Neutrophil Priming Research Articles

TLR Signaling Rescues Fungicidal Activity in Syk-Deficient Neutrophils.

An impaired neutrophil response to pathogenic fungi puts patients at risk for fungal infections with a high risk of morbidity and mortality. Acquired neutrophil dysfunction in the setting of iatrogenic immune modulators can include the inhibition of critical kinases such as spleen tyrosine kinase (Syk). In this study, we used an established system of conditionally immortalized mouse neutrophil progenitors to investigate the ability to augment Syk-deficient neutrophil function against Candida albicans with TLR agonist signaling. LPS, a known immunomodulatory molecule derived from Gram-negative bacteria, was capable of rescuing effector functions of Syk-deficient neutrophils, which are known to have poor fungicidal activity against Candida species. LPS priming of Syk-deficient mouse neutrophils demonstrates partial rescue of fungicidal activity, including phagocytosis, degranulation, and neutrophil swarming, but not reactive oxygen species production against C. albicans, in part due to c-Fos activation. Similarly, LPS priming of human neutrophils rescues fungicidal activity in the presence of pharmacologic inhibition of Syk and Bruton's tyrosine kinase (Btk), both critical kinases in the innate immune response to fungi. In vivo, neutropenic mice were reconstituted with wild-type or Syk-deficient neutrophils and challenged i.p. with C. albicans. In this model, LPS improved wild-type neutrophil homing to the fungal challenge, although Syk-deficient neutrophils did not persist in vivo, speaking to its crucial role on in vivo persistence. Taken together, we identify TLR signaling as an alternate activation pathway capable of partially restoring neutrophil effector function against Candida in a Syk-independent manner.

Circulating neutrophil activation in dogs with naturally occurring spinal cord injury secondary to intervertebral disk herniation.

To investigate the time course of circulating neutrophil priming and activity in dogs with spinal cord injury secondary to intervertebral disk herniation that undergo decompressive surgery. 9 dogs with spinal cord injury and 9 healthy dogs (controls). For dogs with spinal cord injury, blood samples were collected on the day of hospital admission and 3, 7, 30, and 90 days after injury and decompressive surgery. A single blood sample was collected from the control dogs. Flow cytometry analysis was performed on isolated neutrophils incubated with antibody against CD11b and nonfluorescent dihydrorhodamine 123, which was converted to fluorescent rhodamine 123 to measure oxidative burst activity. Expression of CD11b was increased in dogs with spinal cord injury 3 days after injury and decompressive surgery, relative to day 7 expression. Neutrophils expressed high oxidative burst activity both 3 and 7 days after injury and decompressive surgery, compared with activity in healthy dogs. For dogs with spinal cord injury, high CD11b expression 3 days after injury and decompressive surgery was consistent with findings for rodents with experimentally induced spinal cord injury. However, the high oxidative burst activity 3 and 7 days after injury and decompressive surgery was not consistent with data from other species, and additional studies on inflammatory events in dogs with naturally occurring spinal cord injury are needed.

Expansion of Immature Neutrophils During SIV Infection Is Associated With Their Capacity to Modulate T-Cell Function.

In spite of the efficacy of combinational antiretroviral treatment (cART), HIV-1 persists in the host and infection is associated with chronic inflammation, leading to an increased risk of comorbidities, such as cardiovascular diseases, neurocognitive disorders, and cancer. Myeloid cells, mainly monocytes and macrophages, have been shown to be involved in the immune activation observed in HIV-1 infection. However, less attention has been paid to neutrophils, the most abundant circulating myeloid cell, even though neutrophils are strongly involved in tissue damage and inflammation in several chronic diseases, in particular, autoimmune diseases. Herein, we performed a longitudinal characterization of neutrophil phenotype and we evaluated the interplay between neutrophils and T cells in the model of pathogenic SIVmac251 experimental infection of cynomolgus macaques. We report that circulating granulocytes consists mainly of immature CD10- neutrophils exhibiting a prime phenotype during primary and chronic infection. We found that neutrophil priming correlates with CD8+ T cell activation. Moreover, we provide the evidence that neutrophils are capable of modulating CD4+ and CD8+ T-cell proliferation and IFN-γ production in different ways depending on the time of infection. Thus, our study emphasizes the role of primed immature neutrophils in the modulation of T-cell responses in SIV infection.

