investigates the effect of selected LAs on PMNs, comparing a new isolation method with conventional ones. To obtain the PMNs, we performed either gelafundin sedimentation, hypotonic lysis or density gradient centrifugation. Subsequently, PMNs were mixed with different concentrations of bupivacaine, levobupivacaine, lidocaine or ropivacaine. Live cell imaging and flow cytometry were performed to quantify the migration, ROS production, NETosis and antigen expression of PMNs. We found the inhibition of chemotaxis and ROS production by LAs. PMNs showed a strong reduction in time to half maximal NETosis in response to bupivacaine and lidocaine, but not to levobupivacaine and ropivacaine. We also found distinct differences in survival time and migration duration between the isolation methods. This suggests that the careful selection of LAs has a short-term impact on in vitro PMNs.

Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function.

Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The β2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3-linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide-mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.

Effects of Hormone, NEFA and SCFA on the Migration of Neutrophils and the Formation of Neutrophil Extracellular Traps in Dairy Cows.

Simple SummaryPerinatal dairy cows face the challenge of maintaining the resilience of defense against invading pathogens. During the perinatal period, hormonal or metabolic changes cause the decline of immune function of dairy cows and further lead to varying degrees of immunosuppression. The results of this study indicate that, hormones, nonesterified fatty acids (NEFAs) and short-chain fatty acids (SCFAs) can regulate neutrophil migration and the NETs formation of dairy cows in vitro. These results help to further explain the effects of changes in hormone secretion and metabolites on immunosuppression and the increased risk of disease in perinatal dairy cows.Polymorphonuclear neutrophils (PMN) are the first line of defense against the invasion of foreign pathogenic microorganisms and play an essential role in the immune system of dairy cows. The changes in hormone secretion and metabolites of dairy cows during the perinatal period are the key factors that cause immunosuppression and increased risk of diseases. However, the effects of the hormone, nonesterified fatty acid (NEFA), and short-chain fatty acid (SCFA) on the transmammary epithelial migration of dairy cows and the formation of neutrophil extracellular traps (NETs) have rarely been studied. This study explored the effects of hormones, NEFAs and SCFAs on the neutrophil migration and NETs formation of dairy cows in vitro. It was found that P4 and Ac can regulate the transepithelial migration of PMN; SA and Pr can regulate the formation of NETs; E2, OA and Bt can regulate PMN transepithelial migration and NET formation. These results help to further explain the effects of changes in hormone secretion and metabolites on immunosuppression and the increased risk of disease in perinatal dairy cows.

Sevoflurane Dampens Acute Pulmonary Inflammation via the Adenosine Receptor A2B and Heme Oxygenase-1.

Acute respiratory distress syndrome is a life-threatening disease associated with high mortality. The adenosine receptor A2B (Adora2b) provides anti-inflammatory effects, which are also associated with the intracellular enzyme heme oxygenase-1 (HO-1). Our study determined the mechanism of sevoflurane’s protective properties and investigated the link between sevoflurane and the impact of a functional Adora2b via HO-1 modulation during lipopolysaccharide (LPS)-induced lung injury. We examined the LPS-induced infiltration of polymorphonuclear neutrophils (PMNs) into the lung tissue and protein extravasation in wild-type and Adora2b−/− animals. We generated chimeric animals, to identify the impact of sevoflurane on Adora2b of hematopoietic and non-hematopoietic cells. Sevoflurane decreased the LPS-induced PMN-infiltration and diminished the edema formation in wild-type mice. Reduced PMN counts after sevoflurane treatment were detected only in chimeric mice, which expressed Adora2b exclusively on leukocytes. The Adora2b on hematopoietic and non-hematopoietic cells was required to improve the permeability after sevoflurane inhalation. Further, sevoflurane increased the protective effects of HO-1 modulation on PMN migration and microvascular permeability. These protective effects were abrogated by specific HO-1 inhibition. In conclusion, our data revealed new insights into the protective mechanisms of sevoflurane application during acute pulmonary inflammation and the link between sevoflurane and Adora2b, and HO-1 signaling, respectively.

