Regulates Neutrophil Recruitment Research Articles

FRI-493 CLEC14A regulates neutrophil recruitment across liverendothelial cells and correlates with a neutrophil signature in hepatocellular carcinoma

FRI-493 CLEC14A regulates neutrophil recruitment across liverendothelial cells and correlates with a neutrophil signature in hepatocellular carcinoma

C-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

BRP39 Regulates Neutrophil Recruitment in NLRP3 Inflammasome-Induced Liver Inflammation

Background and AimsBRP-39 (Chi3L1) and its human homolog YKL-40, is an established biomarker of liver fibrosis in nonalcoholic steatohepatitis (NASH) patients, but its role in NASH pathogenesis remains unclear. We have recently identified Chi3L1 as one of the top upregulated genes in mice with inducible gain-of-function NOD-like receptor protein 3 (NLRP3) activation that mimics several liver features of NASH. This study aimed to investigate the effects of BRP39 deficiency on NLRP3-induced liver inflammation using Tamoxifen-inducible Nlrp3 knock-in mice sufficient (Nlrp3A350V CRT) and deficient for BRP39 (Nlrp3A350V /BRP-/- CRT). MethodsUsing Tamoxifen-inducible Nlrp3A350V knock-in mice (Nlrp3A350V CRT) mice and Tamoxifen-inducible Nlrp3A350V knock-in BRP39 deficient mice (Nlrp3A350V BRP-/- CRT), we investigated the consequence of BRP39 deficiency influencing NLRP3 induced liver inflammation. ResultsOur results showed that BRP39 deficiency in NLRP3 induced inflammation improved body weight and liver weight. Moreover, liver inflammation, fibrosis and HSC activation were significantly reduced, corresponding to significantly decreased Ly6C+ infiltrating macrophages, CD68+ OPN+ hepatic LAMs (lipid associated macrophages), and activated Ly6G+ and H3Cit+ neutrophils accumulation in the liver. Further investigation revealed that circulatory neutrophils from NLRP3 induced BRP39 deficient mice have impaired chemotaxis and migration ability, and this was confirmed by RNA-bulk sequencing showing reduced immune activation, migration and signaling responses in neutrophils. ConclusionThese data showcase the importance of BRP39 in regulating the NLRP3 inflammasome during liver inflammation and fibrotic NASH by altering cellular activation, recruitment and infiltration during disease progression, and revealing BRP39 to be a potential therapeutic target for future treatment of inflammatory NASH and its associated diseases.

Traditional Chinese Medicine Formulae QY305 Reducing Cutaneous Adverse Reaction and Diarrhea by its Nanostructure.

Traditional Chinese medicine (TCM) is widely used in clinical practice, including skin and gastrointestinal diseases. Here, a potential TCM QY305 (T-QY305) is reported that can modulate the recruitment of neutrophil in skin and colon tissue thus reducing cutaneous adverse reaction and diarrhea induced by epidermal growth factor receptor inhibitors (EGFRIs). On another hand, the T-QY305 formula, through regulating neutrophil recruitment features would highlight the presence of N-QY305, a subunit nanostructure contained in T-QY305, and confirm its role as potentially being the biomaterial conferring to T-QY305 its pharmacodynamic features. Here, the clinical records of two patients are analyzed expressing cutaneous adverse reaction and demonstrate positive effect of T-QY305 on the simultaneous inhibition of both cutaneous adverse reaction and diarrhea in animal models. The satisfying results obtained from T-QY305, lead to further process to the isolation of N-QY305 from T-QY305, in order to demonstrate that the potency of T-QY305 originates from the nanostructure N-QY305. Compared to T-QY305, N-QY305 exhibits higher potency upon reducing adverse reactions. The data represent a promising candidate for reducing cutaneous adverse reaction and diarrhea, meanwhile proposing a new strategy to highlight the presence of nanostructures being the "King" of Chinese medicine formula as the pharmacodynamic basis.

