Increased Neutrophil Migration Research Articles

Modulating effects of heat-killed and live Limosilactobacillus reuteri PSC102 on the immune response and gut microbiota of cyclophosphamide-treated rats.

In the present study, we investigated the potential immunomodulatory effects of heat-killed (hLR) and live Limosilactobacillus reuteri PSC102 (LR; formerly Lactobacillus reuteri PSC102) in RAW264.7 macrophage cells and Sprague-Dawley rats. RAW264.7 murine macrophage cells were stimulated with hLR and LR for 24 h. Cyclophosphamide (CTX)-induced immunosuppressed Sprague-Dawley rats were orally administered with three doses of hLR (L-Low, M-Medium, and H-High) and LR for 3 weeks. The phagocytic capacity, production of nitric oxide (NO), and expression of cytokines in RAW264.7 cells were measured, and the different parameters of immunity in rats were determined. hLR and LR treatments promoted phagocytic activity and induced the production of NO and the expression of iNOS, TNF-α, IL-1β, IL-6, and Cox-2 in macrophage cells. In the in vivo experiment, hLR and LR treatments significantly increased the immune organ indices, alleviated the spleen injury, and ameliorated the number of white blood cells, granulocytes, lymphocytes, and mid-range absolute counts in immunosuppressive rats. hLR and LR increased neutrophil migration and phagocytosis, splenocyte proliferation, and T lymphocyte subsets (CD4+, CD8+, CD45RA+, and CD28+). The levels of immune factors (IL-2, IL-4, IL-6, IL-10, IL-12A, TNF-α, and IFN-γ) in the hLR and LR groups were upregulated compared with those in the CTX-treatment group. hLR and LR treatments could also modulate the gut microbiota composition, thereby increasing the relative abundance of Bacteroidetes and Firmicutes but decreasing the level of Proteobacteria. hLR and LR protected against CTX-induced adverse reactions by modulating the immune response and gut microbiota composition. Therefore, they could be used as potential immunomodulatory agents.

DEL-1, as an anti-neutrophil transepithelial migration molecule, inhibits airway neutrophilic inflammation in asthma.

Neutrophil migration into the airways is a key process in neutrophilic asthma. Developmental endothelial locus-1 (DEL-1), an extracellular matrix protein, is a neutrophil adhesion inhibitor that attenuates neutrophilic inflammation. Levels of DEL-1 were measured in exhaled breath condensate (EBC) and serum in asthma patients by ELISA. DEL-1 modulation of neutrophil adhesion and transepithelial migration was examined in a co-culture model in vitro. The effects of DEL-1-adenoviral vector-mediated overexpression on ovalbumin/lipopolysaccharide (OVA/LPS)-induced neutrophilic asthma were studied in mice in vivo. DEL-1 was primarily expressed in human bronchial epithelial cells and was decreased in asthma patients. Serum DEL-1 concentrations were reduced in patients with severe asthma compared with normal subjects (567.1 ± 75.3 vs. 276.8 ± 29.36 pg/mL, p < .001) and were negatively correlated to blood neutrophils (r = -0.2881, p = .0384) and neutrophil-to-lymphocyte ratio (NLR) (r = -0.5469, p < .0001). DEL-1 concentrations in the EBC of severe asthmatic patients (113.2 ± 8.09 pg/mL) were also lower than normal subjects (193.0 ± 7.61 pg/mL, p < .001) and were positively correlated with the asthma control test (ACT) score (r = 0.3678, p = .0035) and negatively related to EBC IL-17 (r = -0.3756, p = .0131), myeloperoxidase (MPO) (r = -0.5967, p = .0055), and neutrophil elastase (NE) (r = -0.5488, p = .0009) expression in asthma patients. Neutrophil adhesion and transepithelial migration in asthma patients were associated with LFA-1 binding to ICAM-1 and inhibited by DEL-1. DEL-1 mRNA and protein expression in human bronchial epithelial cells were regulated by IL-17. Exogenous DEL-1 inhibited IL-17-enhanced neutrophil adhesion and migration. DEL-1 expression was decreased while neutrophil infiltration was increased in the airway of a murine model of neutrophilic asthma. This was prevented by DEL-1 overexpression. DEL-1 down-regulation leads to increased neutrophil migration across bronchial epithelial cells and is associated with neutrophilic airway inflammation in asthma.