Leukotrienes Are Dispensable for Vaginal Neutrophil Recruitment as Part of the Immunopathological Response During Experimental Vulvovaginal Candidiasis

Recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen is the hallmark of an acute immunopathologic inflammatory response during vulvovaginal candidiasis (VVC) caused by Candida albicans. Recurrent VVC (RVVC) remains a chronic health burden in affected women worldwide despite the use of antifungal therapy. Based on the role leukotrienes (LTs) play in promoting inflammation, leukotriene receptor antagonists (LTRAs) targeted for LTB4 (etalocib) or LTC4, LTD4, and LTE4 (zafirlukast or montelukast) have been shown to reduce inflammation of epithelial tissues. An open-label pilot study using long-term regimens of zafirlukast in women with RVVC indicated the potential for some relief from recurrent episodes. To investigate this clinical observation further, we evaluated the effects of LT antagonistic agents and LT deficiency on the immunopathogenic response in a mouse model of VVC. Results showed that mice given daily intraperitoneal injections of individual LTRAs, starting 2days prior to vaginal inoculation with C. albicans and continuing through 14days post-inoculation, had no measurable reduction in PMN migration. The LTRAs were also ineffective in reducing levels of the hallmark vaginal inflammatory markers (S100A8, IL-1β) and tissue damage (LDH) associated with the immunopathogenic response. Finally, LT-deficient 5-lipoxygenase knockout mice showed comparable levels of vaginal fungal burden and PMN infiltration to wild-type mice following inoculation with a vaginal (ATCC 96113) or laboratory (SC5314) C. albicans isolate. These results indicate that despite some clinical evidence suggestive of off-target efficacy of LTRAs in RVVC, LTs and associated signaling pathways appear to be dispensable in the immunopathogenesis of VVC.

β-Hydroxybutyrate impairs neutrophil migration distance through activation of a protein kinase C and myosin light chain 2 signaling pathway in ketotic cows

Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB <0.6 mM) or clinically ketotic (n = 15, BHB >3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)-a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction-was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.

Trained Immunity Confers Prolonged Protection From Listeriosis.

Trained immunity refers to the ability of the innate immune system exposed to a first challenge to provide an enhanced response to a secondary homologous or heterologous challenge. We reported that training induced with β-glucan one week before infection confers protection against a broad-spectrum of lethal bacterial infections. Whether this protection persists over time is unknown. To tackle this question, we analyzed the immune status and the response to Listeria monocytogenes (L. monocytogenes) of mice trained 9 weeks before analysis. The induction of trained immunity increased bone marrow myelopoiesis and blood counts of Ly6Chigh inflammatory monocytes and polymorphonuclear neutrophils (PMNs). Ex vivo, whole blood, PMNs and monocytes from trained mice produced increased levels of cytokines in response to microbial products and limited the growth of L. monocytogenes. In vivo, following challenge with L. monocytogenes, peripheral blood leukocytes were massively depleted in control mice but largely preserved in trained mice. PMNs were reduced also in the spleen from control mice, and increased in the spleen of trained mice. In transwell experiments, PMNs from trained mice showed increased spontaneous migration and CXCL2/MIP2α-induced chemotaxis, suggesting that training promotes the migration of PMNs in peripheral organs targeted by L. monocytogenes. Trained PMNs and monocytes had higher glycolytic activity and mitochondrial respiration than control cells when exposed to L. monocytogenes. Bacterial burden and dissemination in blood, spleen and liver as well as systemic cytokines and inflammation (multiplex bead assay and bioluminescence imaging) were reduced in trained mice. In full agreement with these results, mice trained 9 weeks before infection were powerfully protected from lethal listeriosis. Altogether, these data suggest that training increases the generation and the antimicrobial activity of PMNs and monocytes, which may confer prolonged protection from lethal bacterial infection.

Abstract 2863: Cancer extracellular vesicles induce lymph node metastasis via neutrophil extracellular traps

