Response Of Polymorphonuclear Neutrophils Research Articles

Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction

BackgroundThe function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals.ResultsBacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging.ConclusionsOur findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples.Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions.Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively.Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Development of a screening protocol to identify persons who are responsive to wood smoke particle-induced airway inflammation with pilot assessment of GSTM1 genotype and asthma status as response modifiers

Background We are currently screening human volunteers to determine their sputum polymorphonuclear neutrophil (PMN) response 6- and 24-hours following initiation of exposure to wood smoke particles (WSP). Inflammatory responders (≥10% increase in %PMN) are identified for their subsequent participation in mitigation studies against WSP-induced airways inflammation. In this report we compared responder status (N = 52) at both 6 and 24 hr time points to refine/expand its classification, assessed the impact of the GSTM1 genotype, asthma status and sex on responder status, and explored whether sputum soluble phase markers of inflammation correlate with PMN responsiveness to WSP. Results Six-hour responders tended to be 24-hour responders and vice versa, but 24-hour responders also had significantly increased IL-1beta, IL-6, IL-8 at 24 hours post WSP exposure. The GSTM1 null genotype significantly (p < 0.05) enhanced the %PMN response by 24% in the 24-hour responders and not at all in the 6 hours responders. Asthma status enhanced the 24 hour %PMN response in the 6- and 24-hour responders. In the entire cohort (not stratified by responder status), we found a significant, but very small decrease in FVC and systolic blood pressure immediately following WSP exposure and sputum %PMNs were significantly increased and associated with sputum inflammatory markers (IL-1beta, IL-6, IL-8, and PMN/mg) at 24 but not 6 hours post exposure. Blood endpoints in the entire cohort showed a significant increase in %PMN and PMN/mg at 6 but not 24 hours. Sex had no effect on %PMN response. Conclusions The 24-hour time point was more informative than the 6-hour time point in optimally and expansively defining airway inflammatory responsiveness to WSP exposure. GSTM1 and asthma status are significant effect modifiers of this response. These study design and subject parameters should be considered before enrolling volunteers for proof-of-concept WSP mitigation studies.

Roles of Polymorphonuclear Neutrophils in Ischemic Brain Injury and Post-Ischemic Brain Remodeling.

Following ischemic stroke, polymorphonuclear neutrophils (PMNs) are rapidly recruited to the ischemic brain tissue and exacerbate stroke injury by release of reactive oxygen species (ROS), proteases and proinflammatory cytokines. PMNs may aggravate post-ischemic microvascular injury by obstruction of brain capillaries, contributing to reperfusion deficits in the stroke recovery phase. Thus, experimental studies which specifically depleted PMNs by delivery of anti-Ly6G antibodies or inhibited PMN brain entry, e.g., by CXC chemokine receptor 2 (CXCR2) or very late antigen-4 (VLA-4) blockade in the acute stroke phase consistently reduced neurological deficits and infarct volume. Although elevated PMN responses in peripheral blood are similarly predictive for large infarcts and poor stroke outcome in human stroke patients, randomized controlled clinical studies targeting PMN brain infiltration did not improve stroke outcome or even worsened outcome due to serious complications. More recent studies showed that PMNs have decisive roles in post-ischemic angiogenesis and brain remodeling, most likely by promoting extracellular matrix degradation, thereby amplifying recovery processes in the ischemic brain. In this minireview, recent findings regarding the roles of PMNs in ischemic brain injury and post-ischemic brain remodeling are summarized.

The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro.

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.

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.