A functionally distinct neutrophil landscape in severe COVID-19 reveals opportunities for adjunctive therapies

Acute respiratory distress syndrome (ARDS) is a life-threatening syndrome, constituted by respiratory failure and diffuse alveolar damage that results from dysregulated local and systemic immune activation, causing pulmonary vascular, parenchymal, and alveolar damage. SARS-CoV-2 infection has become the dominant cause of ARDS worldwide, and emerging evidence implicates neutrophils and their cytotoxic arsenal of effector functions as central drivers of immune-mediated lung injury in COVID-19 ARDS. However, key outstanding questions are whether COVID-19 drives a unique program of neutrophil activation or effector functions that contribute to the severe pathogenesis of this pandemic illness and whether this unique neutrophil response can be targeted to attenuate disease. Using a combination of high-dimensional single-cell analysis and ex vivo functional assays of neutrophils from patients with COVID-19 ARDS, compared with those with non-COVID ARDS (caused by bacterial pneumonia), we identified a functionally distinct landscape of neutrophil activation in COVID-19 ARDS that was intrinsically programmed during SARS-CoV-2 infection. Furthermore, neutrophils in COVID-19 ARDS were functionally primed to produce high amounts of neutrophil extracellular traps. Surprisingly, this unique pathological program of neutrophil priming escaped conventional therapy with dexamethasone, thereby revealing a promising target for adjunctive immunotherapy in severe COVID-19.

TREM-1 is required for enhanced OpZ-induced superoxide generation following priming.

Inflammatory agents, microbial products, or stromal factors pre-activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM-1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF-α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem-1-/- mice were transduced with estrogen regulated Homeobox8 (ER-Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM-1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM-1 and was not altered by priming. In contrast, priming significantly enhanced OpZ-induced oxygen consumption and superoxide production in WT but not Trem-1-/- neutrophils indicating that TREM-1 is required for primed oxidative burst. TREM-1-dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM-1 shedding, and IL-1ß release to the same extent in WT and Trem-1-/- neutrophils. Thus, TREM-1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming.

A rare CTSC mutation in Papillon-Lefèvre Syndrome results in abolished serine protease activity and reduced NET formation but otherwise normal neutrophil function.

Papillon-Lefèvre Syndrome (PLS) is an autosomal recessive monogenic disease caused by loss-of-function mutations in the CTSC gene, thus preventing the synthesis of the protease Cathepsin C (CTSC) in a proteolytically active form. CTSC is responsible for the activation of the pro-forms of the neutrophil serine proteases (NSPs; Elastase, Proteinase 3 and Cathepsin G), suggesting its involvement in a variety of neutrophil functions. In PLS neutrophils, the lack of CTSC protease activity leads to inactivity of the NSPs. Clinically, PLS is characterized by an early, typically pre-pubertal, onset of severe periodontal pathology and palmoplantar hyperkeratosis. However, PLS is not considered an immune deficiency as patients do not typically suffer from recurrent and severe (bacterial and fungal) infections. In this study we investigated an unusual CTSC mutation in two siblings with PLS, a 503A>G substitution in exon 4 of the CTSC gene, expected to result in an amino acid replacement from tyrosine to cysteine at position 168 of the CTSC protein. Both patients bearing this mutation presented with pronounced periodontal pathology. The characteristics and functions of neutrophils from patients homozygous for the 503A>G CTSC mutation were compared to another previously described PLS mutation (755A>T), and a small cohort of healthy volunteers. Neutrophil lysates from patients with the 503A>G substitution lacked CTSC protein and did not display any CTSC or NSP activity, yet neutrophil counts, morphology, priming, chemotaxis, radical production, and regulation of apoptosis were without any overt signs of alteration. However, NET formation upon PMA-stimulation was found to be severely depressed, but not abolished, in PLS neutrophils.

