Neutrophil Migration In Response Research Articles

KRIT1-mediated regulation of neutrophil adhesion and motility.

Loss of Krev interaction-trapped-1 (KRIT1) expression leads to the development of cerebral cavernous malformations (CCM), a disease in which abnormal blood vessel formation compromises the structure and function of the blood-brain barrier. The role of KRIT1 in regulating endothelial function is well-established. However, several studies have suggested that KRIT1 could also play a role in regulating nonendothelial cell types and, in particular, immune cells. In this study, we generated a mouse model with neutrophil-specific deletion of KRIT1 in order to investigate the effect of KRIT1 deficiency on neutrophil function. Neutrophils isolated from adult Ly6Gtm2621(cre)Arte Krit1flox/flox mice had a reduced ability to attach and spread on the extracellular matrix protein fibronectin and exhibited a subsequent increase in migration. However, adhesion to and migration on ICAM-1 was unchanged. In addition, we used a monomeric, fluorescently-labelled fragment of fibronectin to show that integrin activation is reduced in the absence of KRIT1 expression, though β1 integrin expression appears unchanged. Finally, neutrophil migration in response to lipopolysaccharide-induced inflammation in the lung was decreased, as shown by reduced cell number and myeloperoxidase activity in lavage samples from Krit1PMNKO mice. Altogether, we show that KRIT1 regulates neutrophil adhesion and migration, likely through regulation of integrin activation, which can lead to altered inflammatory responses in vivo.

Neutrophil trogocytosis during their trans-endothelial migration: role of extracellular CIRP

BackgroundNeutrophils are the most abundant innate immune cells in the circulating blood, and they act as the first responder against bacterial and fungal infection. However, accumulation of activated neutrophils can cause severe inflammation and tissue damage. Recently, neutrophil trogocytosis or membrane transfer with neighboring cells was reported to modulate immune responses. Extracellular cold-inducible RNA binding protein (eCIRP) is a newly identified damage-associated molecular pattern (DAMP). eCIRP can activate neutrophils to be more pro-inflammatory. This study aimed to identify the role of eCIRP in neutrophil trogocytosis during their trans-endothelial migration.MethodsA trans-endothelial migration (TEM) assay using bone marrow neutrophils and mouse primary lung vascular endothelial cells was conducted using transwell chambers and neutrophil trogocytosis was assessed in vitro. In an in vivo mouse model of acute lung injury, neutrophil trogocytosis was assessed from bronchoalveolar lavage fluid.ResultsIn TEM assay, the trogocytosis of neutrophils occurred during trans-endothelial migration and eCIRP significantly increased the percentage of these neutrophils. The trogocytosed neutrophils acquired the endothelial membrane containing junctional adhesion molecule-C (JAM-C) and VE-cadherin, and these membrane patches were polarized by Mac-1 binding. Furthermore, eCIRP-induced JAM-C positive trogocytosed neutrophils are more pro-inflammatory than the JAM-C negative counterpart. JAM-C positive trogocytosed neutrophils were also observed in the bronchoalveolar lavage fluid of a mouse model of acute lung injury.ConclusionThese data suggest that during the paracellular trans-endothelial migration of neutrophils in response to inflammation, eCIRP induces trogocytosis of neutrophils, and the trogocytosed neutrophils exhibit an exaggerated pro-inflammatory phenotype promoting acute lung injury.

Preclinical safety and efficacy of lentiviral-mediated gene therapy for leukocyte adhesion deficiency type I

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene, which encodes for the CD18 subunit of β2-integrins. Deficient expression of β2-integrins results in impaired neutrophil migration in response to bacterial and fungal infections. Using a lentiviral vector (LV) that mediates a preferential myeloid expression of human CD18 (Chim.hCD18-LV), we first demonstrated that gene therapy efficiently corrected the phenotype of mice with severe LAD-I. Next, we investigated if the ectopic hCD18 expression modified the phenotypic characteristics of human healthy donor hematopoietic stem cells and their progeny. Significantly, transduction of healthy CD34+ cells with the Chim.hCD18-LV did not modify the membrane expression of CD18 nor the adhesion of physiological ligands to transduced cells. Additionally, we observed that the repopulating properties of healthy CD34+ cells were preserved following transduction with the Chim.hCD18-LV, and that a safe polyclonal repopulation pattern was observed in transplanted immunodeficient NOD scid gamma (NSG) mice. In a final set of experiments, we demonstrated that transduction of CD34+ cells from a severe LAD-I patient with the Chim.hCD18-LV restores the expression of β2-integrins in these cells. These results offer additional preclinical safety and efficacy evidence supporting the gene therapy of patients with severe LAD-I.