Caspase-11 deficiency attenuates neutrophil recruitment into the atherosclerotic lesion in apolipoprotein E-deficient mice

Caspase-11 is an inflammatory caspase that triggers an inflammatory response by regulating non-canonical NLRP3 inflammasome activation. Although the deficiency of both caspase-11 and caspase-1, another inflammatory caspase that functions as an executor of the inflammasome, prevents the development of atherosclerosis, the effect of caspase-11 deficiency alone on the development of atherosclerosis has not been fully evaluated. In the present study, we found that caspase-11 deficiency prevented the formation of the necrotic core, whereas it did not affect the development of atherosclerosis in Apoe-deficient mice. Notably, the infiltration of neutrophils into atherosclerotic lesions was attenuated by caspase-11 deficiency. RNA-seq analysis of stage-dependent expression of atherosclerotic lesions revealed that both upregulations of caspase-11 and neutrophil migration are common features of advanced atherosclerotic lesions. Furthermore, similar expression profiles were observed in unstable human plaque. These data suggest that caspase-11 regulates neutrophil recruitment and plaque destabilization in advanced atherosclerotic lesions.

S100A8∕A9 is a valuable biomarker and treatment target to detect and modulate neutrophil involvement in myocardial infarction.

Myocardial infarction (MI) leads to irreversible ischemic damage of the heart muscle and is the leading cause of heart failure. The ischemic cardiac injury triggers a potent local and systemic immune response. In the acute phase post-MI, neutrophils infiltrate the myocardium in large numbers and induce further cardiomyocyte death, expanding the infarcted area. The alarmin S100A8∕A9 is a proinflammatory mediator primarily produced by myeloid cells, with an emerging role in MI. We previously demonstrated that short-term inhibition of S100A8∕A9 during the inflammatory phase of the immune response to MI improves long-term cardiac function. In the present study, we investigated the effects of S100A8∕A9 blockade on myocardial inflammation and post-ischemic myocardial injury in a mouse model of coronary artery ligation. Immunohistochemical (IHC) staining revealed that the presence of S100A9 is strongly correlated with neutrophil infiltration in the myocardium on days 1 and 3 post-MI. A 3-day treatment with the S100A8∕A9 blocker ABR-238901 starting immediately after MI decreased the number of neutrophils and S100A9 presence in the myocardium and had a positive impact on cardiac damage, reducing infarction size. These findings promote S100A9 as an IHC biomarker of neutrophil infiltration and a promising immunomodulatory target to regulate neutrophil recruitment, reduce ischemic injury and promote long-term beneficial cardiac recovery after MI.

Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.

The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.

HRG inhibits liver cancer lung metastasis by suppressing neutrophil extracellular trap formation.

Distant metastasis is a sign of poor prognosis for cancer patients. Extrahepatic liver cancer metastases commonly spread to the lung. Remodelling of the metastatic microenvironment is essential for tumour metastasis. Neutrophil-associated metastatic microenvironment contributes to the early metastatic colonisation of cancer cells in the lung. The lung metastasis models were constructed via treated cancer cells by tail vein injection into mice. And samples of lung were harvested at the indicated time to analyze tumor growth and immune cells in the microenvironment. Tumors and lung metastasis specimens were obtained via surgical operations for research purposes. Neutrophils were obtained from peripheral blood of patients with liver cancer or healthy donors (HD). Hepatocellular carcinoma cells reduce the secretion of histidine-rich glycoprotein (HRG), regulate the recruitment and activation of neutrophils in the metastatic microenvironment and promote the production of neutrophil extracellular traps (NETs), thereby promoting liver cancer lung metastasis. HRG binds to FCγR1 on the neutrophil membrane while inhibiting PI3K and NF-κB activation, thereby reducing IL-8 secretion to reduce neutrophil recruitment. Meanwhile, HRG inhibited IL8-MAPK and NF-κB pathway activation and ROS production, resulting in reduced NETs formation. Our study reveals that liver cancer regulates neutrophil recruitment and NETs formation in the metastatic microenvironment by reducing HRG secretion, thereby promoting tumour lung metastasis. The results of this study will contribute to the development of possible strategies for treating metastases.

Hypoxia inducible-factor 1 alpha regulates neutrophil recruitment during fungal-elicited granulomatous inflammation.