Periodontal Inflammation Primes the Systemic Innate Immune Response

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

The Neutrophil: Constant Defender and First Responder

The role of polymorphonuclear neutrophils (PMNs) in biology is often recognized during pathogenesis associated with PMN hyper- or hypo-functionality in various disease states. However, in the vast majority of cases, PMNs contribute to resilience and tissue homeostasis, with continuous PMN-mediated actions required for the maintenance of health, particularly in mucosal tissues. PMNs are extraordinarily well-adapted to respond to and diminish the damaging effects of a vast repertoire of infectious agents and injurious processes that are encountered throughout life. The commensal biofilm, a symbiotic polymicrobial ecosystem that lines the mucosal surfaces, is the first line of defense against pathogenic strains that might otherwise dominate, and is therefore of critical importance for health. PMNs regularly interact with the commensal flora at the mucosal tissues in health and limit their growth without developing an overt inflammatory reaction to them. These PMNs exhibit what is called a para-inflammatory phenotype, and have reduced inflammatory output. When biofilm growth and makeup are disrupted (i.e., dysbiosis), clinical symptoms associated with acute and chronic inflammatory responses to these changes may include pain, erythema and swelling. However, in most cases, these responses indicate that the immune system is functioning properly to re-establish homeostasis and protect the status quo. Defects in this healthy everyday function occur as a result of PMN subversion by pathological microbial strains, genetic defects or crosstalk with other chronic inflammatory conditions, including cancer and rheumatic disease, and this can provide some avenues for therapeutic targeting of PMN function. In other cases, targeting PMN functions could worsen the disease state. Certain PMN-mediated responses to pathogens, for example Neutrophil Extracellular Traps (NETs), might lead to undesirable symptoms such as pain or swelling and tissue damage/fibrosis. Despite collateral damage, these PMN responses limit pathogen dissemination and more severe damage that would otherwise occur. New data suggests the existence of unique PMN subsets, commonly associated with functional diversification in response to particular inflammatory challenges. PMN-directed therapeutic approaches depend on a greater understanding of this diversity. Here we outline the current understanding of PMNs in health and disease, with an emphasis on the positive manifestations of tissue and organ-protective PMN-mediated inflammation.

Gut-lymph-lung pathway mediates sepsis-induced acute lung injury.

This review attempts to unveil the possible mechanisms underlying how gut lymph affects lung and further gives rise to acute respiratory distress syndrome, as well as potential interventional targets under the condition of ischemia-reperfusion injury. We searched electronic databases including PubMed, MEDLINE, Cochrane Central Register of Controlled Trials, Google Scholar, Web of Science, and Embase to identify relevant literatures published up to December 2019. We enrolled the literatures including the Mesh Terms of “gut lymph or intestinal lymph and acute lung injury or acute respiratory distress syndrome.” Gut is considered to be the origin of systemic inflammation and the engine of multiple organ distress syndrome in the field of critical care medicine, whereas gut lymph plays a pivotal role in initiation of ischemia-reperfusion injury-induced acute respiratory distress syndrome. In fact, in the having been established pathologic model of sepsis leading to multiple organ dysfunction named by Gut Lymph theory, a variety of literatures showed the position and role of changes in gut lymph components in the initiation of systemic inflammatory response, which allows us to screen out potential intervention targets to pave the way for future clinic and basic research.

Modulation of neutrophil extracellular traps release by Klebsiella pneumoniae.

One of the main bactericidal mechanisms of polymorphonuclear neutrophils (PMN) is the release of neutrophil extracellular traps (NETs), which capture and destroy pathogens. Klebsiella pneumoniae (Kpn) producer of carbapenemase (KPC) and belonging to the sequence type 258 (ST258), is a hyper epidemic clone that causes a large number of infections worldwide associated with high persistence and mortality. It is necessary to investigate the interaction of Kpn KPC with the immune system to improve prevention and treatment of infections mediated by this bacterium. Based on the hypothesis that Kpn is able to subvert PMN-mediated death, the aim was to assess whether Kpn KPC ST258 could modulate the bactericidal response of PMN, focusing on NETs formation, compared to another opportunistic pathogen, as Escherichia coli (Eco). The results showed that the release of NETs was absent when PMN were challenged with Kpn KPC, while Eco was a strong inducer of NETosis. Moreover, Kpn KPC was able to inhibit NETosis induced by Eco. The inhibition of Kpn KPC-mediated NETs formation still occurred in spite of exogenous addition of hydrogen peroxide (H2 O2 ), did not involve bacterial-released soluble factors or cell wall components, and was dependent on bacterial viability. Moreover, when degranulation was investigated, we found that Kpn KPC affected only the mobilization of primary granules, which harbor the proteins with more potent bactericidal properties and those related to NETosis. In conclusion, Kpn KPC ST258 effectively managed to evade the PMN response by inhibiting the release of NETs, and primary granule mobilization.