Factor XII promotes the thromboinflammatory response in a rat model of venoarterial extracorporeal membrane oxygenation

BackgroundFactor XII (FXII) is a multifunctional protease capable of activating thrombotic and inflammatory pathways. FXII has been linked to thrombosis in extracorporeal membrane oxygenation (ECMO), but the role of FXII in ECMO-induced inflammatory complications has not been studied. We used novel gene-targeted FXII- deficient rats to evaluate the role of FXII in ECMO-induced thromboinflammation. MethodsFXII-deficient (FXII−/−) Sprague–Dawley rats were generated using CRISPR/Cas9. A minimally invasive venoarterial (VA) ECMO model was used to compare wild-type (WT) and FXII−/− rats in 2 separate experimental cohorts: rats placed on ECMO without pharmacologic anticoagulation and rats anticoagulated with argatroban. Rats were maintained on ECMO for 1 hour or until circuit failure occurred. Comparisons were made with unchallenged rats and rats that underwent a sham surgical procedure without ECMO. ResultsFXII−/− rats were maintained on ECMO without pharmacologic anticoagulation with low resistance throughout the 1-hour experiment. In contrast, WT rats placed on ECMO without anticoagulation developed thrombotic circuit failure within 10 minutes. Argatroban provided a means to maintain WT and FXII−/− rats on ECMO for the 1-hour time frame without thrombotic complications. Analyses of these rats demonstrated that ECMO resulted in increased neutrophil migration into the liver that was significantly blunted by FXII deficiency. ECMO also resulted in increases in high molecular weight kininogen cleavage and complement activation that were abrogated by genetic deletion of FXII. ConclusionsFXII initiates hemostatic system activation and key inflammatory sequelae in ECMO, suggesting that therapies targeting FXII could limit both thromboembolism and inopportune inflammatory complications in this setting.

Comparison between sevoflurane and propofol on immunomodulation in an in vitro model of sepsis.

Patients with sepsis often require sedation and/or anesthesia. Although the immunomodulatory effects of anesthetics have been increasingly recognized, the molecular mechanisms require better elucidation. We compared the effects of sevoflurane with propofol on the expression of pro- and anti-inflammatory biomarkers released by monocytes/macrophages and blood/bronchoalveolar lavage fluid (BALF) neutrophils, the phagocytic capacity of monocytes/ macrophages, and neutrophil migration, as well as mediators associated with alveolar epithelial and endothelial cells obtained from rats with sepsis. Polymicrobial sepsis was induced by cecal ligation and puncture in nine male Wistar rats. After 48 h, animals were euthanized and their monocytes/alveolar macrophages, blood and BALF neutrophils, as well as alveolar epithelial and endothelial cells were extracted, and then exposed to (1) sevoflurane (1 minimal alveolar concentration), (2) propofol (50 μM), or (3) saline, control (CTRL) for 1 h. Sevoflurane reduced interleukin (IL)-6 mRNA expression in monocytes and alveolar macrophages (p = 0.007, p = 0.029), whereas propofol decreased IL-6 mRNA only in alveolar macrophages (p = 0.027) compared with CTRL. Sevoflurane increased IL-10 expression (p = 0.0002) in monocytes compared with propofol and increased IL-10 mRNA and transforming growth factor (TGF)-β mRNA (p = 0.037, p = 0.045) compared with CTRL. Both sevoflurane and propofol did not affect mRNA expression of IL-10 and TGF-β in alveolar macrophages. The phagocytic capacity of monocytes (p = 0.0006) and alveolar macrophages (p = 0.0004) was higher with sevoflurane compared with propofol. Sevoflurane, compared with CTRL, reduced IL-1β mRNA (p = 0.003, p = 0.009) and C-X-C chemokine receptor 2 mRNA (CXCR2, p = 0.032 and p = 0.042) in blood and BALF neutrophils, and increased CXCR4 mRNA only in BALF neutrophils (p = 0.004). Sevoflurane increased blood neutrophil migration (p = 0.015) compared with propofol. Both sevoflurane and propofol increased zonula occludens-1 mRNA (p = 0.046, p = 0.003) in alveolar epithelial cells and reduced Toll-like receptor 4 mRNA (p = 0.043, p = 0.006) in alveolar endothelial cells compared with CTRL. Only propofol reduced surfactant protein B mRNA (p = 0.028) in alveolar epithelial cells. Sevoflurane, compared with propofol, increased anti-inflammatory biomarkers in monocytes, but not in alveolar macrophages, enhanced monocyte/alveolar macrophage phagocytic capacity and increased neutrophil migration in in vitro experimental sepsis. Both propofol and sevoflurane protected lung epithelial and endothelial cells.