Activation and Functional Priming of Blood Neutrophils in Non-Alcoholic Fatty Liver Disease Increases in Non-Alcoholic Steatohepatitis.

IntroductionIn non-alcoholic fatty liver disease (NAFLD), neutrophils in liver infiltrates are activated, which may contribute to disease progression towards non-alcoholic steatohepatitis (NASH). However, the functional status of the blood neutrophils remains unknown and their role in the disease mechanisms is thus uncertain. We therefore characterized activation and function of blood neutrophils in patients with NAFLD in relation to clinical disease markers and the NAFLD plasma milieu.MethodsWe studied 20 patients with NAFLD, among these 6 patients with NASH, and 14 healthy persons. Neutrophil activation, interleukin (IL)-8 production and oxidative burst were measured by flow cytometry on participants´ neutrophils and on healthy neutrophils exposed in vitro to plasma from the study participants.ResultsBlood neutrophils from the NASH patients showed a doubling in their expression of the activation marker CD62L. Also, all NAFLD patients had 50–100% increased expression of CD11b. Functionally, NASH neutrophils had 30% elevated IL-8 production and more than doubled spontaneous oxidative burst. In all NAFLD patients, higher spontaneous oxidative burst was associated with worse liver function. Incubation of healthy neutrophils with NAFLD plasma paradoxically slightly reduced CD62L and CD11b expression, and NASH plasma also reduced the frequency of IL-8-producing neutrophils.ConclusionIn NAFLD, blood neutrophils are activated, and in NASH also functionally primed. This suggests a progressive neutrophil aggressiveness already present with liver fat infiltration. However, NAFLD plasma in vitro, if anything, had the opposite effect on the healthy neutrophils so the NAFLD-related neutrophil activation cannot be attributed to humoral factors and remains unexplained.

Antineutrophil cytoplasmic antibody (ANCA) vasculitis: pathophysiology, diagnosis, and the evolving treatment landscape.

The antineutrophil cytoplasmic antibody (ANCA) vasculitides include several closely related, often severe, multisystem autoimmune diseases characterized by antibodies against serine proteinase 3 (PR3) or myeloperoxidase. Loss of tolerance to these antigens triggers a cascade of events, beginning with the priming of neutrophils by proinflammatory cytokines and complement activation, translocation of ANCA-specific antigens to the plasma membrane, neutrophil hyperactivation, and further activation of the alternative complement pathway, leading to tissue damage and the clinical manifestations of ANCA vasculitis. Due to the heterogeneity in presentation of these diseases, diagnosis is often substantially delayed, leading to poor outcomes. The current treatment pathway for most patients involves induction with cyclophosphamide or rituximab in combination with glucocorticoids, followed by a maintenance phase with rituximab, azathioprine, or methotrexate, during which time glucocorticoids are tapered. Current therapies are often effective in inducing and maintaining remission but are associated with a range of toxicities. Several new therapies are in development for ANCA vasculitis. Avacopan, an orally administered inhibitor of the complement fragment 5a (C5a) receptor, has been assessed in a phase 3 clinical trial and may play a role in reducing the cumulative glucocorticoid dose. Preliminary data suggest that cluster of differentiation (CD) 80 and CD86 blockade with abatacept may also have a role in the management of ANCA vasculitis. There is an unmet need for additional therapeutic options for patients with these diseases.