The effects of functional platelet-derived extracellular mitochondria on the inflammatory phenotype of neutrophils

Abstract Unregulated inflammation has been associated with chronic inflammatory auto-immune diseases, such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. Polymorphonuclear neutrophils (PMNL) are amongst the immune cells involved in the acute inflammatory response used to fight bacterial infections. Once activated, PMNL release inflammatory mediators in the extracellular milieu to recruit various immune cells required to fight the invading pathogens. Platelets (PLTs) are also implicated in the body’s inflammatory response. Interestingly, activated PLTs can release fully functional mitochondria in the extracellular milieu. Known as the powerhouse of the cell, the mitochondria share similar characteristics with bacteria. Therefore, we hypothesize that PLTs-derived mitochondria present in the extracellular milieu, acting in a similar way as bacteria, induce a sterile inflammatory response that involves the PMNL. The objective of this study was to investigate the sterile inflammatory response of PMNL caused by the exposure of PLTs-derived extracellular mitochondria. Following the co-incubation of PMNL with various physiological doses of PLTs-derived mitochondria, a characterization of the interaction between PMNL and PLTs-derived mitochondria and an investigation of the inflammatory proprieties of PMNL was performed. We have shown that PLTs-derived mitochondria associate with PMNL, resulting in a 2.47-fold mitochondrial-dependent increase in oxygen consumption. Data also shows that PLTs-derived mitochondria significantly induce the release of PMNL microparticles. The knowledge gained from this study provides insight into the mechanism of sterile inflammation in auto-immune diseases.

Functional Platelet‐derived Mitochondria Induce the Release of Neutrophil Microparticles

INTRODUCTIONInflammation is an essential process of the host defense against infections, illness, or tissue injuries. However, unregulated inflammation has been associated with chronic inflammatory auto‐immune diseases, such as rheumatoid arthritis, multiple sclerosis and atherosclerosis. Polymorphonuclear neutrophils (PMNL) are amongst the immune cells involved in the acute inflammatory response used to fight bacterial infections. Once activated, PMNL release inflammatory mediators, enzymes and large quantities of microparticles in the extracellular milieu to recruit various immune cells required to fight the invading pathogens. Recent evidence also shows that platelets (PLTs), well known for their coagulation proprieties, are also implicated in the body’s inflammatory response. Interestingly, activated PLTs can release fully functional mitochondria in the extracellular milieu. Known as the powerhouse of the cell, the mitochondria share similar characteristics with bacteria. Therefore, we hypothesize that PLTs‐derived mitochondria present in the extracellular milieu, acting in a similar way as bacteria, induce a sterile inflammatory response that involves the PMNL.OBJECTIVESThe main objective of this study is to investigate the sterile inflammatory response of PMNL caused by the exposure of PLTs‐derived extracellular mitochondria.METHODSBlood was obtained from healthy consenting donors, then PMNL and PLTs‐derived mitochondria were isolated and purified. Following the co‐incubation of PMNL with various physiological doses of PLTs‐derived mitochondria, a characterization of the interaction between PMNL and PLTs‐derived mitochondria and an investigation of the inflammatory proprieties of PMNL was performed using flow cytometry, confocal microscopy, and high‐resolution respirometry.RESULTSOur data demonstrates that PLTs‐derived mitochondria associate with PMNL as quickly as 2 hours. This results in an increase of the PMNL intracellular calcium content and a 2.47‐fold mitochondrial‐dependent increase in oxygen consumption. Data also shows that PLTs‐derived mitochondria significantly induce, in a dose‐dependent manner, the release of PMNL microparticles.CONCLUSIONSThis study provides a new insight into the role of PLTs‐derived mitochondria in the context of sterile inflammation. Specifically, the characterization of its interaction with PMNL reveals a novel modulator in the fundamental inflammatory response. The knowledge gained from this study provides insight into the mechanism of sterile inflammation in auto‐immune diseases.Support or Funding InformationCanadian Institutes of Health Research, New Brunswick Health Research Foundation, New Brunswick Innovation Foundation

In Vitro Assay for Sensitive Determination of Human Blood PMN Responses.