Hv1/VSOP regulates neutrophil directional migration and ERK activity by tuning ROS production.

High-level reactive oxygen species (ROS) production in neutrophils is tightly regulated, as it can damage host cells. Neutrophils also undergo low-level ROS production when stimulated by cytokines or chemoattractants, but its biologic significance remains largely unknown. Voltage-gated proton channels (Hv1/VSOP) activity reportedly supports ROS production in neutrophils; however, we show here that Hv1/VSOP balances ROS production to suppress neutrophil directional migration in the presence of low concentrations of N-formyl-Met-Leu-Phe (fMLF). Neutrophils derived from Hvcn1 gene knockout mice produced more ROS than neutrophils from wild-type mice in the stimulation with fMLF at concentration of 1µM and nonstimulus condition. They also exhibited stronger chemotactic responses to low concentrations of fMLF than did wild-type neutrophils. Receptor sensitivity to fMLF and evoked Ca2+ responses did not differ between Hv1/VSOP-deficient and wild-type neutrophils. Activation of ERK, but not p38, was enhanced and prolonged during the increased ROS production seen after fMLF stimulation in Hv1/VSOP-deficient neutrophils. Inhibiting ROS production suppressed the enhanced ERK activation in Hv1/VSOP-deficient neutrophils and their directional migration. These results indicate that Hv1/VSOP balances ROS production to reduce ERK signaling and suppress excessive neutrophil migration in response to fMLF. Our findings thus reveal a novel role for ROS in the directional migration of neutrophils.

Microfluidic skin chip with vasculature for recapitulating the immune response of the skin tissue.

There is a considerable need for cell-based in vitro skin models for studying dermatological diseases and testing cosmetic products, but current in vitro skin models lack physiological relevance compared to human skin tissue. For example, many dermatological disorders involve complex immune responses, but current skin models are not capable of recapitulating the phenomena. Previously, we reported development of a microfluidic skin chip with a vessel structure and vascular endothelial cells. In this study, we cocultured dermal fibroblasts and keratinocytes with vascular endothelial cells, human umbilical vascular endothelial cells. We verified the formation of a vascular endothelium in the presence of the dermis and epidermis layers by examining the expression of tissue-specific markers. As the vascular endothelium plays a critical role in the migration of leukocytes to inflammation sites, we incorporated leukocytes in the circulating media and attempted to mimic the migration of neutrophils in response to external stimuli. Increased secretion of cytokines and migration of neutrophils was observed when the skin chip was exposed to ultraviolet irradiation, showing that the microfluidic skin chip may be useful for studying the immune response of the human tissue.

Modulation of neutrophil chemotaxis by Aggregatibacter actinomycetemcomitans leukotoxin

ABSTRACTObjective: Aggressive periodontitis (AgP) is frequently associated with the presence of Aggregatibacter actinomycetemcomitans, a Gram-negative pathogen, which expresses leukotoxin (Ltx) as a virulence factor. Ltx is known to disturb neutrophil function and to activate these cells. This study was aimed at investigating the ability of Ltx to alter the chemotactic behaviour of neutrophils, the most abundant leukocyte in periodontitis.Methods: Neutrophils from healthy blood donors were isolated and stimulated or primed with low concentrations of Ltx (1, 5 and 10 ng/mL). Neutrophil migration in response to Ltx gradients and to the biofilm-derived chemotactic agent fMLP after Ltx-priming was monitored by real-time video microscopy using an Insall chamber.Results: Although Ltx alone enhanced neutrophil movement at all concentrations, low directional accuracy was observed compared to the positive control (fMLP). Ltx priming led to directional movement towards fMLP with significantly enhanced speed. In case of 10 ng/mL Ltx, however, this chemotactic response towards fMLP appeared to be delayed.Conclusion: The results indicate that Ltx induces non-directional neutrophil movement, whilst enhancing migration towards fMLP. Clincially, this may lead to the presence of a higher number of activated neutrophils in the periodontal tissues, potentially causing more pronounced host tissue damage.