Abstract In most human cancers, regional lymph nodes (LNs) are the first sites of metastasis. In addition to being an important part of the tumor staging system, with the advent of novel therapies, lymph node metastasis has become a crucial clinical intervention point before distant metastasis, the leading cause of cancer-associated deaths. To initiate metastasis, the conditions of LNs need to be optimized for tumor cell deposition and growth. This process is believed to be mediated by the activation of immune cells including polymorphonuclear neutrophils (PMNs), and tumor derived factors, such as extracellular vesicles (EVs). Indeed, tumor derived EVs (tEVs) were shown to prepare sentinel LNs for increased melanoma metastasis, however, the cellular mechanism is not well defined. Early observations suggest that PMNs and neutrophil extracellular traps (NETs), DNA comprising structures that are extruded in response to inflammatory cues, are associated with adverse oncologic outcomes. Moreover, PMNs exhibit considerable plasticity to tEVs, as gastric tEVs can polarize PMN toward a pro-tumor (N2) phenotype and induce NET formation. Thus, one potential mechanism of increased LN metastasis is that tEVs recruit PMNs and propend NETs formation. Here, we show that lymphatic PMN accumulation is associated with higher rates of LN metastasis in human esophageal cancer patients. Furthermore, we demonstrate that LN PMN accumulation is mediated by tEVs-lymphatic interaction both in vitro and in vivo. Finally, we demonstrate that lymphatic PMN facilitate metastasis through the accumulation of PMN prior to tumor ingress. Using Boyden chamber assays, we observed an increase in PMN migration towards tEVs educated lymphatic endothelial cells (LECs). Moreover, ELISA showed tEVs educated LEC increased secretion of the PMN chemoattractants CXCL4 and CXCL8. Additionally, through confocal microscopy and immunofluorescence, we observed that tEVs induced PMN recruitment to LNs and NETs released in vivo in a dose-dependent manner. Finally, using transgenic pad4-/- knockout mice, which are unable to generate NETs, we showed that the absence of NETs led to decreased LN metastasis. Together, these findings highlight the role of tumor derived tEVs both as PMN recruiters to LNs and NETs inducers. By further investigating the detailed mechanism and the efficiency of NETs targeting agents, this project will lead to major advances in the management of cancer patients. Citation Format: Xin SU, Ariane Brassard, Ramin Rohanizadeh, Iqraa Dhoparee-Doomah, Betty Giannias, France Bourdeau, Veena Sangwan, Roni F. Rayes, Jonathan D. Spicer, Lorenzo E. Ferri, Jonathan J. Cools-Lartigue. Cancer extracellular vesicles induce lymph node metastasis via neutrophil extracellular traps [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2863.

The influence of extracellular tissue on neutrophil function and its possible linkage to inflammatory diseases

Background: Migration, production of reactive oxygen species (ROS), release of myeloperoxidase (MPO), and NETosis are functional immunological reactions of elementary importance for polymorphonuclear neutrophils (PMN).Unregulated inflammatory response of PMN within tissues plays a key role in the pathophysiology of several diseases. However, little is known about the behavior of PMN after migration through blood vessel walls. Therefore, we investigated the influence of the extracellular matrix (ECM) on PMN function. Materials and Methods: We established an in vitro chemotaxis model of type I and III collagen, fibrin, and herbal agarose tissues using µ‐slide chemotaxis devices and N‐formylmethionine‐leucyl‐phenylalanine (fMLP). PMN within the matrices were assessed with a fluorescent time‐lapse microscope for live‐cell imaging. Results: PMN function was obviously influenced by the ECM. Type III collagen had an inhibitory effect on PMN migration regarding track length, direction, and targeting. Type III collagen also had an accelerating effect on neutrophil ROS production. Agarose had an inhibitory effect on MPO release and fibrin a retarding effect on NETosis. Conclusion: Because of the high abundance of type III collagen in lung and skin matrices, the interaction of PMN with the respective matrix could be an important mechanism in the pathophysiology of acute respiratory distress syndrome and pyoderma gangrenosum.

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers. We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

CX3CR1 Depletion Promotes the Formation of Platelet-Neutrophil Complexes and Aggravates Acute Peritonitis.

Peritonitis is a life-threatening condition on intensive care units. Inflammatory cytokines and their receptors drive inflammation, cause the formation of platelet-neutrophil complexes (PNCs) and therefore the migration of polymorphonuclear neutrophils (PMNs) into the inflamed tissue. CX3CL1 and its receptor CX3CR1 are expressed in various cells, and promote inflammation. The shedding of CX3CL1 is mediated by a disintegrin and metalloprotease (ADAM) 17. The role of the CX3CL1-CX3CR1 axis in acute peritonitis remains elusive. In zymosan-induced peritonitis, we determined the formation of PNCs in the blood and the expression of PNC-related molecules on PNCs. PMN migration into the peritoneal lavage was evaluated in wild-type (WT) and CX3CR1-/- animals by flow cytometry. CX3CL1, ADAM17, and the expression of various inflammatory cytokines were detected. Further, we determined the inflammation-associated activation of the intracellular transcription factor extracellular signal-regulated kinase 1/2 (ERK1/2) by Western blot. The PMN accumulation in the peritoneal lavage and the PNC formation in the circulation were significantly raised in CX3CR1-/- compared with WT animals. The expression of PNC-related selectins on PNCs was significantly increased in the blood of CX3CR1-/- animals, as well as cytokine levels. Further, we observed an increased activation of ERK1/2 and elevated ADAM17 expression in CX3CR1-/- during acute inflammation. Selective ERK1/2 inhibition ameliorated inflammation-related increased ADAM17 expression. A CX3CR1 deficiency raised the release of inflammatory cytokines and increased the PNC formation respectively PMN migration via an elevated ERK1/2 activation during acute peritonitis. Further, we observed a link between the ERK1/2 activation and an elevated ADAM17 expression on PNC-related platelets and PMNs during inflammation. Our data thus illustrate a crucial role of CX3CR1 on the formation of PNCs and regulating inflammation in acute peritonitis.