Acetate sensing by GPR43 alarms neutrophils and protects from severe sepsis

Bacterial sepsis is a major cause of mortality resulting from inadequate immune responses to systemic infection. Effective immunomodulatory approaches are urgently needed but it has remained elusive, which targets might be suitable for intervention. Increased expression of the G-protein-coupled receptor GPR43, which is known to govern intestinal responses to acetate, has been associated with sepsis patient survival but the mechanisms behind this observation have remained unclear. We show that elevated serum acetate concentrations prime neutrophils in a GPR43-dependent fashion, leading to enhanced neutrophil chemotaxis, oxidative burst, cytokine release and upregulation of phagocytic receptors. Consequently, acetate priming improved the capacity of human neutrophils to eliminate methicillin-resistant Staphylococcus aureus. Acetate administration increased mouse serum acetate concentrations and primed neutrophils. Notably, it rescued wild-type mice from severe S. aureus sepsis and reduced bacterial numbers in peripheral organs by several magnitudes. Acetate treatment improved the sepsis course even when applied several hours after onset of the infection, which recommends GPR43 as a potential target for sepsis therapy. Our study indicates that the severity of sepsis depends on transient neutrophil priming by appropriate blood acetate concentrations. Therapeutic interventions based on GPR43 stimulation could become valuable strategies for reducing sepsis-associated morbidity and mortality.

A cross-sectional cohort study of the activity and turnover of neutrophil granulocytes in juvenile idiopathic arthritis

BackgroundThe inflammatory process in juvenile idiopathic arthritis (JIA) involves both the innate and the adaptive immune system. The turnover and activity of neutrophil granulocytes may be reflected by proteins secreted from primary or secondary granules and from the cytoplasm of sequestered cells. Our primary aim was to compare the levels of the secondary neutrophil granule protein human neutrophil lipocalin (HNL), in JIA patients and controls, and to explore a possible priming of neutrophils through parallel analyses in plasma and serum. A secondary aim was to relate the levels of HNL to two other well-studied leukocyte proteins, S100A8/A9 and myeloperoxidase (MPO), as well as to clinical aspects of JIA.MethodsThe concentrations of the three biomarkers in serum, two of them also in plasma, were measured using enzyme-linked immunosorbent assay in 37 children with JIA without medical treatment, in high disease activity based on juvenile arthritis disease activity score 27 (JADAS27), 32 children on medical treatment, mainly in lower disease activity, and 16 healthy children. We assessed for differences between two groups using the Mann-Whitney U test, and used the Kruskal-Wallis test for multiple group comparisons. Spearman rank correlation, linear and multiple regression analyses were used for evaluation of associations between biomarker concentrations and clinical scores.ResultsThe concentrations of HNL and MPO in serum were significantly increased in children with JIA (p < 0.001, p = 0.002) compared with healthy children, but we found no difference in the plasma levels of HNL and MPO between children with JIA and controls. The serum concentrations of MPO and HNL were unaffected by medical treatment, but S100A8/A9 was reduced by medical treatment and correlated with JADAS27 in both univariate (r = 0.58, p < 0.001) and multivariate (r = 0.59, p < 0.001) analyses.ConclusionsNeutrophil granulocytes in children with JIA are primed to release primary and secondary granule proteins, without relation to medical treatment, whereas signs of increased turnover and sequestration of neutrophil granulocytes are reduced by treatment. Levels of neutrophil-originating proteins in serum most likely reflect underlying disease activities of JIA.

Extrathoracic multiple trauma dysregulates neutrophil function and exacerbates pneumonia-induced lung injury.