Gβ1 is required for neutrophil migration in zebrafish

Signaling mediated by G protein-coupled receptors (GPCRs) is essential for the migration of cells toward chemoattractants. The recruitment of neutrophils to injured tissues in zebrafish larvae is a useful model for studying neutrophil migration and trafficking in vivo. Indeed, the study of this process led to the discovery that PI3Kγ is required for the polarity and motility of neutrophils, features that are necessary for the directed migration of these cells to wounds. However, the mechanism by which PI3Kγ is activated remains to be determined. Here we show that signaling by specifically the heterotrimeric G protein subunit Gβ1 is critical for neutrophil migration in response to wounding. In embryos treated with small-molecule inhibitors of Gβγ signaling, neutrophils failed to migrate to wound sites. Although both the Gβ1 and Gβ4 isoforms are expressed in migrating neutrophils, only deficiency for the former (morpholino-based knockdown) interfered with the directed migration of neutrophils towards wounds. The Gβ1 deficiency also impaired the ability of cells to change cell shape and reduced their general motility, defects that are similar to those in neutrophils deficient for PI3Kγ. Transplantation assays showed that the requirement for Gβ1 in neutrophil migration is cell autonomous. Finally, live imaging revealed that Gβ1 is required for polarized activation of PI3K, and for the actin dynamics that enable neutrophil migration. Collectively, our data indicate that Gβ1 signaling controls proper neutrophil migration by activating PI3K and modulating actin dynamics. Moreover, they illustrate a role for a specific Gβ isoform in chemotaxis in vivo.

Fms-like tyrosine kinase-3 ligand increases resistance to burn wound infection through effects on plasmacytoid dendritic cells

BackgroundPatients experiencing large thermal injuries are susceptible to opportunistic infections that can delay recovery and lead to sepsis. Dendritic cells (DC) are important for the detection of pathogens and activation of the innate and acquired immune responses. DCs are significantly decreased in burn patients early after injury, and the development of sepsis is associated with persistent DC depletion. In a murine model of burn wound infection, the enhancement of DCs after injury by treatment with the DC growth factor Fms-like tyrosine kinase-3 ligand (FL) enhances neutrophil migration to infection, improves bacterial clearance, and increases survival in a DC-dependent manner. FL expands the production of both conventional DCs (cDC) and plasmacytoid DCs (pDC). It has been established that cDCs are required for some of the protective effects of FL after burn injury. This study was designed to determine the contribution of the pDC subset.MethodsMice were subjected to full-thickness scald burns under deep anesthesia and were provided analgesia. pDCs were depleted by injection of anti-plasmacytoid dendritic cell antigen-1 antibodies. Survival, bacterial clearance, and neutrophil responses in vivo and in vitro were measured.ResultsDepletion of preexisting pDCs, but not FL-expanded pDCs, abrogated the beneficial effects of FL on survival, bacterial clearance, and neutrophil migration in response to burn wound infection. This requisite role of pDCs for FL-mediated enhancement of neutrophil migratory capacity is not due to direct effects of pDCs on neutrophils. cDCs, but not pDCs, directly increased neutrophil migratory capacity after co-culture.ConclusionsThe protective effects of FL treatment after burn injury are mediated by both pDCs and cDCs. Pharmacological enhancement of both DC subtypes by FL is a potential therapeutic intervention to enhance immune responses to infection and improve outcome after burn injury.

Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity

Aquaporins (AQPs, AQP0-12) are membrane water/glycerol channel proteins that participate in various cellular functions in many cell types. AQP9 is expressed in several immune cells including neutrophils; however, its role in cutaneous immune response remains unknown. Here we show the involvement of AQP9 in skin allergic inflammation using AQP9 knockout (AQP9−/−) mice and the contact hypersensitivity (CHS) model. First, AQP9−/− mice showed impairment of CHS responses to 2,4-dinitrofluorobenzene (DNFB) compared with wild-type (WT) mice. The sensitization phase of CHS was examined by evaluating the immune cells accumulated in skin draining lymph nodes (LNs) after DNFB application. Adoptive transfer of AQP9−/− LNs cells into the ears of WT recipients resulted in impaired CHS response, indicating the defect of sensitization in AQP9−/−. IL-17 production was decreased in AQP9−/− compared with WT LNs cells, though the T cell proliferation and IFN-gamma production levels were comparable. WT mice showed high neutrophil accumulation in LNs at 18 h after DNFB application, but it was diminished in AQP9−/− mice. In accordance, we found the attenuation of AQP9−/− neutrophil migration to LNs after DNFB application. Furthermore, the neutrophil migration in response to CCR7 ligands was suppressed in AQP9−/− using chemotaxis chambers. Finally, we found that the administration of Gr-1 (neutrophil) antibody during the sensitization phase suppressed IL-17 production from LNs cells as well as CHS response in WT mice, suggesting the implication of neutrophils for IL-17 production during the development of CHS. These data suggest that AQP9-expressing neutrophils play an important role in the sensitization phase of CHS through their migration function.

Effects of Aggregatibacter actinomycetemcomitans leukotoxin on neutrophil migration and extracellular trap formation

BackgroundAggressive periodontitis is associated with the presence of Aggregatibacter actinomycetemcomitans, a leukotoxin (Ltx)-producing periodontal pathogen. Ltx has the ability to lyse white blood cells including neutrophils.ObjectivesThis study was aimed at investigating the interactions between neutrophils and Ltx with regard to the chemotactic properties of Ltx and the release of neutrophil extracellular traps (NETs).MethodsNeutrophils from healthy blood donors were isolated and incubated for 30 min and 3 h with increasing concentrations of Ltx (1, 10, and 100 ng/mL) as well as with A. actinomycetemcomitans strains (NCTC 9710 and HK 1651) producing different levels of Ltx. Formation of NETs and cell lysis were assessed by microscopy, fluorescence-based assays, and measurement of released lactate dehydrogenase. Neutrophil migration in response to different Ltx gradients was monitored by real-time video microscopy, and image analysis was performed using ImageJ software.ResultsAlthough Ltx (10 and 100 ng/mL) and the leukotoxic A. actinomycetemcomitans strain HK 1651 lysed some neutrophils, other cells were still capable of performing NETosis in a concentration-dependent manner. Low doses of Ltx and the weakly leukotoxic strain NCTC 9710 did not lead to neutrophil lysis, but did induce some NETosis. Furthermore, all three concentrations of Ltx enhanced random neutrophil movement; however, low directional accuracy was observed compared with the positive control (fMLP).ConclusionsThe results indicate that Ltx acts both as a neutrophil activator and also causes cell death. In addition, Ltx directly induces NETosis in neutrophils prior to cell lysis. In future studies, the underlying pathways involved in Ltx-meditated neutrophil activation and NETosis need to be investigated further.

Localizing the lipid products of PI3Kγ in neutrophils.

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM).We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs.

Research highlights: measuring and manipulating cell migration

Microfabricated systems and microfluidic tools are well-suited to interface with cells because of the matching length scales. In this issue, we highlight three recent papers in which unique tools were used to control or measure cell migration. Cell migration is a key biological process involved in normal physiology (e.g. in embryonic development, immune response, and wound healing) which can go awry in diseases such as cancer. We highlight work applying electric fields and surface patterning to direct the collective migration of epithelial cells using galvanotaxis, in which surprisingly larger patches of cells respond more uniformly to electric fields. Such a platform may yield insights into or be co-opted to control tissue formation. We also describe recent results on developing a simple system to measure the migration of neutrophils in response to chemoattractants and on using this system to discriminate between neutrophils from asthmatic and non-asthmatic patients. Finally, a micropillar system is highlighted in which the epithelial-to-mesenchymal transition is quantified at the single-cell level, and may aid in our understanding of the plastic process of progression to a malignant phenotype. Cell migration is a wonderfully complex process in which microscale systems can not only contribute to a better understanding, but might also improve its recording and manipulation for practical applications.

Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense.

Neutrophils are key innate immune effector cells that are essential to fighting bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagn1 (encoding Jagunal homolog 1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis identified marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity in Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection and impaired formation of cytotoxic granules, as well as defective myeloperoxidase release and killing of Candida albicans. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Notably, GM-CSF also restored the defective fungicidal activity of bone marrow cells from humans with JAGN1 mutations. These data directly identify Jagn1 (JAGN1 in humans) as a new regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for humans.

The evolution and appearance of C3 duplications in fish originate an exclusive teleost c3 gene form with anti-inflammatory activity.

The complement system acts as a first line of defense and promotes organism homeostasis by modulating the fates of diverse physiological processes. Multiple copies of component genes have been previously identified in fish, suggesting a key role for this system in aquatic organisms. Herein, we confirm the presence of three different previously reported complement c3 genes (c3.1, c3.2, c3.3) and identify five additional c3 genes (c3.4, c3.5, c3.6, c3.7, c3.8) in the zebrafish genome. Additionally, we evaluate the mRNA expression levels of the different c3 genes during ontogeny and in different tissues under steady-state and inflammatory conditions. Furthermore, while reconciling the phylogenetic tree with the fish species tree, we uncovered an event of c3 duplication common to all teleost fishes that gave rise to an exclusive c3 paralog (c3.7 and c3.8). These paralogs showed a distinct ability to regulate neutrophil migration in response to injury compared with the other c3 genes and may play a role in maintaining the balance between inflammatory and homeostatic processes in zebrafish.

SRF is required for neutrophil migration in response to inflammation

AbstractSerum response factor (SRF) is a ubiquitously expressed transcription factor and master regulator of the actin cytoskeleton. We have previously shown that SRF is essential for megakaryocyte maturation and platelet formation and function. Here we elucidate the role of SRF in neutrophils, the primary defense against infections. To study the effect of SRF loss in neutrophils, we crossed Srffl/fl mice with select Cre-expressing mice and studied neutrophil function in vitro and in vivo. Despite normal neutrophil numbers, neutrophil function is severely impaired in Srf knockout (KO) neutrophils. Srf KO neutrophils fail to polymerize globular actin to filamentous actin in response to N-formyl-methionine-leucine-phenylalanine, resulting in significantly disrupted cytoskeletal remodeling. Srf KO neutrophils fail to migrate to sites of inflammation in vivo and along chemokine gradients in vitro. Polarization in response to cytokine stimuli is absent and Srf KO neutrophils show markedly reduced adhesion. Integrins play an essential role in cellular adhesion, and although integrin expression levels are maintained with loss of SRF, integrin activation and trafficking are disrupted. Migration and cellular adhesion are essential for normal cell function, but also for malignant processes such as metastasis, underscoring an essential function for SRF and its pathway in health and disease.

Sa1765 Defective Autophagy Contributes to Impaired Bacterial Clearance of SAMP1/YITFC Mice