Chronic pulmonary aspergillosis (CPA) is a devastating disease with increasing prevalence worldwide. The characteristic granulomatous-like inflammation poses as the major setback to effective antifungal therapies by limiting drug access to fungi. These inflammatory lung structures are reported to be severely hypoxic; nevertheless, the underlying mechanisms whereby these processes contribute to fungal persistence remain largely unknown. Hypoxia-inducible factor 1 alpha (HIF-1α), besides being the major cellular response regulator to hypoxia, is a known central immune modulator. Here, we used a model of Aspergillus fumigatus airway infection in myeloid-restricted HIF-1α knock-out (mHif1α-/- ) mice to replicate the complex structures resembling fungal granulomas and evaluate the contribution of HIF-1α to antifungal immunity and disease development. We found that fungal-elicited granulomas in mHif1α-/- mice had significantly smaller areas, along with extensive hyphal growth and increased lung fungal burden. This phenotype was associated with defective neutrophil recruitment and an increased neutrophil death, therefore highlighting a central role for HIF-1α-mediated regulation of neutrophil function in the pathogenesis of chronic fungal infection. These results hold the promise of an improved capacity to manage the progression of chronic fungal disease and open new avenues for additional therapeutic targets and niches of intervention.

Interleukin-22 regulates neutrophil recruitment in ulcerative colitis and is associated with resistance to ustekinumab therapy

The function of interleukin-22 (IL-22) in intestinal barrier homeostasis remains controversial. Here, we map the transcriptional landscape regulated by IL-22 in human colonic epithelial organoids and evaluate the biological, functional and clinical significance of the IL-22 mediated pathways in ulcerative colitis (UC). We show that IL-22 regulated pro-inflammatory pathways are involved in microbial recognition, cancer and immune cell chemotaxis; most prominently those involving CXCR2+ neutrophils. IL-22-mediated transcriptional regulation of CXC-family neutrophil-active chemokine expression is highly conserved across species, is dependent on STAT3 signaling, and is functionally and pathologically important in the recruitment of CXCR2+ neutrophils into colonic tissue. In UC patients, the magnitude of enrichment of the IL-22 regulated transcripts in colonic biopsies correlates with colonic neutrophil infiltration and is enriched in non-responders to ustekinumab therapy. Our data provide further insights into the biology of IL-22 in human disease and highlight its function in the regulation of pathogenic immune pathways, including neutrophil chemotaxis. The transcriptional networks regulated by IL-22 are functionally and clinically important in UC, impacting patient trajectories and responsiveness to biological intervention.

796 ECRG4 regulates neutrophil responsiveness to proinflammatory signals during recruitment to infection

Rapid neutrophil recruitment to cutaneous injury is critical for controlling local infection and preventing systemic dissemination. Neutrophils are mobilized from bone marrow reserves and home to vasculature near the source of inflammation, where they extravasate and migrate to the site of injury. We found that Esophageal Cancer Related Gene 4 (ECRG4), a leukocyte surface protein, amplifies early neutrophil recruitment to injury and hypothesized that ECRG4 may be important for the inflammatory response to infection through its ability to regulate neutrophil recruitment.

Loss of CXC-Chemokine Receptor 1 Expression in Chorioamnionitis Is Associated with Adverse Perinatal Outcomes.

Background: Chorioamnionitis complicates about 1–5% of deliveries at term and causes about one-third of stillbirths. CXC-chemokine receptor 1 (CXCR1) binds IL-8 with high affinity and regulates neutrophil recruitment. We aimed to determine the immunoexpression of CXCR1 in placentas with chorioamnionitis, and its association with adverse perinatal outcomes. Methods: A total of 101 cases of chorioamnionitis and 32 cases of non-chorioamnionitis were recruited over a period of 2 years. CXCR1 immunohistochemistry was performed, and its immunoexpression in placentas was evaluated. The adverse perinatal outcomes included intrauterine death, poor APGAR score, early neonatal death, and respiratory complications. Results: Seventeen cases (17/101, 16.8%) with chorioamnionitis presented as preterm deliveries. Lung complications were more common in mothers who were >35 years (p = 0.003) and with a higher stage in the foetal inflammatory response (p = 0.03). Notably, 24 cases (23.8%) of histological chorioamnionitis were not detected clinically. Interestingly, the loss of CXCR1 immunoexpression in the umbilical cord endothelial cells (UCECs) was significantly associated with foetal death (p = 0.009). Conclusion: The loss of CXCR1 expression in UCECs was significantly associated with an increased risk of adverse perinatal outcomes and could be used as a biomarker to predict adverse perinatal outcomes in chorioamnionitis. Further study is warranted to study the pathophysiology involved in the failure of CXCR1 expression in these cells.

Cardioprotective Effect of circ_SMG6 Knockdown against Myocardial Ischemia/Reperfusion Injury Correlates with miR-138-5p-Mediated EGR1/TLR4/TRIF Inactivation.