Polymorphonuclear neutrophils (PMNs) are the most common leukocytes in the circulation and exhibit a wide range of distinct cellular phenomena as part of their microbicidal killing activities, including degranulation, phagocytosis, reactive oxygen species (ROS) production, adhesion, chemotaxis and production of PMN Extracellular Traps (NETs). As a simple in vitro test of PMN functional responses in human blood we have developed a multicolor flow cytometry-based assay of PMN cluster of differentiation (CD) surface marker expression. Short incubations of whole human blood can be performed in the presence of a wide range of agonists or inhibitors, followed by sensitive detection of changes in CD marker expression. This protocol has the advantage that small amounts of human blood are necessary and there are no PMN isolation steps, which can alter PMN activation status.

The Gut Microbiome Regulates Psychological Stress-Induced Inflammation in Sickle Cell Disease

Psychological stress (PS) is reported to precipitate vaso-occlusive episides (VOE) in sickle cell disease (SCD), but the mechanisms by which it does, are largely unknown. We subjected humanized (Berkeley) SCD mice to restraint stress and analyzed VOE using intravital microscopy. We found that stress exacerbated VOE as shown by reduced venular blood flow rates (control: 494 ± 30 × 103 pL/s; stress: 380 ± 20 × 103 pL/s; n = 25 - 43; p = 0.009) and reduced survival time (median survival time in control and stress groups: 394 min and 175 min, respectively; p = 0.02, Log-rank test). As previous studies show that adherent polymorphonuclear neutrophils (PMNs), especially aged PMNs marked by low CD62L and high CXCR4 expression, contribute to VOEs, we evaluated the behavior of leukocytes in live mice and observed a robust PMN response manifested by significantly increased adhesion and interactions with sickle RBC in stressed SCD animals. Flow cytometry analysis revealed a striking increase in aged PMNs in stressed SCD mice (control: 0.7 ± 0.1 × 106/ml; stress: 2.9 ± 0.3 × 106/ml; n = 3 - 4; p = 0.001), suggesting that the aged PMN expansion induced by stress may lead to the exacerbation of VOE in stressed SCD mice. Since aged PMN numbers are regulated by microbiota under steady state, we investigated whether response to PS also required the presence of the microbiota. Depletion of microbiota with broad-spectrum antibiotics (ABX) in SCD mice significantly prevented stress-induced aged PMN expansion (control: 4.8 ± 1.2 × 106/ml; ABX: 1.2 ± 0.3× 106/ml; n = 4 - 5; p = 0.0006), which was further confirmed using C57BL/6 germ-free (GF) mice (control: 1.1 ± 0.3 × 106/ml; GF: 0.2 ± 0.1 × 106/ml; n = 4; p = 0.003). Interestingly, analysis of inflammatory cytokines revealed a microbiota-dependent augmentation of IL-17A in the stressed mice. To investigate the role of IL-17A in the stress response, we neutralized IL-17A using an antibody (Ab) in C57BL/6 mice and found that IL-17A inhibition significantly dampened the aged PMN expansion induced by stress (isotype control Ab: 0.6 ± 0.1 × 106/ml; anti-IL-17A: 0.1 ± 0.0 × 106/ml; n = 4 - 5; p = 0.0004), which was also confirmed using IL-17A-/- mice. Further analyses revealed that Th17 lymphocytes were the major IL-17A-producing cells contributing to the stress response. In addition, depletion of microbiota with ABX or neutralization of IL-17A significantly alleviated stress-exacerbated VOE as measured by reduced PMN activation, improved blood flow rate and prolonged SCD mouse survival. As segmented filamentous bacteria (SFB) commensals were previously found to induce a Th17 response, we next tested the hypothesis that SFB was the driving bacteria in the stress response. Indeed, we found that PS-induced aged PMN expansion was greater in SFB+ mice (from Taconic) than in SFB- mice (from Jackson lab) (Taconic: 0.64 ± 0.11× 106/ml; Jax: 0.13 ± 0.02 × 106/ml; n = 4 - 5; p = 0.005). Depletion of SFB using vancomycin, a drug that eliminates Gram+ bacteria (including SFB), significantly reduced stress-induced aged PMN expansion. Furthermore, colonization of GF mice with SFB augmented aged PMN expansion after stress compared to stress control GF mice (control GF: 0.08 ± 0.02 × 106/ml; GF + SFB: 0.34 ± 0.10× 106/ml; n = 4 - 5; p = 0.04), suggesting that SFB was the key bacteria promoting aged PMN expansion during stress. The stress mechanisms involved the hypothalamic-pituitary-adrenal (HPA) axis since the blockade of glucocorticoid (GC) production using metyrapone or following adrenalectomy significantly prevented aged PMN expansion in stressed mice (PBS: 0.5 ± 0.1 × 106/ml; metyrapone: 0.1 ± 0.1× 106/ml; n = 5; p = 0.02. sham: 1.2 ± 0.2 × 106/ml; adrenalectomy: 0.4 ± 0.2 × 106/ml; n = 4 - 5; p = 0.01). Moreover, PS and dexamethasone greatly increased the gut permeability. Blockade of GC synthesis using metyrapone significantly alleviated stress-induced exacerbation of VOE manifested by reduced inflammation, improved the blood flow rate and prolonged SCD mice survival (median survival time in control and metyrapone groups: 155 min and 200 min, respectively; p = 0.02, Log-rank test). Overall, our results suggest that stress activation of the HPA axis increases the intestinal permeability, thereby promoting exposure to commensals that stimulate Th17 cells to produce IL-17A, which enables the expansion of aged polymorphonuclear neutrophils driving vaso-occlusion in SCD. Disclosures Frenette: Albert Einstein College of Medicine, Inc: Patents &amp; Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.