IL1B triggers inflammatory cytokine production in bovine oviduct epithelial cells and induces neutrophil accumulation via CCL2.

The oviduct is essential for reproduction. We previously showed that oviduct epithelial cells (OECs) isolated from aged cows expressed higher levels of inflammatory cytokines, including interleukin (IL) 1A and IL1B. In addition, aging is associated with tissue dysfunction and cellular senescence via a senescence-associated secretory phenotype (SASP) and immune cell accumulation. We investigated whether IL1A or IL1B causes SASP production, cellular senescence, and inflammatory responses in bovine OECs. The OECs were isolated from bovine oviducts from young (mean 50.3months) and aged cows (mean 157.0months) and cultured. Treatment with IL1A or IL1B induced SASP production (IL8, IL6, TNFA, and CCL2) and mRNA expression of cell adhesion molecules in bovine OECs, but both IL1s did not induce cellular senescence in OECs and migration of polymorphonuclear neutrophils (PMNs). Cultured medium of OECs treated with IL1s, especially IL1B, dramatically induced PMN migration. Treatment with the CCL2 inhibitor, but not IL8 or its receptor CXCR2 inhibitors, significantly reduced immune cell migration in IL1B-treated OEC-cultured medium. Treatment with IL1B increased PMN adhesion to OECs, resulting in further SASP production in OECs due to a PMN-OEC interaction. We suggest that senescence-associated IL1s cause SASP production in bovine OECs and CCL2 induced by IL1B is essential for the migration of immune cells to OECs. Specifically, IL1B regulates PMN migration and adhesion to bovine OECs, and PMNs accelerate inflammatory cytokine production from bovine OECs via a direct interaction. These phenomena may contribute to chronic oviductal inflammation, resulting in subfertility.

Fasciola hepatica induces weak NETosis and low production of intra- and extracellular ROS in exposed bovine polymorphonuclear neutrophils

Fasciola hepatica is the causative agent of fasciolosis, a worldwide distributed zoonotic disease, leading to hepatitis in humans and livestock. Newly excysted juveniles (NEJ) of F. hepatica are the first invasive stages to encounter leukocytes of host innate immune system in vivo. Among leukocytes, polymorphonuclear neutrophils (PMN) are the most abundant granulocytes of blood system and first ones to migrate into infection sites. PMN are able to cast neutrophil extracellular traps (NETs), also known as NETosis, consisting of nuclear DNA, decorated with histones, enzymes and antimicrobial peptides, which can entrap and eventually kill invasive parasites. Given that only few large parasitic helminths have been identified as potent NETosis inducers, here we studied for first time whether different F. hepatica stages can also trigger NETosis. Therefore, isolated bovine PMN were co-cultured with viable F. hepatica-NEJ, -metacercariae, -eggs and soluble antigen (FhAg). Interestingly, all stages failed to induce considerable levels of NETosis as detected by immunofluorescence- and scanning electron microscopy (SEM) analyses. NEJ remained motile until the end of incubation period. In line, NETosis quantification via nuclear area expansion (NAE) analysis revealed NEJ as weak NETosis inducers. However, bovine PMN frequently displaced towards motile NEJ and were found attached to NEJ surfaces. Functional PMN chemotaxis assays using vital F. hepatica-NEJ revealed a slight increase of PMN migration when compared to non-exposed controls. Additional experiments on intra- and extracellular reactive oxygen species (ROS) production revealed that soluble FhAg failed to induce ROS production of exposed PMN. Finally, mitochondrial oxygen consumption rates (OCR), extracellular acidification rates (ERAC) and proton production rates (PPR) were not significantly increased in FhAg-stimulated PMN. In summary, data suggest that F. hepatica might effectively evade PMN activation and NETosis by secreting parasite-specific molecules to either resolve NETs or to impair NETosis signaling pathways. We call for future molecular analysis not only on F. hepatica-derived NETosis modulation but also on its possible role in fasciolosis-associated pathology in vivo.