The lymphatic endothelium-derived follistatin: activin A axis regulates neutrophil motility in response to Pseudomonas aeruginosa.

The lymphatic system plays an active role during infection, however the role of lymphatic-neutrophil interactions in host-defense responses is not well understood. During infection with pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus and Yersinia pestis, neutrophils traffic from sites of infection through the lymphatic vasculature, to draining lymph nodes to interact with resident lymphocytes. This process is poorly understood, in part, due to the lack of in vitro models of the lymphatic system. Here we use a 3D microscale lymphatic vessel model to examine neutrophil-lymphatic cell interactions during host defense responses to pathogens. In previous work, we have shown that follistatin is secreted at high concentrations by lymphatic endothelial cells during inflammation. Follistatin inhibits activin A, a member of the TGF-β superfamily, and, together, these molecules form a signaling pathway that plays a role in regulating both innate and adaptive immune responses. Although follistatin and activin A are constitutively produced in the pituitary, gonads and skin, their major source in the serum and their effects on neutrophils are poorly understood. Here we report a microfluidic model that includes both blood and lymphatic endothelial vessels, and neutrophils to investigate neutrophil-lymphatic trafficking during infection with P. aeruginosa. We found that lymphatic endothelial cells produce secreted factors that increase neutrophil migration toward P. aeruginosa, and are a significant source of both follistatin and activin A during Pseudomonas infection. We determined that follistatin produced by lymphatic endothelial cells inhibits activin A, resulting in increased neutrophil migration. These data suggest that the follistatin:activin A ratio influences neutrophil trafficking during infection with higher ratios increasing neutrophil migration.

Neutrophil-Derived COX-2 has a Key Role during Inflammatory Hyperalgesia.

Inflammation is a vital process for the injured tissue restoration and one of its hallmarks is inflammatory hyperalgesia. The cyclooxygenase (COX) pathway is strongly related to the inflammatory and painful process. Usually, the COX-1 isoform is described as homeostatic, while COX-2 is characterized as inducible in inflammatory conditions. Although it is well known that neutrophil cells are the first to arrive at the inflamed site and the major source of COX-2 is still unknown, the specific role of neutrophil-derived COX-2 in the pain process is. Thus, in the present study, we demonstrate for the first time that neutrophil-derived COX-2 plays a key role in peripheral inflammatory hyperalgesia. Conditional knockout mice for COX-2 in neutrophils (COX-2fl/fl: Mrp8cre±) exhibited higher pain sensitivity after carrageenan (CG) injection and long-lasting IL-1β-induced hyperalgesia compared with the control group (COX-2fl/fl). Also, CG-induced inflammation in COX-2fl/fl: Mrp8cre± mice showed COX-1 overexpression, and increased neutrophil migration and pro-inflammatory cytokines (e.g., IL-1β and CXCL1). These findings revealed that neutrophil COX-2 has an important role in the regulation of inflammatory hyperalgesia.

Hypoxia regulates cytokines expression and neutrophils migration by ERK signaling in zebrafish

Normal dissolved oxygen in water is essential for maintaining the physiological functions of fish, but environmental pollution, such as eutrophication can lead to a decrease in oxygen content in water. How this reduction of dissolved oxygen in water affects the immune functions of fish and the potential regulatory mechanisms have not been thoroughly elucidated. In this study, we made full use of the aquatic model animal zebrafish to explore this question. In a model of LPS-induced inflammation, we found that hypoxia induced by infusing nitrogen into water increased the expression of pro-inflammatory cytokines, such as il-1β, il-6, and il-8. In vivo imaging also showed that hypoxia significantly increased neutrophil migration to the site of caudal fin injury in the transgenic line. Subsequently, we found that the phosphorylation level of ERK protein was significantly activated upon hypoxia and proved the roles of ERK signaling in the expression of pro-inflammatory cytokines and neutrophil migration in zebrafish. This study indicated that reduced water oxygen significantly increases the inflammatory response of the zebrafish.