Klebsiella pneumoniae ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

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

Differential response of human blood leukocytes to brushite, monetite, and calcium polyphosphate biomaterials.

Calcium phosphate-based biomaterials are extensively used for bone replacement and regeneration in orthopedic, dental, and maxillofacial surgical applications. The injury induced by surgical implantation of bone replacement graft materials initiates a cascade of host responses, starting with blood-biomaterial contact, protein adsorption on the material surface, blood coagulation, and leukocyte responses. During the initial acute inflammatory response, polymorphonuclear neutrophils (PMNs) and monocytes, abundant circulating leukocytes of the myeloid lineage, are recruited to the site of inflammation. In addition to responding to pathogenic challenges, these cells respond to particulate substances within the body including crystals of monosodium urate (MSU). Host responses toward grafts impact short- and long-term success in tissue engineering and regenerative applications. Although multinucleated osteoclasts, formed by monocyte/macrophage fusion, are generally thought to be responsible for resorption of implant biomaterials, the ability of different biomaterials to trigger PMNs, which are invariably present at the early stages after implant surgery, and are abundant in the oral cavity, has never been tested. In this article, we present analysis of the response of human blood-derived PMNs and monocytes toward brushite, monetite, and calcium polyphosphate (CPP) biomaterial substrates and compare this to the response to MSU crystals, the latter serving as a positive control. Employing multicolor flow cytometry to look at PMN and monocyte cell surface markers of activation to gauge the response to different biomaterials, we found that both types of myeloid cells are highly activated after exposure to brushite, monetite, and MSU but not CPP. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:253-262, 2020.

Zearalenone Induces Estrogen-Receptor-Independent Neutrophil Extracellular Trap Release in Vitro.

Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin synthesized in Fusarium species, mainly Fusarium graminearum and Fusarium culmorum, and it has strong estrogenic activity and causes genotoxic effects, reproductive disorders, and immunosuppressive effects. Neutrophil extracellular trap (NET) has been studied for many years. Initially, NET was considered a form of the innate response that combats invading microorganisms. However, NET is involved in a series of pathophysiological mechanisms, including thrombosis, tissue necrosis, autoinflammation, and even autoimmunity. We recently found that polymorphonuclear neutrophils response to ZEA exposure by undergoing NET formation. However, the molecular mechanisms involved in this process remain poorly characterized. Here, we analyze whether estrogen receptors (ERs) can affect NET formation after ZEA stimulation. The involvement of ERs is investigated with the selective ER antagonists. Moreover, we investigate the mechanisms of NET formation using immunofluorescence staining, fluorescence microplate, and western blot analysis. Our results show that ERs (ERα and ERβ) are not involved in ZEA-induced NET formation, but reactive oxygen species (ROS), extracellular signal-regulated kinase (ERK), and p38 are postulated to be involved. Specifically, we provide data demonstrating that ZEA-induced ROS may promote activation of ERK and p38 as well as subsequent NET release. We are the first to demonstrate this new mechanism of ZEA-induced NET formation, which may help in understanding the role of ZEA in overexposure diseases and provide a relevant basis for therapeutic applications.