Increased neutrophil recruitment represents a hallmark event in myocardial ischemia/reperfusion (I/R) injury due to the ensuing inflammatory response. Circular RNAs (circRNAs) are important regulatory molecules involved in cell physiology and pathology. Herein, we analyzed the role of a novel circRNA circ_SMG6 in the regulation of neutrophil recruitment following I/R injury, which may associate with the miR-138-5p/EGR1/TLR4/TRIF axis. Myocardial I/R injury was modeled in vivo by ligation of the left anterior descending (LAD) artery followed by reperfusion in mice and in vitro by exposing a cardiomyocyte cell line (HL-1) to hypoxia/reoxygenation (H/R). Gain- and loss-of-function experiments were performed to evaluate the effect of the circ_SMG6/miR-138-5p/EGR1/TLR4/TRIF axis on cardiac functions, myocardial infarction, myocardial enzyme levels, cardiomyocyte activities, and neutrophil recruitment. We found that the EGR1 expression was increased in myocardial tissues of I/R mice. Knockdown of EGR1 was found to attenuate I/R-induced cardiac dysfunction and infarction area, pathological damage, and cardiomyocyte apoptosis. Mechanistic investigations showed that circ_SMG6 competitively bound to miR-138-5p and consequently led to upregulation of EGR1, thus facilitating myocardial I/R injury in mice and H/R-induced cell injury. Additionally, ectopic EGR1 expression augmented neutrophil recruitment and exacerbated the ensuing I/R injury, which was related to the activated TLR4/TRIF signaling pathway. Overall, our findings suggest that circ_SMG6 may deteriorate myocardial I/R injury by promoting neutrophil recruitment via the miR-138-5p/EGR1/TLR4/TRIF signaling. This pathway may represent a potential therapeutic target in the management of myocardial I/R injury.

Extracellular Vesicle Associated miRNAs Regulate Signaling Pathways Involved in COVID-19 Pneumonia and the Progression to Severe Acute Respiratory Corona Virus-2 Syndrome

BackgroundExtracellular vesicles (EVs) are mediators of cell-to-cell communication in inflammatory lung diseases. They function as carriers for miRNAs which regulate mRNA transcripts and signaling pathways after uptake into recipient cells. We investigated whether miRNAs associated with circulating EVs regulate immunologic processes in COVID-19.MethodsWe prospectively studied 20 symptomatic patients with COVID-19 pneumonia, 20 mechanically ventilated patients with severe COVID-19 (severe acute respiratory corona virus-2 syndrome, ARDS) and 20 healthy controls. EVs were isolated by precipitation, total RNA was extracted, profiled by small RNA sequencing and evaluated by differential gene expression analysis (DGE). Differentially regulated miRNAs between groups were bioinformatically analyzed, mRNA target transcripts identified and signaling networks constructed, thereby comparing COVID-19 pneumonia to the healthy state and pneumonia to severe COVID-19 ARDS.ResultsDGE revealed 43 significantly and differentially expressed miRNAs (25 downregulated) in COVID-19 pneumonia when compared to controls, and 20 miRNAs (15 downregulated) in COVID-19 ARDS patients in comparison to those with COVID-19 pneumonia. Network analysis for comparison of COVID-19 pneumonia to healthy controls showed upregulated miR-3168 (log2FC=2.28, padjusted<0.001), among others, targeting interleukin-6 (IL6) (25.1, 15.2 - 88.2 pg/ml in COVID-19 pneumonia) and OR52N2, an olfactory smell receptor in the nasal epithelium. In contrast, miR-3168 was significantly downregulated in COVID-19 ARDS (log2FC=-2.13, padjusted=0.003) and targeted interleukin-8 (CXCL8) in a completely activated network. Toll-like receptor 4 (TLR4) was inhibited in COVID-19 pneumonia by miR-146a-5p and upregulated in ARDS by let-7e-5p.ConclusionEV-derived miRNAs might have important regulative functions in the pathophysiology of COVID-19: CXCL8 regulates neutrophil recruitment into the lung causing epithelial damage whereas activated TLR4, to which SARS-CoV-2 spike protein binds strongly, increases cell surface ACE2 expression and destroys type II alveolar cells that secrete pulmonary surfactants; both resulting in pulmonary-capillary leakage and ARDS. These miRNAs may serve as biomarkers or as possible therapeutic targets.