Forty percent of critically ill trauma patients will develop an infectious complication. Pneumonia is the most common cause of death of trauma patients surviving their initial insult. We previously demonstrated that polytrauma (PT), defined as two or more severe injuries in at least two areas of the body, induces emergency hematopoiesis characterized by accelerated myelopoiesis in the bone marrow and increased myeloid cell frequency in the peripheral tissues. We hypothesized that PT alone induces priming of neutrophils, resulting in hyperactivation upon secondary exposure to bacteria and causing acute lung injury and increased susceptibility to secondary exposure to Pseudomonas aeruginosa pneumonia. C57BL/6 mice were subjected to PT consisting of a lower extremity pseudofracture, liver crush injury, and 15% blood-volume hemorrhage. Pneumonia was induced by intratracheal injection of 5 × 106 CFU live P. aeruginosa or 1 × 107 of heat-killed P. aeruginosa (HKPA). For reactive oxygen species (ROS), studies polymorphonuclear neutrophils (PMNs) were isolated by immunomagnetic bead negative selection and stimulated ex-vivo with HKPA. Reactive oxygen species production was measured by immunofluorescence. For histology, lung sections were stained by hematoxylin-eosin and analyzed by a blinded grader. Polytrauma induced persistent changes in immune function at baseline and to secondary infection. Pneumonia after injury resulted in increased mortality (60% vs. 5% p < 0.01). Blood neutrophils from PT mice had higher resting (unstimulated) ROS production than in naive animals (p < 0.02) demonstrating priming of the neutrophils following PT. After intratracheal HKPA injection, bronchoalveolar lavage PMNs from injured mice had higher ROS production compared with naive mice (p < 0.01), demonstrating an overexuberant immunopathologic response of neutrophils following PT. Polytrauma primes neutrophils and causes immunopathologic PMN ROS production, increased lung injury and susceptibility to secondary bacterial pneumonia. These results suggest that trauma-induced immune dysfunction can cause immunopathologic response to secondary infection and suggests neutrophil-mediated pulmonary damage as a therapeutic target for posttrauma pneumonia.

Mature neutrophils acquire a T cell suppressor phenotype in the ovarian cancer microenvironment characterized by disruption of key T cell signaling and metabolic responses

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Despite being an immunogenic tumor, single-agent checkpoint inhibitor therapy in the recurrent setting has been disappointing, raising the notion of suppressive factors in the tumor microenvironment (TME) as obstacles to immunotherapy. We previously observed that neutrophils acquire a complement dependent T cell suppressor phenotype in the TME, characterized by inhibition of T cell proliferation and activation through mechanisms distinct from myeloid-derived suppressor cells. Using ascites fluid supernatants (ASC) from patients with EOC as an authentic component of the TME, we identified ASC-activated neutrophils require multiple neutrophil effector functions including NADPH oxidase activation, SNARE-mediated exocytosis and degranulation. ASC-activated neutrophils also adhered to T cells and caused trogocytosis of T cell membranes. These injury and signaling cues resulted in T cell immunoparalysis, characterized by impaired CD3/CD28 stimulated Zap70 phosphorylation, NFAT translocation, IL2 production, glucose uptake, mitochondrial function, and mTOR activation. We also observed that in anti-CD3/CD28 pre-activated T cells, addition of neutrophils + ASC resulted significant reduction in Zap70 phosphorylation. Our results demonstrate complement-dependent priming of neutrophils in the TME inducing a T cell non-responsiveness distinct from established checkpoint pathways and identify targets for immunotherapy. Further studies will focus on the effects of neutrophil suppressors on the TCR signaling cascade and cell stress response pathways that may enable T cell survival in response to neutrophil damage at the expense of activation.

Pinaverium Bromide Attenuates Lipopolysaccharide-Induced Excessive Systemic Inflammation via Inhibiting Neutrophil Priming