Inhibition of Prostaglandin F2α Receptors Exaggerates HCl-Induced Lung Inflammation in Mice.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe respiratory disorders that are caused by aspiration, sepsis, trauma, and pneumonia. A clinical feature of ALI/ARDS is the acute onset of severe hypoxemia, and the mortality rate, which is estimated at 38–50%, remains high. Although prostaglandins (PGs) are detected in the bronchoalveolar lavage fluid of patients with ALI/ARDS, the role of PGF2α in ALI remains unclear. We aimed to clarify the role of PGF2α/PGF2α receptor (FP) signaling in acid-induced ALI using an FP receptor antagonist, AL8810. Intratracheal injection of hydrochloric acid (HCl) increased neutrophil migration into the lungs, leading to respiratory dysfunction. Pre-administration of AL8810 further increased these features. Moreover, pre-treatment with AL8810 enhanced the HCl-induced expression of pro-inflammatory cytokines and neutrophil migratory factors in the lungs. Administration of HCl decreased the gene expression of lung surfactant proteins, which was further reduced by co-administration of AL8810. Administration of AL8810 also increased lung edema and reduced mRNA expression of epithelial sodium channel in the lungs, indicating that AL8810 reduced fluid clearance. Furthermore, AL8810 also increased lipopolysaccharide-induced expression of adhesion molecules such as intracellular adhesion molecule-1 and E-selectin in human umbilical vein endothelial cells. These results indicate that inhibition of FP receptors by AL8810 exacerbated HCl-induced ALI.

Aronia melanocarpa Extract Fermented by Lactobacillus plantarum EJ2014 Modulates Immune Response in Mice.

The present study aimed to assess the immunomodulatory effects of fermented Aronia melanocarpa extract (FAME) on RAW 264.7 cells and BALB/c mice. Aronia melanocarpa fruit was fermented with Lactobacillus plantarum EJ2014 by adding yeast extract and monosodium glutamate for 9 days at 30 °C to produce γ-aminobutyric acid (GABA). After fermentation, significant GABA production was noted, along with minerals, polyphenols, and flavonoids (p < 0.05). The polyphenol content was confirmed by liquid chromatography with tandem mass spectrometry (LC–MS/MS) analysis. RAW 264.7 cells were stimulated with lipopolysaccharide (LPS, 1 μg/mL) in the presence or absence of FAME, and proinflammatory cytokine contents were measured by qPCR. In the in vivo experiment, female BALB/c mice were administered 125, 250, and 500 mg/kg of FAME for 21 days. FAME treatment increased neutrophil migration and phagocytosis (p < 0.05). It also increased splenocyte proliferation, CD4+ and CD8+ T-cell expression, and lymphocyte proliferation. Furthermore, it increased IFN-γ, IL-2, and IL-4 cytokine levels in a dose-dependent manner (p < 0.05). However, it decreased TNF-α and IL-6 levels (p < 0.05). These results indicate that FAME fortified with GABA including bioactive compounds exerts anti-inflammatory effects by inhibiting proinflammatory cytokines in RAW 264.7 cells and modulates immune response in mice. Thus, FAME could be a potential therapeutic agent for inflammatory disorders.

PH and Proton Sensor GPR65 Determine Susceptibility to Atopic Dermatitis.

pH sensing by GPR65 regulates various inflammatory conditions, but its role in skin remains unknown. In this study, we performed a phenome-wide association study and report that the T allele of GPR65-intronic single-nucleotide polymorphism rs8005161, which reduces GPR65 signaling, showed a significant association with atopic dermatitis, in addition to inflammatory bowel diseases and asthma, as previously reported. Consistent with this genetic association in humans, we show that deficiency of GPR65 in mice resulted in markedly exacerbated disease in the MC903 experimental model of atopic dermatitis. Deficiency of GPR65 also increased neutrophil migration in vitro. Moreover, GPR65 deficiency in mice resulted in higher expression of the inflammatory cytokine TNF-α by T cells. In humans, CD4+ T cells from rs8005161 heterozygous individuals expressed higher levels of TNF-α after PMA/ionomycin stimulation, particularly under pH 6 conditions. pH sensing by GPR65 appears to be important for regulating the pathogenesis of atopic dermatitis.