Inhibition of Protein Disulfide Isomerase Ameliorates Vaso-Occlusion and Thrombosis in a Mouse Model of Sickle Cell Disease

IntroductionSickle cell disease (SCD) results from a single mutation in the β-globin gene. Altered erythrocyte physiology in SCD results in severe vascular complications, including vaso-occlusion (VOC) and painful crises along with various other manifestations, such as thrombosis and end organ damage. Extracellular protein disulfide isomerase (PDI) plays a critical role in the regulation of neutrophil recruitment, platelet activation and fibrin formation, processes implicated in the thrombo-inflammatory vasculopathy in SCD. We investigated the effect of a PDI inhibitor, isoquercetin, in combination with hydroxyurea, in TNFα-induced VOC and thrombosis in a mouse model of SCD. In addition, the effect of chronic PDI inhibition by isoquercetin on thrombo-inflammatory vasculopathy was also examined.MethodsHumanized TOWNES knock-in mice, containing human normal (βA/βA, control) or sickle (βS/βS, SCD) hemoglobin β chains, were used as a mouse model of SCD. PDI inhibition in the plasma was achieved by oral gavage or daily diet feeding of isoquercetin. Intravital microscopy was performed to determine vaso-occlusive events in the venules and thrombus formation in the arterioles in TNFα-stimulated mice. Plasma levels of PDI and parameters for coagulation and vascular inflammation were determined by ELISA. Neutrophil accumulation in the organs was determined as the myeloperoxidase (MPO) levels in tissue homogenates.ResultsCompared to control mice, SCD mice have elevated plasma PDI levels. Gavage of isoquercetin resulted in reduced PDI activity in the plasma of treated mice (38±10% inhibition, p <0.05). Isoquercetin ingestion increased leukocyte rolling flux fraction in TNFα-treated SCD mice as monitored using intravital microscopy, whereas the influence on leukocyte adhesion and shear rate was minimal. When a reversal agent for PDI inhibitor, b'x (the isolated substrate binding domain of PDI) was co-infused, the effect of isoquercetin on leukocyte rolling was reversed, suggesting this effect of isoquercetin is mediated by PDI inhibition. We also determined the effect of isoquercetin in combination with hydroxyurea, a treatment with established efficacy in SCD, on the vaso-occlusive events in our model. We confirmed that hydroxyurea treatment elevated the plasma nitric oxide level as determined by plasma nitrite/nitrate content (11.5±1.9 μM vs 5.0±1.9 μM, p <0.05). Consistent with previous studies, hydroxyurea decreased leukocyte adhesion while the rolling flux fraction was not changed. However, combined treatment with isoquercetin and hydroxyurea in SCD mice increased the rolling flux fraction (15±3% vs 6±1%, p <0.05), decreased leukocyte adhesion (158±13 mm-2 vs 289±24 mm-2, p <0.05), and increased vessel shear rate (302±18 s-1 vs 122±11 s-1, p <0.05). We investigated in vivo thrombus formation in the arterioles of TNFα-treated SCD mice using intravital microscopy following laser injury. Compared to the control mice, SCD mice have significantly enhanced fibrin generation but normal platelet accumulation. Both isoquercetin and hydroxyurea inhibited TNFα-induced platelet accumulation (62±36% AUC-Area Under the Curve with isoquercetin and 48±17% AUC with hydroxyurea of untreated SCD) and fibrin generation (30±6% AUC with isoquercetin and 44±9% AUC with hydroxyurea of untreated SCD). To study the effect of chronic isoquercetin treatment in SCD, control and SCD mice were fed isoquercetin for 4 weeks. Daily consumption of isoquercetin resulted in reduction of plasma PDI activity (18±5% inhibition, p <0.05). Consistent with the intravital microscopic data, chronic PDI inhibition reduced neutrophil migration and accumulation into the lung, as determined by the MPO level (481±85 ng/mg vs 829±140 ng/mg, p <0.05) in the tissue homogenate. Chronic treatment with isoquercetin also reduced plasma fibrinogen degradation products (FDP) (102±15 μg/ml vs 183±25 μg/ml, p <0.05) in SCD mice. In addition, a correlation (Pearson r=0.651) was observed between reducing plasma FDP and lung MPO levels.ConclusionsAcute and chronic PDI inhibition by isoquercetin may provide beneficial effect on vascular complications in a mouse model of SCD. PDI inhibition has the potential to ameliorate thrombo-inflammatory syndromes associated with SCD. These studies justify a longitudinal trial of PDI inhibitors in SCD. DisclosuresNo relevant conflicts of interest to declare.