Abstract Dominant infiltration of neutrophils is a hallmark of many inflammatory diseases, especially in septic shock. IL-1β as one of the most early released proinflammatory cytokine in neutrophil, plays a pivotal role in the progress of sepsis. In this study, we built a high-throughput–compatible drug screen assay platform based on our newly constructed reporter C57BL/6 mice, pIL1-DsRed, expressing the DsRed gene under the control of the IL-1β promoter. After screening 1200 U.S. Food and Drug Administration–approved compounds, we found that pinaverium bromide (PVB) significantly suppressed the DsRed expression of primed neutrophil and improved the survival rate of mice given LPS in an endotoxin challenge analogous to sepsis, regardless of whether PVB was administered before or after LPS. PVB also protected the liver and lung from LPS-induced damage and reduced organ-specific inflammatory responses. PVB decreased the production of IL-1β, IL-6, and CXCL1 mRNA in the lungs of LPS-treated mice and decreased the serum levels of liver transaminases (alanine aminotransferase and aspartate aminotransferase) at multiple time points and doses tested. PVB can significantly suppress primed neutrophil-specific respiratory bursts and migration as well. Lastly, PVB affected neutrophils’ gene expression and phenotypic changes during neutrophil priming. PVB downregulated GM-CSF–induced expression of CD54 and dectin-2 (markers of fully primed neutrophils) at both mRNA and protein levels during late-phase neutrophil priming. In summary, we demonstrated that PVB can be used as a potential therapeutic agent for sepsis by inhibiting neutrophil priming.

Neutrophils from hereditary hemochromatosis patients are protected from iron excess and are primed

AbstractIron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, neutrophil function may be severely altered in conditions of iron overload, as observed in chronically transfused patients. Therefore, a tight regulation of neutrophil iron homeostasis seems to be critical for avoiding iron toxicity. Hepcidin is the key iron regulator in organisms; however, no studies have investigated its role in maintaining neutrophil iron homeostasis or characterized neutrophil function in patients with hereditary hemochromatosis (HH), a common iron overload genetic disorder that results from a defect in hepcidin production. To explore these issues, we studied 2 mouse models of iron overload: an experimentally induced iron overload model (EIO), in which hepcidin is increased, and a genetic HH model of iron overload with a deletion of hepatic hepcidin. We found that iron-dependent increase of hepatic hepcidin results in neutrophil intracellular iron trapping and consecutive defects in oxidative burst activity. In contrast, in both HH mouse models and HH patients, the lack of hepcidin expression protects neutrophils from toxic iron accumulation. Moreover, systemic iron overload correlated with a surprising neutrophil priming and resulted in a more powerful oxidative burst. Indeed, important factors in neutrophil priming and activation, such as tumor necrosis factor α (TNF-α), VCAM-1, and ICAM-1 are increased in the plasma of HH patients and are associated with an increase in HH neutrophil phagocytosis capacity and a decrease in L-selectin surface expression. This is the first study to characterize neutrophil iron homeostasis and associated functions in patients with HH.

Innate Priming of Neutrophils Potentiates Systemic Multiorgan Injury.

Excessive inflammatory reactions mediated by first-responder cells such as neutrophils contribute to the severity of multiorgan failure associated with systemic injury and infection. Systemic subclinical endotoxemia due to mucosal leakage may aggravate neutrophil activation and tissue injury. However, mechanisms responsible for neutrophil inflammatory polarization are not well understood. In this study, we demonstrate that subclinical low-dose endotoxemia can potently polarize neutrophils into an inflammatory state in vivo and in vitro, as reflected in elevated expression of adhesion molecules such as ICAM-1 and CD29, and reduced expression of suppressor molecule CD244. When subjected to a controlled administration of gut-damaging chemical dextran sulfate sodium, mice conditioned with subclinical dose LPS exhibit significantly elevated infiltration of neutrophils into organs such as liver, colon, and spleen, associated with severe multiorgan damage as measured by biochemical as well as histological assays. Subclinical dose LPS is sufficient to induce potent activation of SRC kinase as well as downstream activation of STAT1/STAT5 in neutrophils, contributing to the inflammatory neutrophil polarization. We also demonstrate that the administration of 4-phenylbutyric acid, an agent known to relieve cell stress and enhance peroxisome function, can reduce the activation of SRC kinase and enhance the expression of suppressor molecule CD244 in neutrophils. We show that i.v. injection of 4-phenylbutyric acid conditioned neutrophils can effectively reduce the severity of multiorgan damage in mice challenged with dextran sulfate sodium. Collectively, our data, to our knowledge, reveal novel inflammatory polarization of neutrophils by subclinical endotoxemia conducive for aggravated multiorgan damage as well as potential therapeutic intervention.