Frontline Science: Staphylococcus aureus promotes receptor-interacting protein kinase 3- and protease-dependent production of IL-1β in human neutrophils

Microbial infection elicits robust immune responses that initially depend on polymorphonuclear neutrophils (PMN), which ingest and kill invading bacteria. However, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) remain viable within PMN and prompt their lysis with concomitant release of damage-associated molecular patterns and proinflammatory cytokines that promote additional inflammation. Here, we show that ultrapure human PMN (>99.8% pure) that have ingested CA-MRSA released interleukin (IL)-1β but not IL-18. The ingested CA-MRSA needed to be viable, and phagocytosis alone was insufficient to stimulate IL-1β secretion from PMN fed CA-MRSA. In contrast to PMN response to the canonical NLRP3 inflammasome agonist nigericin, IL-1β secretion by PMN fed CA-MRSA occurred independently of NLRP3 inflammasome or caspase-1 activation and required instead active receptor-interacting protein kinase 3 (RIPK3) but not RIPK1. Furthermore, inhibition of neutrophil serine proteases blocked pro-IL-1β cleavage in PMN fed CA-MRSA. Taken together, our data suggest that with respect to secretion of IL-1β and IL-18, PMN differ from human macrophages and exhibit agonist-specific responses. After phagocytosis of CA-MRSA, human PMN secreted IL-1β through a previously unrecognized mechanism dependent on RIPK3 and serine proteases but independent of canonical NLRP3 inflammasome and caspase-1 activation.

Novel Mechanism behind the Immunopathogenesis of Vulvovaginal Candidiasis: "Neutrophil Anergy".

For over 3 decades, investigators have studied the pathogenesis of vulvovaginal candidiasis (VVC) and recurrent VVC (RVVC) through clinical studies and animal models. While there was considerable consensus that susceptibility was not associated with any apparent deficiencies in adaptive immunity, protective immune mechanisms and the role of innate immunity remained elusive. It was not until an innovative live-challenge design was conducted in women that a fuller understanding of the natural history of infection/disease was achieved. These studies revealed that symptomatic infection is associated with recruitment of polymorphonuclear neutrophils (PMNs) into the vaginal lumen. Subsequent studies in the established mouse model demonstrated that infiltrating PMNs were incapable of reducing the fungal burden, which supported the hypothesis that VVC/RVVC was an immunopathology, whereby Candida and the host response drive symptomatic disease. Further studies in mice revealed the requirement for C. albicans hyphae and identified pattern recognition receptors (PRRs) and proinflammatory mediators responsible for the PMN response, all of which are critical pieces of the immunopathogenesis. However, a mechanism explaining PMN dysfunction at the vaginal mucosa remained an enigma. Ultimately, by employing mouse strains resistant or susceptible to chronic VVC, it was determined that heparan sulfate (HS) in the vaginal environment of susceptible mice serves as a competitive ligand for Mac-1 on PMNs, which effectively renders the PMNs incapable of binding to Candida to initiate killing. Hence, the outcome of symptomatic VVC/RVVC is postulated to be dependent on a PMN-mediated immunopathogenic response involving HS that effectively places the neutrophils in a state of functional anergy.

Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems

Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to target reactive oxygen and nitrogen species during polymorphonuclear neutrophil inflammatory burst following injury. Here, we review the following 1) the broad effect of myeloperoxidase on normal physiology, 2) the difference between myeloperoxidase and other peroxidases, 3) the current optical probes available for use as surrogates for direct measures of myeloperoxidase-derived oxidants, and 4) the range of preclinical options for imaging myeloperoxidase accumulation at sites of inflammation in mice. We also stress the advantages and drawbacks of each of these methods, the pharmacokinetic considerations that may limit probe use to strictly cell cultures for some reactive oxygen and nitrogen species, rather than in vivo utility as indicators of myeloperoxidase function. Taken together, our review should shed light on the fundamental rational behind these techniques for measuring myeloperoxidase activity and polymorphonuclear neutrophil response after injury toward developing safe myeloperoxidase inhibitors as potential therapy for chronic obstructive pulmonary disease and rheumatoid arthritis.