Therapeutic Anticoagulation with Argatroban and Heparins Reduces Granulocyte Migration: Possible Impact on ECLS-Therapy?

Introduction Anticoagulants such as argatroban and heparins (low-molecular-weight and unfractionated) play an immense role in preventing thromboembolic complications in clinical practice. Nevertheless, they can also have a negative effect on the immune system. This study is aimed at investigating the influence of these substances on polymorphonuclear neutrophils (PMNs), whose nonspecific defense mechanisms can promote thrombogenesis. Methods Blood samples from 30 healthy volunteers were investigated, whereby PMNs were isolated by density gradient centrifugation and incubated with 0.8 μg/mL of argatroban, 1.0 U/mL of low-molecular-weight heparin (LMWH), 1.0 U/mL of unfractionated heparin (UFH), or without drug (control). A collagen-cell mixture was prepared and filled into 3D μ-slide chemotaxis chambers (IBIDI® GmbH, Germany). Stimulation was initiated by using a chemokine gradient of n-formyl-methionine-leucyl-phenylalanine (fMLP), and microscopic observation was conducted for 4.5 hours. The cells' track length and track straightness, as well as the number of attracted granulocytes, level of ROS (reactive oxygen species) production, and NET (neutrophil extracellular traps) formation, were analyzed and categorized into migration distances and time periods. Results All three anticoagulants led to significantly reduced PMN track lengths, with UFH having the biggest impact. The number of tracks observed in the UFH group were significantly reduced compared to the control group. Additionally, the UFH group demonstrated a significantly lower track straightness compared to the control. ROS production and NET formation were unaffected. Conclusion Our data provide evidence that anticoagulants have an inhibitory effect on the extent of PMN migration and chemotactic migration efficiency, thus indicating their potential immune-modulatory and prothrombotic effects.

IL-33/ST2 pathway regulates neutrophil migration and predicts outcome in patients with severe alcoholic hepatitis

Severe alcoholic hepatitis (SAH) is associated with a high risk of infection. The IL-33/ST2 pathway is involved in sepsis control but data regarding its role in alcohol-related liver disease (ALD) are lacking. We aimed to characterize the role of IL-33/ST2 in the polymorphonuclear neutrophils (PMNs) of patients with ALD and SAH. Serum and circulating neutrophils were collected from patients with SAH, alcoholic cirrhosis and healthy controls. We quantified IL-33/ST2 pathway activity and CXCR2 at baseline and after exposure to IL-33. We also determined the migration capacity of PMNs. The decoy receptor of IL-33 (soluble ST2 [sST2]) was increased in SAH vs. cirrhosis and controls, demonstrating the defect in this pathway during ALD. The sST2 level was associated with response to treatment, 2-month survival, infection-free survival and probability of infection in SAH. Endotoxemia was weakly correlated with sST2. GRK2, a negative regulator of CXCR2, was overexpressed in PMNs of patients with SAH and cirrhosis and was decreased by IL-33. CXCR2 levels on PMNs were lower in SAH vs. cirrhosis and controls. Treatment with IL-33 partially restored CXCR2 expression in SAH and cirrhosis. PMN migration upon IL-8 was lower in patients with SAH and cirrhosis vs. controls. Treatment with IL-33 partially restored migration in those with SAH and cirrhosis. Interestingly, the migration capacity of PMNs and the response to IL-33 were enhanced in responders to corticosteroids (Lille <0.45) compared to non-responders. The IL33/ST2 pathway is defective in SAH and predicts outcome. This defect is associated with decreased CXCR2 expression on the surface of PMNs and lower migration capacity, which can be corrected by IL-33, especially in patients responding to steroids. These results suggest that IL-33 has therapeutic potential for SAH and its infectious complications. The neutrophils of patients with severe alcoholic hepatitis are associated with a defect in the IL-33/ST2 pathway. This defect is associated with lower migration capacities in neutrophils and a higher probability of getting infected. Administration of IL-33 to the neutrophils at least partly restores this defect and may be effective at reducing the risk of infection in patients with severe alcoholic hepatitis.