Mannose-binding lectin and associate serine protease complex modulates neutrophil respiratory burst and gene expression in Capra hircus.

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

Synthetic Oligodeoxynucleotides in the Regulation of Leukotriene Synthesis in Human Neutrophils

Polymorphonuclear leukocytes (PMNLs, neutrophils) play a central role in the defense against microbial infections. In the immune response to bacterial and fungal infections neutrophils eliminate pathogens through phagocytosis. During phagocytosis neutrophils produce physiologically active compounds called leukotrienes. Neutrophil production of leukoriene B4 is responsible for a second wave of neutrophil recruitment during inflammation. In the activation of neutrophils, not only the bacteria themselves play a role, but also pathogen associated molecular patterns, in particular, bacterial DNA and its fragments. Neutrophil priming with bacterial products makes neutrophils more responsive to activating stimuli. Synthetic CpG‐oligodeoxynucleotides (CpG‐ODNs) mimic microbial DNA and are now widely used to study DNA‐mediated cellular events. In our study we compared the effects of synthetic oligodeoxynucleotides (ODNs) of different structure on leukotriene synthesis in neutrophils during Salmonella bacteria phagocytosis. We treated PMNLs with opsonized S. typhimurium at a ratio of bacteria:PMNLs in the range 20–25:1. Leukotrienes and other 5‐lipoxygenase products were detected after priming neutrophils by 30 min incubation with 1 μM ODN and further stimulation for 15 min with opsonized Salmonella bacteria. The neutrophil exposure to A‐class CpG‐ODN, with phosphodiester inner core, inhibited leukotriene synthesis in neutrophil interaction with bacteria Salmonella typhimurium. Sequences, containing the human telomeric (TTAGGG)n repeats, on the contrary, activated leukotriene synthesis. Effects of A‐class CpG‐ODNs were sensitive to inhibitors of endosomal acidification and probably induced intracellular signaling through binding to toll like receptor TLR9. Synthetic DNA fragments containing stimulating CpG motifs initiate TLR9 signals, which lead to activation of the transcription factor NF‐kB. In our assays NF‐kB inhibitor BAY11‐7082 abolished the effects of A‐class CpG ODNs and increased the effects of telomeric ODNs on leukotriene synthesis. The results of our study revealed that the signal to inhibition of leukotriene synthesis by A‐class CpG‐ODNs and to activation of leukotriene synthesis by telomeric ODNs may be due to interaction of both classes of ODNs with TLR9 on neutrophils.Ethics statement. The authors prepared neutrophils from the blood of healthy volunteers. Blood was collected via venous puncture, as approved by the Ministry of Public Health Service of the Russian Federation. Experimental and the subject consent procedures were approved by the Ethics Committee of the A. N. Belozersky Institute of Physico‐Chemical Biology, Moscow State University. Fully informed consent was obtained, and all investigations were conducted according to the principles laid down in the Declaration of Helsinki.Support or Funding InformationThis study was funded by a grant from the Russian Foundation for Basic Research (17‐04‐01491)

Uric acid-mediated inflammasome activation in IL-6 primed innate immune cells is regulated by baricitinib