Polymicrobial stimulation of human fetal membranes induce neutrophil activation and neutrophil extracellular trap release

Preterm birth is a major contributor to neonatal mortality and morbidity. While the causes of preterm birth remain incompletely understood, infection is a major risk factor, and chorioamnionitis is commonly observed. Chorioamnionitis is characterized by inflammation and neutrophil infiltration of the fetal membranes (FM). We recently reported that human FMs which had been exposed to low levels of bacterial lipopolysaccharide (LPS) recruit neutrophils and activate them, increasing their secretion of pro-inflammatory cytokines, degranulation of myeloperoxidase (MPO), and release of neutrophil extracellular traps (NETs). Herein, we demonstrate that conditioned media (CM) from viral dsRNA (Poly(I:C))-stimulated FMs also increased neutrophil migration, and induced the secretion of inflammatory IL-8 and the release of NETs. Furthermore, CM from FMs stimulated by a combination of bacterial LPS and Poly(I:C) augmented neutrophil NET release, compared to CM from FMs stimulated with either Poly(I:C) or LPS alone. NETs induced by FMs exposed to Poly(I:C), with or without LPS, were released and degraded quicker than those induced by resting or LPS-stimulated FM-CM. These findings indicate that FMs exposed to viral dsRNA promote neutrophil recruitment, activation and NET formation, similar to FMs exposed to bacterial LPS alone. However, in response to FM polymicrobial stimulation the levels and kinetics of NET release are augmented. This work builds upon our understanding of how infections at the maternal-fetal interface may affect neutrophil function.

Murine Myeloid Progenitors Attenuate Immune Dysfunction Induced by Hemorrhagic Shock.

SummaryHemorrhagic shock induces an aberrant immune response characterized by simultaneous induction of a proinflammatory state and impaired host defenses. The objective of this study was to evaluate the impact of conditionally immortalized neutrophil progenitors (NPs) on this aberrant immune response. We employed a mouse model of hemorrhagic shock, followed by the adoptive transfer of NPs and subsequent inoculation of Staphylococcus aureus to induce pneumonia. We observed that transplant of NPs decreases the proportion of host neutrophils that express programmed death ligand 1 and intercellular adhesion molecule 1 in the context of prior hemorrhage. Following hemorrhage, NP transplant decreased proinflammatory cytokines in the lungs, increased neutrophil migration into the airspaces, and enhanced bacterial clearance. Further, hemorrhagic shock improved NP engraftment in the bone marrow. These results suggest that NPs hold the potential for use as a cellular therapy in the treatment and prevention of secondary infection following hemorrhagic shock.

Rheumatoid Arthritis Synovial Fluid Neutrophils Drive Inflammation Through Production of Chemokines, Reactive Oxygen Species, and Neutrophil Extracellular Traps.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting synovial joints. Neutrophils are believed to play an important role in both the initiation and progression of RA, and large numbers of activated neutrophils are found within both synovial fluid (SF) and synovial tissue from RA joints. In this study we analyzed paired blood and SF neutrophils from patients with severe, active RA (DAS28>5.1, n=3) using RNA-seq. 772 genes were significantly different between blood and SF neutrophils. IPA analysis predicted that SF neutrophils had increased expression of chemokines and ROS production, delayed apoptosis, and activation of signaling cascades regulating the production of NETs. This activated phenotype was confirmed experimentally by incubating healthy control neutrophils in cell-free RA SF, which was able to delay apoptosis and induce ROS production in both unprimed and TNFα primed neutrophils (p<0.05). RA SF significantly increased neutrophil migration through 3μM transwell chambers (p<0.05) and also increased production of NETs by healthy control neutrophils (p<0.001), including exposure of myeloperoxidase (MPO) and citrullinated histone-H3-positive DNA NETs. IPA analysis predicted NET production was mediated by signaling networks including AKT, RAF1, SRC, and NF-κB. Our results expand the understanding of the molecular changes that take place in the neutrophil transcriptome during migration into inflamed joints in RA, and the altered phenotype in RA SF neutrophils. Specifically, RA SF neutrophils lose their migratory properties, residing within the joint to generate signals that promote joint damage, as well as inflammation via recruitment and activation of both innate and adaptive immune cells. We propose that this activated SF neutrophil phenotype contributes to the chronic inflammation and progressive damage to cartilage and bone observed in patients with RA.