Nrf2 downregulates zymosan-induced neutrophil activation and modulates migration

Polymorphonuclear neutrophils (PMNs) are the first line of defense against pathogens and their activation needs to be tightly regulated in order to limit deleterious effects. Nrf2 (Nuclear factor (erythroïd-derived 2)-like 2) transcription factor regulates oxidative stress and/or represses inflammation in various cells such as dendritic cells or macrophages. However, its involvement in PMN biology is still unclear. Using Nrf2 KO mice, we thus aimed to investigate the protective role of Nrf2 in various PMN functions such as oxidative burst, netosis, migration, cytokine production and phagocytosis, mainly in response to zymosan. We found that zymosan induced Nrf2 accumulation in PMNs leading to the upregulation of some target genes including Hmox-1, Nqo1 and Cat. Nrf2 was able to decrease zymosan-induced PMN oxidative burst; sulforaphane-induced Nrf2 hyperexpression confirmed its implication. Tnfα, Ccl3 and Cxcl2 gene transcription was decreased in zymosan-stimulated Nrf2 KO PMNs, suggesting a role for Nrf2 in the regulation of proinflammatory cytokine production. However, Nrf2 was not involved in phagocytosis. Finally, spontaneous migration of Nrf2 KO PMNs was lower than that of WT PMNs. Moreover, in response to low concentrations of CXCL2 or CXCL12, Nrf2 KO PMN migration was decreased despite similar CXCR2 and CXCR4 expression and ATP levels in PMNs from both genotypes. Nrf2 thus seems to be required for an optimal migration. Altogether these results suggest that Nrf2 has a protective role in several PMN functions. In particular, it downregulates their activation in response to zymosan and is required for an adequate migration.

Effects of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil activity in dairy cows with subclinical hypocalcemia1.

Hypocalcemia in dairy cows is often associated with inflammation-related disorders such as metritis and mastitis. The protein encoded by the Ca2+ release-activated calcium modulator 1 (ORAI1) gene is a membrane Ca2+ channel subunit that is activated when Ca2+ stores are depleted. Polymorphonuclear neutrophils (PMNL) have a crucial role in the defense against infection through migration, adhesion, chemotaxis, phagocytosis, and reactive oxygen species (ROS) production in response to pathogens. Whether hypocalcemia affects the activity of PMNL and if ORAI1 is involved remains unknown. To address this, PMNL were isolated at 3 d of calving from dairy cows diagnosed as clinically healthy (n = 20, CONTROL) or with plasma concentration of calcium < 2.0 mmol/L as a criterion for diagnosis of subclinical hypocalcemia (n = 20, HYPOCAL). PMNL isolated from both groups of cows were treated with or without the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin, Ca2+ ionophore Ionomycin, and ORAI1 blocker 2APB. The intracellular Ca2+ concentration, ORAI1 abundance, ROS, phagocytosis rate, migration, and adhering capacity of treated PMNL were evaluated. Some of the in vitro assays also included use of small interfering ORAI1 RNA (siORAI1), 100 nM 1,25(OH)2D3, or 100 nM parathyroid hormone (PTH). Intracellular Ca2+ concentration was markedly lower in HYPOCAL. In addition, ORAI1 was detected in PMNL plasma membrane via FACS and was markedly lower in cows with HYPOCAL. Migration, adhesion capacity, and phagocytosis rate of PMNL were lower in response to HYPOCAL. Furthermore, plasma and PMNL concentration of nucleosome assembly protein (NAP2) and pro-platelet basic protein (CXCL7) was markedly lower with HYPOCAL. All these changes were associated with lower ROS production by PMNL. Thapsigargin and ionomycin treatment in vitro increased ORAI1 expression, migration of PMNL, adhering capacity, phagocytosis rate, and ROS production; conversely, those effects were abrogated by siORAI1 and ORAI1 inhibitor 2APB treatment. Also cytosolic Ca2+ concentration and ORAI1 abundance were increased by 1,25(OH)2D3 and PTH supplementation. Overall, the data indicate that failure of PMNL to uptake Ca2+ due to downregulation of ORAI1 during subclinical hypocalcemia is a factor contributing to impaired PMNL function. In addition, plasma PTH or 1,25(OH)2D3 could regulate ORAI1 and also participate in the regulation of PMNL activity.