Objectives Gout is an inflammatory arthropathy caused by the deposition of monosodium urate (MSU). The synthesis and release of IL-1β is crucial for MSU-induced synovial inflammation. The aim of the present study was to investigate the mechanism of MSU crystal-induced autoinflammatory processes. Methods In vitro studies were used to evaluate the role of IL-6 in inflammasome activation in human neutrophils cultured with MSU crystals. Human neutrophils were stimulated with MSU in the presence or absence of IL-6 priming to determine NLRP3 inflammasome activation and subsequent cleaved caspase-1 induction or IL-1β production. Results IL-6 or MSU stimulation alone did not result in the efficient IL-1β production from human neutrophils. However, MSU stimulation induced marked IL-1β production from IL-6-primed neutrophils. Pretreatment with baricitinib, which blocks IL-6 receptor signaling, prevented MSU-induced cleaved caspase-1 or IL-1β induction in IL-6-primed neutrophils. Tocilizumab pretreatment also inhibited MSU-mediated IL-1β production from IL-6-primed neutrophils. Conclusion Priming of human neutrophils with IL-6 promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These results suggest that an endogenous cytokine, IL-6, is involved in MSU-mediated NLRP3 inflammasome activation and subsequent IL-1β production from innate immune cells and has a crucial role in MSU crystal-induced synovial inflammation. These findings provide insights into uric acid-mediated autoinflammation in the innate immune system.

TRPV4-A Missing Link Between Mechanosensation and Immunity.

Transient receptor potential vanilloid-type 4 (TRPV4) cation channel is widely expressed in all tissues as well as in immune cells and its function as mechanosensitive Ca2+ channel seems to be conserved throughout all mammalian species. Of late, emerging evidence has implicated TRPV4 in the activation and differentiation of innate immune cells, especially in neutrophils, monocytes, and macrophages. As such, TRPV4 has been shown to mediate neutrophil adhesion and chemotaxis, as well as production of reactive oxygen species in response to pro-inflammatory stimuli. In macrophages, TRPV4 mediates formation of both reactive oxygen and nitrogen species, and regulates phagocytosis, thus facilitating bacterial clearance and resolution of infection. Importantly, TRPV4 may present a missing link between mechanical forces and immune responses. This connection has been exemplary highlighted by the demonstrated role of TRPV4 in macrophage activation and subsequent induction of lung injury following mechanical overventilation. Mechanosensation via TRPV4 is also expected to activate innate immune cells and establish a pro-inflammatory loop in fibrotic diseases with increased deposition of extracellular matrix (ECM) and substrate stiffness. Likewise, TRPV4 may be activated by cell migration through the endothelium or the extracellular matrix, or even by circulating immune cells squeezing through the narrow passages of the pulmonary or systemic capillary bed, a process that has recently been linked to neutrophil priming and depriming. Here, we provide an overview over the emerging role of TRPV4 in innate immune responses and highlight two distinct modes for the activation of TRPV4 by either mechanical forces (“mechanoTRPV4”) or by pathogens (“immunoTRPV4”).

Neutrophil priming as a risk factor for surgical site infection in patients with colon cancer treated by laparoscopic surgery

BackgroundThe purpose of this study is to identify perioperative marker predicting postoperative surgical site infection (SSI) including with anastomotic leakage (AL) in curative colon cancer patients, laparoscopically.MethodsIn total, 135 colon cancer patients (stage I–III) undergoing curative laparoscopic surgery between January 2004 and December 2013 were enrolled in this study. We collected data on clinicopathological factors, laboratory data on pre and postoperative day 3 (POD3) and tumor markers levels to assess the relation to surgical site infection (SSI) including with anastomotic leakage (AL).ResultsSSI and AL occurred in 16 cases (5.6%) and 4 cases (3%), respectively. SSI and AL were not association with clinicopathological factors. Within laboratory data and tumor markers preoperatively, high neutrophil counts were significantly associated with SSI (P < 0.05) and AL (P < 0.01), respectively. Area under curves (AUC) of SSI and AL were 0.656 and 0.854, respectively. In addition, high neutrophil counts on POD3 also were significantly associated with SSI (P < 0.01) and AL (P < 0.01), respectively. Area under curves (AUC) of SSI and AL were 0.747 and 0.832, respectively.ConclusionNeutrophil count on pre and POD3 are potentially valuable indicators of SSI including with AL in colon cancer patients undergoing curative surgery laparoscopically.