scholarly journals Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

2020 ◽  
Vol 133 (17) ◽  
pp. jcs248880 ◽  
Author(s):  
Wenqing Zhou ◽  
Alan Y. Hsu ◽  
Yueyang Wang ◽  
Ramizah Syahirah ◽  
Tianqi Wang ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Neutrophil Function ◽  
Mitochondrial Outer Membrane ◽  
Hematopoietic Tissue ◽  
Mitofusin 2 ◽  
Sheer Stress ◽  
Intracellular Ca

ABSTRACTNeutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (MFN2) regulates neutrophil homeostasis and chemotaxis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue, trapped in the vasculature in zebrafish embryos, and not capable of chemotaxis. Consistent with this, human neutrophil-like cells that are deficient for MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis on 2D surfaces. Deletion of MFN2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mechanistically, MFN2-deficient neutrophil-like cells display disrupted mitochondria–ER interaction, heightened intracellular Ca2+ levels and elevated Rac activation after chemokine stimulation. Restoring a mitochondria–ER tether rescues the abnormal Ca2+ levels, Rac hyperactivation and chemotaxis defect resulting from MFN2 depletion. Finally, inhibition of Rac activation restores chemotaxis in MFN2-deficient neutrophils. Taken together, we have identified that MFN2 regulates neutrophil migration via maintaining the mitochondria–ER interaction to suppress Rac activation, and uncovered a previously unrecognized role of MFN2 in regulating cell migration and the actin cytoskeleton.This article has an associated First Person interview with the first authors of the paper.

scholarly journals Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeleton

2019 ◽  
Author(s):  
Wenqing Zhou ◽  
Alan Y. Hsu ◽  
Yueyang Wang ◽  
Tianqi Wang ◽  
Jacob Jeffries ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Neutrophil Function ◽  
Mouse Embryonic Fibroblast ◽  
Mitochondrial Outer Membrane ◽  
Null Mouse ◽  
Hematopoietic Tissue ◽  
Mitofusin 2

AbstractNeutrophils rely on glycolysis for energy production. How mitochondria regulate neutrophil function is not fully understood. Here, we report that mitochondrial outer membrane protein Mitofusin 2 (Mfn2) regulates neutrophil homeostasis in vivo. Mfn2-deficient neutrophils are released from the hematopoietic tissue and trapped in the vasculature in zebrafish embryos. Human neutrophil-like cells deficient with MFN2 fail to arrest on activated endothelium under sheer stress or perform chemotaxis. Deletion of Mfn2 results in a significant reduction of neutrophil infiltration to the inflamed peritoneal cavity in mice. Mfn2, but not Mfn1, -null mouse embryonic fibroblast cells have altered actin structure and are impaired in wound closure. MFN2-deficient neutrophil-like cells display heightened intracellular calcium levels and Rac activation after chemokine stimulation. Mechanistically, MFN2 maintains mitochondria-ER interaction. Restoring mitochondria-ER tether rescues the chemotaxis defect and Rac activation resulted from MFN2 depletion. Finally, inhibition of Rac restores chemotaxis in MFN2-deficient neutrophils. Altogether, we identified that MFN2 regulates neutrophil migration via suppressing Rac activation and uncovered a previously unrecognized role of MFN2 in regulating the actin cytoskeleton.

scholarly journals Proof-of-concept clinical trial of etokimab shows a key role for IL-33 in atopic dermatitis pathogenesis

2019 ◽  
Vol 11 (515) ◽  
pp. eaax2945 ◽  
Author(s):  
Yi-Ling Chen ◽  
Danuta Gutowska-Owsiak ◽  
Clare S. Hardman ◽  
Melanie Westmoreland ◽  
Teena MacKenzie ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Therapeutic Potential ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Severity Index ◽  
Fundamental Biology ◽  
Targeted Inhibition