Background: The commensal microbiota are known to regulate the activity of neutrophils and other leukocytes in the intestine, but the impact of the microbiota on systemic neutrophil activity remains unknown. Serum amyloid A (Saa) is a circulating HDL-associated apolipoprotein and acute phase protein. Saa genes are expressed by multiple tissues and cell types and are markedly induced by diverse stimuli including the gut microbiota. However, the precise roles of Saa in inflammation remain elusive because both pro-and anti-inflammatory actions have been reported. Aim: Assess the effects of microbiota colonization and Saa induction on systemic neutrophil development and function. Methods: The effect of the microbiota on neutrophil localization and migration was evaluated using germ-free (GF) and conventionalized (CONVD) transgenic zebrafish expressing GFP in neutrophils (Tg(mpx:GFP)) Flow cytometry was used to quantify and sort GFP(+) neutrophils for qRTPCR ofmpxmRNA levels. Neutrophil migrationwas assessed using tracking analysis of GFP(+) neutrophils using Volocity. Neutrophil recruitment was assessed using a tail-wounding assay. Morpholinos targeting saa were used on GF and CONVD fish to evaluate the role of saa on neutrophil biology. In vitro studies were performed using PAC-2 zebrafish embryonic fibroblast cells and peritoneal isolated murine neutrophils. NF-κB pathway activation was measured in SAA stimulated PAC-2 cells by western blot and through transfection of a pikbaa:Luc reporter. Murine neutrophil migration in response to SAA in the presence of an NF-κB inhibitor was assessed using a transwell system. Results: The presence of a microbiota resulted in increased neutrophil number (~3 fold, p<0.05) and myeloperoxidase expression (~5 fold, p<0.05)), and altered neutrophil localization and migratory behaviors (~2 fold increased velocity and increased directional migration). These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to the injury site. Morpholino knockdown experiments revealed saamediates microbial stimulation of tissue-specific neutrophil migratory behaviors. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB (~3 fold, p<0.001), and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression.Conclusions:These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signaling axis in mediating neutrophil migratory responses.

Sa1767 STAT3 Activation in CD4+ Lymphocytes Shapes Epithelial Host Defense During Infectious Colitis

Background: The commensal microbiota are known to regulate the activity of neutrophils and other leukocytes in the intestine, but the impact of the microbiota on systemic neutrophil activity remains unknown. Serum amyloid A (Saa) is a circulating HDL-associated apolipoprotein and acute phase protein. Saa genes are expressed by multiple tissues and cell types and are markedly induced by diverse stimuli including the gut microbiota. However, the precise roles of Saa in inflammation remain elusive because both pro-and anti-inflammatory actions have been reported. Aim: Assess the effects of microbiota colonization and Saa induction on systemic neutrophil development and function. Methods: The effect of the microbiota on neutrophil localization and migration was evaluated using germ-free (GF) and conventionalized (CONVD) transgenic zebrafish expressing GFP in neutrophils (Tg(mpx:GFP)) Flow cytometry was used to quantify and sort GFP(+) neutrophils for qRTPCR ofmpxmRNA levels. Neutrophil migrationwas assessed using tracking analysis of GFP(+) neutrophils using Volocity. Neutrophil recruitment was assessed using a tail-wounding assay. Morpholinos targeting saa were used on GF and CONVD fish to evaluate the role of saa on neutrophil biology. In vitro studies were performed using PAC-2 zebrafish embryonic fibroblast cells and peritoneal isolated murine neutrophils. NF-κB pathway activation was measured in SAA stimulated PAC-2 cells by western blot and through transfection of a pikbaa:Luc reporter. Murine neutrophil migration in response to SAA in the presence of an NF-κB inhibitor was assessed using a transwell system. Results: The presence of a microbiota resulted in increased neutrophil number (~3 fold, p<0.05) and myeloperoxidase expression (~5 fold, p<0.05)), and altered neutrophil localization and migratory behaviors (~2 fold increased velocity and increased directional migration). These effects of the microbiota on neutrophil homeostasis were accompanied by an increased recruitment of neutrophils to the injury site. Morpholino knockdown experiments revealed saamediates microbial stimulation of tissue-specific neutrophil migratory behaviors. In vitro studies revealed that zebrafish cells respond to Saa exposure by activating NF-κB (~3 fold, p<0.001), and that Saa-dependent neutrophil migration requires NF-κB-dependent gene expression.Conclusions:These results implicate the commensal microbiota as an important environmental factor regulating diverse aspects of systemic neutrophil development and function, and reveal a critical role for a Saa-NF-κB signaling axis in mediating neutrophil migratory responses.