High Fat Feeding Primes Mice to Binge Alcohol‐induced Liver Injury: Critical Role of Cathelicidin as a Chemoattractant

BackgroundAlcohol consumption and obesity are known synergistic risk factors of steatohepatitis. Combination of high fat diet (HFD) and acute alcohol synergistically induced acute liver inflammation via enhanced hepatic neutrophil infiltration. CRAMP (Cathelicidin‐related antimicrobial peptide), the murine ortholog of LL‐37, which is the only known member of human cathelicidin antimicrobial peptide family, possess both anti‐ and pro‐inflammatory activity. Our previous studies showed that CRMAP deficiency exacerbated binge‐on‐chronic alcohol‐induced liver injury. Here we aimed to investigate the role of CRAMP in alcohol‐induced liver injury in HFD fed mice, and to explore the underlying mechanism.MethodsCRAMP KO mice (Camp−/−) and wild type (WT) mice were fed control diet (CD) or HFD for 10 weeks, a bolus of alcohol was gavaged 9 hours before sample harvesting.ResultsTen‐week HFD + acute alcohol markedly induced liver steatosis and injury in WT mice, reflected by significantly increased liver macrosteatosis and serum levels of AST and ALT. Immunofluorescent assay showed a significantly stronger staining of Ly6G in the livers of mice treated with HFD + acute alcohol compared to CD feeding + acute alcohol, indicating an increased neutrophil infiltration. Interestingly, HFD+acute alcohol treatment significantly increased hepatic protein levels of CRAMP, which co‐localized with Ly6G‐positive neutrophils. In addition, hepatic mRNA expression of chemokines, Mcp‐1 and Cxcl‐2, was markedly increased. Depletion of Camp gene (Camp−/−) markedly reduced HFD + acute alcohol‐induced liver steatosis, injury and neutrophil infiltration, along with reduced expression of Mcp‐1. Importantly, feeding Camp−/− mice with HFD + acute alcohol significantly reduced the mRNA expression level of Cxcr‐2, a known CRAMP receptor. In vitro study showed that synthetic CRAMP peptide increased neutrophil migration, which was dependent on Cxcr‐2 expression upon treatment by fatty acid and ethanol. In addition, significantly decreased mRNA levels of hepatic Tgf‐β and Col‐1a1 were found in Camp−/− mice compared to WT mice, suggesting that CRAMP may play a role in hepatic stellate cells activation.ConclusionsHFD + acute alcohol treatment synergistically induces liver injury through systemic upregulation of chemoattractant CRAMP and subsequent enhancement of hepatic neutrophil infiltration. CRAMP deficiency protects against acute alcohol‐induced steatohepatitis in the HFD primed mice.Support or Funding InformationSupported by grants from NIHSchematic hypothesis of CRAMP‐mediated neutrophils infiltration in HFD and alcohol‐induced liver injury in miceFigure 1

Inhibition of Aerobic Glycolysis Promotes Neutrophil to Influx to the Infectious Site Via CXCR2 in Sepsis.

Recent evidences suggest that metabolic reprogramming plays an important role in the regulation of innate inflammatory response; however, the specific mechanism is unclear. In this study, we found that glycolytic inhibitor 2-deoxyglucose (2-DG) significantly improved the survival rate in cecal ligation and puncture (CLP)-induced septic mice. 2-DG-treated mice developed increased neutrophil migration to the infectious site and more efficient bacterial clearance than untreated mice. 2-DG reversed the down-regulation of chemokine receptor 2 (CXCR2) and the impaired chemotaxis induced by CLP in mice or lipopolysaccharides (LPS) in human neutrophils. Furthermore, 2-DG reversed the down-regulation of CXCR2 in neutrophils by decreasing the expression of G protein-coupled receptor kinase-2 (GRK2), a serin-threonine protein kinase that mediated the internalization of chemokine receptors, which was induced via the inhibition of extracellular regulated protein kinases (ERK) phosphorylation and the promotion of P38 phosphorylation. Finally, SB225002, a CXCR2 antagonist, partially blocked the protective effects of 2-DG in sepsis. Together, we found a novel mechanism for the migration of neutrophils regulated by metabolism and suggested that aerobic glycolysis might be a potential target of intervention in sepsis.