Targeted inhibition of cytokine pathways provides opportunities to understand fundamental biology in vivo in humans. The IL-33 pathway has been implicated in the pathogenesis of atopy through genetic and functional associations. We investigated the role of IL-33 inhibition in a first-in-class phase 2a study of etokimab (ANB020), an IgG1 anti–IL-33 monoclonal antibody, in patients with atopic dermatitis (AD). Twelve adult patients with moderate to severe AD received a single systemic administration of etokimab. Rapid and sustained clinical benefit was observed, with 83% achieving Eczema Area and Severity Index 50 (EASI50), and 33% EASI75, with reduction in peripheral eosinophils at day 29 after administration. We noted significant reduction in skin neutrophil infiltration after etokimab compared with placebo upon skin challenge with house dust mite, reactivity to which has been implicated in the pathogenesis of AD. We showed that etokimab also inhibited neutrophil migration to skin interstitial fluid in vitro. Besides direct effects on neutrophil migration, etokimab revealed additional unexpected CXCR1-dependent effects on IL-8–induced neutrophil migration. These human in vivo findings confirm an IL-33 upstream role in modulating skin inflammatory cascades and define the therapeutic potential for IL-33 inhibition in human diseases, including AD.

Inflammatory Disease and Abortive Platelet Shedding Caused by a Mutation in a Pivotal Regulator of Actin Dynamics in the redears Mouse.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1606-1606
Author(s):  
Monica J. Justice ◽  
Ben T. Kile ◽  
Lanette S. Woodward
Keyword(s):  
Actin Cytoskeleton ◽  
Inflammatory Disease ◽  
Critical Role ◽  
Neutrophil Infiltration ◽  
Blood Platelet ◽  
Forward Genetics ◽  
Actin Dynamics ◽  
Actin Depolymerization ◽  
Function Discovery

Abstract Mouse mutagenesis using forward genetics is valuable as a gene function discovery tool. We are looking for blood defects in a large ENU mutagenesis screen, and have isolated many new mouse mutants that reveal new mechanisms in hematopoiesis. One mutant mouse strain, called redears, is an intriguing model of inflammatory disease and thrombocytopenia. Animals homozygous for the redears (rd) mutation develop spontaneous inflammatory lesions of the ears and tail characterized by neutrophil infiltration and peripheral neutrophila. Unexpectedly, blood platelet numbers are dramatically reduced in rd/rd animals. A thorough analysis of platelet biogenesis shows that the platelet precursor cell, the megakaryocyte, undergoes abnormal maturation, which results in gross morphological abnormalities, increased ploidy and abortive platelet shedding. Here we report a mutation in a novel gene related to the yeast actin-interacting protein Aip1 in rd/rd mice. In yeast, Aip1 interacts with, and increases the activity of cofilin, a key regulator of actin depolymerization. Our data confirm that actin dynamics are dysregulated in rd/rd megakaryocytes and neutrophils. The massive cytoplasmic reorganization that is required for megakaryocyte maturation and platelet shedding has long been assumed to depend on the actin cytoskeleton. Intriguingly, recent studies suggest the process is caspase-dependent, and represents a form of ‘para-apoptosis’. With this in mind, we found that chemotaxis and apoptosis are perturbed in rd/rd neutrophils, suggesting that neutrophils are playing a key role in driving the inflammation. Disrupted actin depolymerization would provide an explanation for chemotactic deficiencies. Further, recent evidence implicating cofilin and other actin regulators in the initiation of apoptosis would suggest that this novel protein may play an essential role in neutrophil cell death. Thus, the redears mouse not only provides the first in vivo demonstration of the critical role of the actin cytoskeleton in megakaryocyte development and platelet production, but also represents a unique reagent to examine the relationship between actin dynamics, cellular maturation, inflammation and apoptosis. Our ongoing mutagenesis efforts continue to reveal new developmental mechanisms. New mutants, genetic tools, and resources can be found at www.mouse-genome.bcm.tmc.edu

scholarly journals Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2188-2195 ◽  
Author(s):  
RC Woodman ◽  
PH Reinhardt ◽  
S Kanwar ◽  
FL Johnston ◽  
P Kubes
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Neutrophil Migration ◽  
Specific Inhibitor ◽  
Neutrophil Infiltration ◽  
Cathepsin G ◽  
Neutrophil Adhesion ◽  
Human Neutrophil Elastase

Abstract The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

scholarly journals Metoprolol exerts a non-class effect against ischaemia–reperfusion injury by abrogating exacerbated inflammation

2020 ◽  
Vol 41 (46) ◽  
pp. 4425-4440 ◽  
Author(s):  
Agustín Clemente-Moragón ◽  
Mónica Gómez ◽  
Rocío Villena-Gutiérrez ◽  
Doménica V Lalama ◽  
Jaime García-Prieto ◽  
...  
Keyword(s):  
Conformational Changes ◽  
In Silico ◽  
Adrenergic Receptor ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Ischaemia Reperfusion Injury ◽  
Ischaemia Reperfusion ◽  
Β Blockers