Srf Is Required For Neutrophil Migration In Response To Inflammation

Serum Response Factor (SRF) is a ubiquitously expressed transcription factor and master regulator of the actin cytoskeleton. We have previously shown, that SRF is essential for megakaryocyte maturation and platelet formation and function. Here we elucidate the role of SRF in neutrophils, the primary defense against infections. To study the effect of loss of SRF in neutrophils, we crossed Srffl/fl mice with select Cre-expressing mice and studied neutrophil function in vitro and in vivo. Despite normal neutrophil numbers, neutrophil function is severely impaired in mice in whom Srf is selectively deleted in the hematopoietic system (Srf KO). Srf KO neutrophils fail to migrate to sites of inflammation in vivo. In a model mimicking lung infection by nebulization of lipopolysaccharide (LPS), significantly fewer neutrophils are recruited to the inflamed lungs 4 and 24 hours after LPS administration as evident by cell numbers retrieved in the bronchoalveolar lavage fluid (BAL, total cells: WT 0.568 ± 0.093x106 vs. KO 0.128 ± 0.024x106 at 4 hours and WT 1.337 ± 0.369x106 vs. KO 0.347 ± 0.045x106 at 24 hours; p <0.005 at 4 hours, p < 0.05 at 24 hours). Similarly, in an in vivo peritonitis model, where all other immune cells normally express Srf, significantly fewer Srf KO than WT neutrophils are recruited to the inflamed peritoneal space resulting in significantly reduced Srf KO neutrophil numbers in the peritoneal lavage fluid. To directly assess neutrophil migration and chemotaxis we performed in vitro transwell assays and assessed neutrophil migration in the Dunn chamber assay. Srf KO neutrophils show a significant migration defect in response to fMLP and KC. We next assessed actin polymerization in Srf WT and KO neutrophils. Srf KO neutrophils fail to polymerize globular actin to filamentous actin in response to fMLP. Neutrophil polarization in response to cytokine stimuli is markedly decreased. In addition, Srf KO neutrophils show markedly reduced adhesion. Integrins play an essential role in neutrophil adhesion and migration. Srf KO neutrophils show normal expression of the integrin LFA1 (CD11a/CD18), however, Mac1 (CD11b/CD18) expression is markedly increased on the cell surface as assessed by flow cytometry and Immunofluorescent staining, despite reduced mRNA expression. We find that trafficking of Mac1, essential for directed cell migration, is disrupted in Srf KO neutrophils, likely contributing to the migratory defect. Migration and cellular adhesion are essential for normal cell function, but also for malignant processes such as metastasis, thus underscoring an essential function for SRF and its pathway in health and disease. Disclosures:No relevant conflicts of interest to declare.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Cyclophilin A cooperates with MIP-2 to augment neutrophil migration

BackgroundChemokines contribute to inflammatory responses by inducing leukocyte migration and extravasation. In addition, chemoattractants other than classical chemokines can also be present. Many chemokines have been demonstrated to cooperate, leading to an augmentation in leukocyte recruitment and providing a potential role for the presence of multiple chemoattractants. Extracellular cyclophilins are a group of alternative chemotactic factors, which can be highly elevated during various inflammatory responses and, as we have previously shown, can contribute significantly to neutrophil recruitment in an animal model of acute lung inflammation. In the current studies we investigated whether the most abundant extracellular cyclophilin, CypA, has the capacity to function in partnership with 2 classical chemokines known to be secreted in the same model, macrophage inflammatory protein (MIP)-2/CXCL2 and keratinocyte chemoattractant (KC)/CXCL1.MethodsNeutrophil migration in response to combinations of CypA and MIP-2 or CypA and KC was measured by in vitro chemotaxis assays. Biochemical responses of neutrophils incubated with the combinations of chemoattractants were determined by changes in chemokine receptor internalization and actin polymerization measured by flow cytometry, and changes in intracellular calcium mobilization measured with a calcium sensitive fluorochrome.ResultsA combination of CypA and MIP-2, but not KC, augmented neutrophil migration. Based on the level of augmentation, the cooperation between CypA and MIP-2 appeared to be synergistic. Evidence that CypA and MIP-2 cooperate at the biochemical level was demonstrated by increases in receptor internalization, calcium mobilization, and actin polymerization.ConclusionThese findings provide evidence for the capacity of extracellular cyclophilins to interact with classical chemokines, resulting in greater and more efficient leukocyte recruitment.