The voltage-gated potassium channel KV1.3 regulates neutrophil recruitment during inflammation.

AimsNeutrophil trafficking within the vasculature strongly relies on intracellular calcium signalling. Sustained Ca2+ influx into the cell requires a compensatory efflux of potassium to maintain membrane potential. Here, we aimed to investigate whether the voltage-gated potassium channel KV1.3 regulates neutrophil function during the acute inflammatory process by affecting sustained Ca2+ signalling.Methods and resultsUsing in vitro assays and electrophysiological techniques, we show that KV1.3 is functionally expressed in human neutrophils regulating sustained store-operated Ca2+ entry through membrane potential stabilizing K+ efflux. Inhibition of KV1.3 on neutrophils by the specific inhibitor 5-(4-Phenoxybutoxy)psoralen (PAP-1) impaired intracellular Ca2+ signalling, thereby preventing cellular spreading, adhesion strengthening, and appropriate crawling under flow conditions in vitro. Using intravital microscopy, we show that pharmacological blockade or genetic deletion of KV1.3 in mice decreased neutrophil adhesion in a blood flow dependent fashion in inflamed cremaster muscle venules. Furthermore, we identified KV1.3 as a critical component for neutrophil extravasation into the inflamed peritoneal cavity. Finally, we also revealed impaired phagocytosis of Escherichia coli particles by neutrophils in the absence of KV1.3.ConclusionWe show that the voltage-gated potassium channel KV1.3 is critical for Ca2+ signalling and neutrophil trafficking during acute inflammatory processes. Our findings do not only provide evidence for a role of KV1.3 for sustained calcium signalling in neutrophils affecting key functions of these cells, they also open up new therapeutic approaches to treat inflammatory disorders characterized by overwhelming neutrophil infiltration.

Deficiency of Dietary Fiber Modulates Gut Microbiota Composition, Neutrophil Recruitment and Worsens Experimental Colitis.

Ulcerative colitis is an inflammatory disease of the colon that is associated with colonic neutrophil accumulation. Recent evidence indicates that diet alters the composition of the gut microbiota and influences host–pathogen interactions. Specifically, bacterial fermentation of dietary fiber produces metabolites called short-chain fatty acids (SCFAs), which have been shown to protect against various inflammatory diseases. However, the effect of fiber deficiency on the key initial steps of inflammation, such as leukocyte–endothelial cell interactions, is unknown. Moreover, the impact of fiber deficiency on neutrophil recruitment under basal conditions and during inflammation in vivo is unknown. Herein, we hypothesized that a fiber-deficient diet promotes an inflammatory state in the colon at baseline and predisposes the host to more severe colitis pathology. Mice fed a no-fiber diet for 14 days showed significant changes in the gut microbiota and exhibited increased neutrophil-endothelial interactions in the colonic microvasculature. Although mice fed a no-fiber diet alone did not have observable colitis-associated symptoms, these animals were highly susceptible to low dose (0.5%) dextran sodium sulphate (DSS)-induced model of colitis. Supplementation of the most abundant SCFA, acetate, prevented no-fiber diet-mediated enrichment of colonic neutrophils and colitis pathology. Therefore, dietary fiber, possibly through the actions of acetate, plays an important role in regulating neutrophil recruitment and host protection against inflammatory colonic damage in an experimental model of colitis.

IκB Kinase-β Regulates Neutrophil Recruitment Through Activation of STAT3 Signaling in the Esophagus