Abstract Aims Clinical guidelines recommend early intravenous β-blockers during ongoing myocardial infarction; however, it is unknown whether all β-blockers exert a similar cardioprotective effect. We experimentally compared three clinically approved intravenous β-blockers. Methods and results Mice undergoing 45 min/24 h ischaemia–reperfusion (I/R) received vehicle, metoprolol, atenolol, or propranolol at min 35. The effect on neutrophil infiltration was tested in three models of exacerbated inflammation. Neutrophil migration was evaluated in vitro and in vivo by intravital microscopy. The effect of β-blockers on the conformation of the β1 adrenergic receptor was studied in silico. Of the tested β-blockers, only metoprolol ameliorated I/R injury [infarct size (IS) = 18.0% ± 0.03% for metoprolol vs. 35.9% ± 0.03% for vehicle; P < 0.01]. Atenolol and propranolol had no effect on IS. In the three exacerbated inflammation models, neutrophil infiltration was significantly attenuated only in the presence of metoprolol (60%, 50%, and 70% reductions vs. vehicle in myocardial I/R injury, thioglycolate-induced peritonitis, and lipopolysaccharide-induced acute lung injury, respectively). Migration studies confirmed the particular ability of metoprolol to disrupt neutrophil dynamics. In silico analysis indicated different intracellular β1 adrenergic receptor conformational changes when bound to metoprolol than to the other two β-blockers. Conclusions Metoprolol exerts a disruptive action on neutrophil dynamics during exacerbated inflammation, resulting in an infarct-limiting effect not observed with atenolol or propranolol. The differential effect of β-blockers may be related to distinct conformational changes in the β1 adrenergic receptor upon metoprolol binding. If these data are confirmed in a clinical trial, metoprolol should become the intravenous β-blocker of choice for patients with ongoing infarction.

scholarly journals Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2188-2195
Author(s):  
RC Woodman ◽  
PH Reinhardt ◽  
S Kanwar ◽  
FL Johnston ◽  
P Kubes
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Neutrophil Migration ◽  
Specific Inhibitor ◽  
Neutrophil Infiltration ◽  
Cathepsin G ◽  
Neutrophil Adhesion ◽  
Human Neutrophil Elastase

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

scholarly journals Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration duringStreptococcus pneumoniaeLung Infection

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Andrew Zukauskas ◽  
Randall J. Mrsny ◽  
Paula Cortés Barrantes ◽  
Jerrold R. Turner ◽  
John M. Leong ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Mortality Rates ◽  
Positive Bacterium ◽  
Immunomodulatory Agents ◽  
Content Type ◽  
Mouse Lungs

ABSTRACTStreptococcus pneumoniaeremains a source of morbidity and mortality in both developed and underdeveloped nations of the world. Disease can manifest as pneumonia, bacteremia, and meningitis, depending on the localization of infection. Interestingly, there is a correlation in experimental murine infections between the development of bacteremia and influx of neutrophils into the pulmonary lumen. Reduction of this neutrophil influx has been shown to improve survivability during infection. In this study, we usein vitrobiotinylation and neutrophil transmigration andin vivomurine infection to identify a system in which two epithelium-localized ATP-binding cassette transporters, MRP1 and MRP2, have inverse activities dictating neutrophil transmigration into the lumen of infected mouse lungs. MRP1 effluxes an anti-inflammatory molecule that maintains homeostasis in uninfected contexts, thus reducing neutrophil infiltration. During inflammatory events, however, MRP1 decreases and MRP2 both increases and effluxes the proinflammatory eicosanoid hepoxilin A3. If we then decrease MRP2 activity during experimental murine infection withS. pneumoniae, we reduce both neutrophil infiltration and bacteremia, showing that MRP2 coordinates this activity in the lung. We conclude that MRP1 assists in depression of polymorphonuclear cell (PMN) migration by effluxing a molecule that inhibits the proinflammatory effects of MRP2 activity.IMPORTANCEStreptococcus pneumoniaeis a Gram-positive bacterium that normally inhabits the human nasopharynx asymptomatically. However, it is also a major cause of pneumonia, bacteremia, and meningitis. The transition from pneumonia to bacteremia is critical, as patients that develop septicemia have ~20% mortality rates. Previous studies have shown that while neutrophils, a major bacterium-induced leukocyte, aid inS. pneumoniaeelimination, they also contribute to pathology and may mediate the lung-to-blood passage of the bacteria. Herein, we show that epithelium-derived MRP1 and MRP2 efflux immunomodulatory agents that assist in controlling passage of neutrophils during infection and that limiting neutrophil infiltration produced less bacteremia and better survival during murine infection. The importance of our work is twofold: ours is the first to identify an MRP1/MRP2 axis of neutrophil control in the lung. The second is to provide possible therapeutic targets to reduce excess inflammation, thus reducing the chances of developing bacteremia during pneumococcal pneumonia.