Regulation of murine arthritis by systemic, spinal, and intra-articular adrenoceptors.

The regulation of the immune system by the sympathetic nervous system is allowing the design of novel treatments for inflammatory disorders such as arthritis. In this study, we have analyzed the effects of α- and β-adrenoceptor agonists injected subcutaneously, intrathecally, or intra-articularly in zymosan-induced arthritis. Murine arthritis was induced by intra-articular (knee joint) injection of zymosan. α1 (phenylephrine), α2 (clonidine), β1 (dobutamine), or β2 (salbutamol)-adrenoceptor agonists were injected subcutaneously (sc), intrathecally (it), or intra-articularly (ia) to activate peripheral, spinal, or intra-articular adrenoceptors and to study their effects on articular edema formation and neutrophil migration into the synovial cavity. Treatments with phenylephrine did not affect the edema formation, but it increased neutrophil migration when injected subcutaneously (155.3%) or intra-articularly (187.7%). Treatments with clonidine inhibited neutrophil migration (59.9% sc, 68.7% it, 42.8% ia) regardless of the route of administration, but it inhibited edema formation only when injected intrathecally (66.7%) or intra-articularly (36%) but not subcutaneously. Treatments with dobutamine inhibited both edema (42.0% sc, 69.5% it, 61.6% ia) and neutrophil migration (28.4% sc, 70.3% it, 82.4% ia) in a concentration dependent manner. Likewise, all the treatments with salbutamol also inhibited edema formation (89.9% sc, 62.4% it, 69.8% ia) and neutrophil migration (76.6% sc, 39.1% it, 71.7% ia). Whereas the β-adrenoceptor agonists induced anti-inflammatory effects regardless of their route of administration, α1- and α2-adrenoceptor agonists induced either pro- and anti-inflammatory effects, respectively.

Abstract # 3187 The effect of cytokine stimulation on astrocyte chemokine production and neutrophil chemoattraction

Systemic inflammation can exacerbate symptoms of many neurological diseases. This effect may be facilitated by either aberrant glial responses, or the trafficking of peripheral immune cells to the CNS. The later involves the production of chemokines, which themselves are capable of acting on neurons. In this study, primary astrocytes were stimulated with IL-1α, IL-1β, TNF, IL-1α/TNF or IL-1β/TNF and the levels of twenty-eight secreted chemokines were semi-quantitatively determined using a Proteome Profiler array and densitometry. Pharmacological inhibitors were used to determine the effects of NFκB, p38 MAPK, JNK, and ERK1/2 pathway activation on chemokine production. Finally, neutrophil migration assays were performed to demonstrate functionality. Chemokine production was dependent on stimulation condition with IL-1α/TNF and IL-1β/TNF challenge being the most efficient. The most abundantly produced chemokines were CCL2, CCL5, CCL12, CXCL1, CXCL5 and CXCL10. No single pharmacological inhibitor abolished chemokine secretion by astrocytes, implicating redundancy in the transcriptional activation at the level of the promotor. In agreement with the known capacity for CXCL1 and CXCL5 to promote neutrophil trafficking, cytokine treated astrocyte increased neutrophil migration. Intriguingly, pretreatment of astrocytes with a pharmacological inhibitor of TGF-βactivated kinase 1 ameliorated cytokine mediated neutrophil trafficking. Together, these results suggest that astrocytes likely contribute to trafficking of peripheral cells to the brain during systemic inflammation.