Background & AimsThe epithelial barrier is the host’s first line of defense against damage to the underlying tissue. Upon injury, the epithelium plays a critical role in inflammation. The IκB kinase β (IKKβ)/nuclear factor-κB pathway was shown to be active in the esophageal epithelium of patients with esophageal disease. However, the complex mechanisms by which IKKβ signaling regulates esophageal disease pathogenesis remain unknown. Our prior work has shown that expression of a constitutively active form of IKKβ specifically in esophageal epithelia of mice (IkkβcaEsophageal Epithelial Cell-Knockin (EEC-KI)) is sufficient to cause esophagitis.MethodsWe generated ED-L2/Cre;Rosa26-Ikkβca+/L;Stat3L/L (IkkβcaEEC-KI;Stat3Esophageal Epithelial Cell Knockout (EEC-KO)) mice, in which the ED-L2 promoter activates Cre recombinase in the esophageal epithelium, leading to constitutive activation of IKKβ and loss of Stat3. Esophageal epithelial tissues were collected and analyzed by immunostaining, RNA sequencing, quantitative real-time polymerase chain reaction assays, flow cytometry, and Western blot. IkkβcaEEC-KI mice were treated with neutralizing antibodies against interleukin (IL)23p19 and IL12p40.ResultsHere, we report that IkkβcaEEC-KI mice have increased activation of epithelial Janus kinase 2/STAT3 signaling. Stat3 deletion in IkkβcaEEC-KI mice attenuated the neutrophil infiltration observed in IkkβcaEEC-KI mice and resulted in decreased expression of genes related to immune cell recruitment and activity. Blocking experiments in IkkβcaEEC-KI mice showed that STAT3 activation and subsequent neutrophil recruitment are dependent on IL23 secretion.ConclusionsOur study establishes a novel interplay between IKKβ and STAT3 signaling in epithelial cells of the esophagus, where IKKβ/IL23/STAT3 signaling controls neutrophil recruitment during the onset of inflammation. GEO accession number: GSE154129.

Control of neutrophil influx during peritonitis by transcriptional cross-regulation of chemokine CXCL1 by IL-17 and IFN-γ.

Neutrophil infiltration is a hallmark of peritoneal inflammation, but mechanisms regulating neutrophil recruitment in patients with peritoneal dialysis (PD)-related peritonitis are not fully defined. We examined 104 samples of PD effluent collected during acute peritonitis for correspondence between a broad range of soluble parameters and neutrophil counts. We observed an association between peritoneal IL-17 and neutrophil levels. This relationship was evident in effluent samples with low but not high IFN-γ levels, suggesting a differential effect of IFN-γ concentration on neutrophil infiltration. Surprisingly, there was no association of neutrophil numbers with the level of CXCL1, a key IL-17-induced neutrophil chemoattractant. We investigated therefore the production of CXCL1 by human peritoneal mesothelial cells (HPMCs) under in vitro conditions mimicking clinical peritonitis. Stimulation of HPMCs with IL-17 increased CXCL1 production through induction of transcription factor SP1 and activation of the SP1-binding region of the CXCL1 promoter. These effects were amplified by TNFα. In contrast, IFN-γ dose-dependently suppressed IL-17-induced SP1 activation and CXCL1 production through a transcriptional mechanism involving STAT1. The SP1-mediated induction of CXCL1 was also observed in HPMCs exposed to PD effluent collected during peritonitis and containing IL-17 and TNFα, but not IFN-γ. Supplementation of the effluent with IFN-γ led to a dose-dependent activation of STAT1 and a resultant inhibition of SP1-induced CXCL1 expression. Transmesothelial migration of neutrophils in vitro increased upon stimulation of HPMCs with IL-17 and was reduced by IFN-γ. In addition, HPMCs were capable of binding CXCL1 at their apical cell surface. These observations indicate that changes in relative peritoneal concentrations of IL-17 and IFN-γ can differently engage SP1-STAT1, impacting on mesothelial cell transcription of CXCL1, whose release and binding to HPMC surface may determine optimal neutrophil recruitment and retention during peritonitis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

ECRG4 regulates neutrophil recruitment and CD44 expression during the inflammatory response to injury.

The complex molecular microenvironment of the wound bed regulates the duration and degree of inflammation in the wound repair process, while its dysregulation leads to impaired healing. Understanding factors controlling this response provides therapeutic targets for inflammatory disease. Esophageal cancer-related gene 4 (ECRG4) is a candidate chemokine that is highly expressed on leukocytes. We used ECRG4 knockout (KO) mice to establish that the absence of ECRG4 leads to defective neutrophil recruitment with a delay in wound healing. An in vitro human promyelocyte model identified an ECRG4-mediated suppression of the hyaluronic acid receptor, CD44, a key receptor mediating inflammation resolution. In ECRG4 KO mouse leukocytes, there was an increase in CD44 expression, consistent with a model in which ECRG4 negatively regulates CD44 levels. Therefore, we propose a previously unidentified mechanism in which ECRG4 regulates early neutrophil recruitment and subsequent CD44-mediated resolution of inflammation.