scholarly journals Molecular Insights Into Neutrophil Biology From the Zebrafish Perspective: Lessons From CD18 Deficiency

2021 ◽  
Vol 12 ◽  
Author(s):  
Almke Bader ◽  
Jincheng Gao ◽  
Thibaud Rivière ◽  
Bettina Schmid ◽  
Barbara Walzog ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Neutrophil Migration ◽  
Amino Acid Sequences ◽  
Sterile Inflammation ◽  
Type I ◽  
Hematopoietic Tissue ◽  
Sequence Alignments ◽  
Zebrafish Larvae ◽  
Inflamed Tissue

Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the β2 integrin family (CD11/CD18) are critically required for the initial neutrophil adhesion to the inflamed endothelium and several post-adhesion steps allowing their extravasation into the inflamed tissue. Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of β2 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of β2 integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the β subunit of β2 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.

Mice Lacking the Signaling Molecule, CalDAG-GEFI, Represent a Mouse Model for Leukocyte Adhesion Deficiency Type III.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 674-674
Author(s):  
Wolfgang Bergmeier ◽  
Tobias Goerge ◽  
Hong-Wei Wang ◽  
Stephen M. Cifuni ◽  
Jill R. Crittenden ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Leukocyte Adhesion ◽  
Thrombus Formation ◽  
Neutrophil Infiltration ◽  
Neutrophil Function ◽  
Calcium Flux ◽  
Leukocyte Adhesion Deficiency ◽  
Normal Surface

Abstract Defective inside-out activation of β 1, β 2, and β 3 integrins in platelets and leukocytes is a main characteristic of patients with leukocyte adhesion deficiency (LAD)-III syndrome. We have recently shown that CalDAG-GEFI, a member of the CalDAG-GEF/RasGRP family of intracellular signaling molecules that catalyzes the exchange of GTP for GDP bound to Rap1, plays a key role for the activation of α IIbβ 3 in murine platelets. Here we studied the role of CalDAG-GEFI for neutrophil function as well as the activation of β 1 integrins in platelets. Neutrophils from CalDAG-GEFI−/ − mice showed normal surface expression of key adhesion receptors such as L-selectin, PSGL-1, or β 1/β 2 integrins. Calcium flux, degranulation, and oxygen radical formation were similar in wild-type (WT) and mutant cells. In contrast, β 2 integrin-mediated adhesion to fibrinogen was significantly reduced in cells lacking CalDAG-GEFI when compared to controls. In vivo, CalDAG-GEFI-deficient neutrophils showed normal rolling along stimulated venules, while firm adhesion was almost completely inhibited. A similar defect in firm adhesion was observed in WT mice pre-treated with blocking antibodies against β 2 integrins. To determine the role of CalDAG-GEFI in neutrophil emigration, inflammation was induced in the peritoneum or the skin. In both models, neutrophil infiltration was significantly reduced in CalDAG-GEFI−/ − mice when compared to controls. We further demonstrate that CalDAG-GEFI regulates the activation of β 1 integrins in platelets and that CalDAG-GEFI-deficiency leads to a complete inhibition of arterial thrombus formation in mice. Due to its central role in the activation of β 1, β 2, and β 3 integrins, we propose CalDAG-GEFI as a candidate gene defective in LAD-III patients.

Srf Is Required For Neutrophil Migration In Response To Inflammation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 319-319
Author(s):  
Ashley Taylor ◽  
Wenwen Tang ◽  
Emanuela Bruscia ◽  
Ping-Xia Zhang ◽  
Aiping Lin ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Neutrophil Migration ◽  
Lavage Fluid ◽  
Neutrophil Function ◽  
Cellular Adhesion ◽  
Immunofluorescent Staining

Abstract 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.

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