Interaction with an endothelial lumen increases neutrophil lifetime and motility in response to P aeruginosa

AbstractNeutrophil infiltration into tissues is essential for host defense and pathogen clearance. Although many of the signaling pathways involved in the transendothelial migration of neutrophils are known, the role of the endothelium in regulating neutrophil behavior in response to infection within interstitial tissues remains unclear. Here we developed a microscale 3-dimensional (3D) model that incorporates an endothelial lumen, a 3D extracellular matrix, and an intact bacterial source to model the host microenvironment. Using this system, we show that an endothelial lumen significantly increased neutrophil migration toward a source of Pseudomonas aeruginosa. Surprisingly, we found neutrophils, which were thought to be short-lived cells in vitro, migrate for up to 24 hours in 3D in the presence of an endothelial lumen and bacteria. In addition, we found that endothelial cells secrete inflammatory mediators induced by the presence of P aeruginosa, including granulocyte-macrophage colony-stimulating factor (GM-CSF), a known promoter of neutrophil survival, and interleukin (IL)-6, a proinflammatory cytokine. We found that pretreatment of neutrophils with a blocking antibody against the IL-6 receptor significantly reduced neutrophil migration to P aeruginosa but did not alter neutrophil lifetime, indicating that secreted IL-6 is an important signal between endothelial cells and neutrophils that mediates migration. Taken together, these findings demonstrate an important role for endothelial paracrine signaling in neutrophil migration and survival.

Importance of the Complement Alternative Pathway in Serum Chemotactic Activity During Sepsis.

Serum chemotactic activity is important in regulating neutrophil migration. The ability of neutrophils to migrate to infectious site is crucial for host effective pathogen control, but unregulated neutrophil activation can also cause tissue damage. During bacterial sepsis, the complement alternative pathway (AP) is massively activated in blood and tissues and reportedly contributes to sepsis pathogenesis. Complement factor B (FB) is an essential component of the AP activation. However, the impact of FB/AP activation on blood chemotactic activity during bacterial infection is unclear. In this study, we found that sera of septic mice following cecal ligation and puncture (CLP) had much higher chemotactic activities on neutrophils than those of sham animals. Compared with wild-type (WT) mice, FB mice had significantly attenuated serum chemotactic activity, under both nonseptic and septic conditions. Moreover, sera with the activated AP by zymosan and cobra venom factor (CVF) in vitro induced a significant increase in neutrophil migration compared with sera without the AP activation. Complement activation generates complement cleavage fragment such as Ba, C3a, and C5a. To delineate the contribution of these downstream effectors, we incubated AP-active sera (AP activated by zymosan/CVF) or sera from sham and septic mice with anti-C5a or mAb1379 (anti-Ba) neutralizing antibody. We found that anti-C5a, but not mAb1379, markedly attenuated the neutrophil chemotactic effect of the AP-activated sera and that of septic sera. Taking together, these data suggest that the complement AP activation during bacterial sepsis plays a pivotal role in promoting blood chemotactic activity through a C5a-dependent mechanism.

The correlation between the concentration of faecal calprotectin and markers of inflammation in children with gastrointestinal disorders

Introduction: Calprotectin is an acute phase protein that occurs in large amounts in the granules and cytosolic fluid of neutrophils, and in smaller amounts in monocytes and macrophages. In bowel inflammation, increased neutrophil migration from the circulation to the intestinal lumen is observed as a consequence of mucosal damage. This leads to the release of a significant amount of calprotectin by activated leukocytes in intestine, thereby increasing its concentration in the faeces. This allows the non-invasive assessment of intestinal inflammation. Other markers useful in patients with bowel inflammation are erythrocyte sedimentation rate and C-reactive protein. Aim: The aim of the study was to evaluate the correlation between the concentration of faecal calprotectin (FC) and other markers of inflammation, such as C-reactive protein (CRP) and ferritin in serum, erythrocyte sedimentation rate (ESR) and white blood cells (WBC) count and in children with Crohn’s disease and ulcerative colitis. Material and methods: This study is based on a retrospective analysis of laboratory results of 370 children suspected of inflammatory bowel disease (IBD). Pearson’s coefficient was used to assess the correlation between the parameters. Results: There was a positive correlation between concentration of FC and parameters such as CRP (r=0.16; p=0.0345), ESR (r=0.38; p&lt;0.0001) and WBC (r=0.24; p=0.0008) in children with IBD. There was also a negative correlation between concentration of FC and ferritin (r=-0.24; p=0.0089) in children with IBD. Conclusion: In this study, ESR turned out to be more adequate than CRP and WBC in detecting inflammation in patients with IBD.