scholarly journals In vivo imaging of hepatic neutrophil migration in severe alcoholic hepatitis with 111In-radiolabelled leucocytes

2018 ◽  
Vol 38 (4) ◽  
Author(s):  
Jonathan R. Potts ◽  
Neda Farahi ◽  
Mark R. Howard ◽  
Mark R. Taylor ◽  
Sarah Heard ◽  
...  
Keyword(s):  
Alcoholic Hepatitis ◽  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Post Injection ◽  
Leucocyte Scintigraphy ◽  
Novel Treatments ◽  
Severe Alcoholic Hepatitis ◽  
Normal Controls ◽  
H 30

The study’s aim was to image severe alcoholic hepatitis (SAH) using 111In-labelled leucocytes with two objectives in mind: firstly for non-invasive diagnosis and secondly to provide a platform for experimental therapies aiming to inhibit intrahepatic neutrophil migration. 111In-leucocyte scintigraphy was performed 30 min and 24 h post-injection in 19 patients with SAH, 14 abstinent patients with alcohol-related cirrhosis and 11 normal controls. Eleven with SAH and seven with cirrhosis also had 99mTc-nanocolloid scintigraphy. Change in hepatic 111In radioactivity was expressed as decay-corrected 24 h:30 min count ratio and, in SAH, compared with histological grading of steatohepatitis and expression of granulocyte marker, CD15. Hepatic microautoradiography on biopsy specimens obtained 24 h post-injection of 111In-leucocytes was performed in one patient. Median 24 h:30 min hepatic 111In activity ratio was higher in SAH (2.5 (interquartile range (IQR): 1.7–4.0) compared with cirrhotics and normal controls (1.0 (0.8–1.1) and 0.8 (0.7–0.9) respectively, P<0.0001). In SAH, it correlated with CD15 expression (r = 0.62, P=0.023) and was higher in marked compared with mild/moderate steatohepatitis (4.0 (3.0–4.6) compared with 1.8 (1.5–2.6), P=0.006). Hepatic-to-splenic 99mTc count rate ratio was reduced in SAH (0.5 (0.4–1.4)) compared with cirrhotics (2.3( 0.6–3.0)) and three historic normal controls (4.2 (3.8–5.0); P=0.003), consistent with impaired hepatic reticuloendothelial function. Scintigraphic findings in SAH included prominent lung radioactivity at 30 min, likely the result of neutrophil primimg. Microautoradiography demonstrated cell-associated 111In in areas of parenchymal neutrophil infiltration. In conclusion, 111In-leucocyte scintigraphy can non-invasively diagnose SAH and could provide a platform for evaluation of novel treatments aiming to inhibit intrahepatic neutrophil migration.

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.

scholarly journals TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration

2015 ◽  
Vol 309 (1) ◽  
pp. G30-G41 ◽  
Author(s):  
Yoon Seok Roh ◽  
Bi Zhang ◽  
Rohit Loomba ◽  
Ekihiro Seki
Keyword(s):  
Liver Injury ◽  
Alcoholic Hepatitis ◽  
Early Stage ◽  
Neutrophil Infiltration ◽  
Wild Type ◽  
Ethanol Feeding ◽  
Binge Ethanol ◽  
Deficient Mice ◽  
Cxcl1 Expression

Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.

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 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 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 &lt; 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 Intravenous antagomiR-494 lessens brain-infiltrating neutrophils by increasing HDAC2-mediated repression of multiple MMPs in experimental stroke

2019 ◽  
Author(s):  
Fangfang Li ◽  
Haiping Zhao ◽  
Guangwen Li ◽  
Sijia Zhang ◽  
Rongliang Wang ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Neuronal Damage ◽  
Neutrophil Migration ◽  
Glucose Deprivation ◽  
Neutrophil Infiltration ◽  
Predictive Biomarker ◽  
Experimental Stroke ◽  
Mmp Genes

Abstract Background: Neutrophil infiltration and phenotypic transformation are believed to contribute to neuronal damage and clinical outcome in ischemic stroke. Emerging evidence suggests that HDAC2 is an epigenetic regulator of inflammatory cells. Here, we investigated whether miR-494 affects HDAC2-mediated neutrophil infiltration and phenotypic shift. Methods: The miR-494 levels in neutrophils from AIS patients were detected by real-time PCR. C57BL/6J mice were subjected to transient middle cerebral artery occlusion, and the N1/N2 neutrophil shift was examined. Cortical neurons were subjected to oxygen-glucose deprivation and stimulated with supernatant from differently treated neutrophils or were cocultured with neutrophils; neuronal injury was detected, and ChIP-Seq was performed to clarify which genes are the binding targets of HDAC2. Finally, a transwell assay was conducted to examine neutrophil migration. Results: Compared to the control subjects, AIS patients had increased neutrophil expression of miR-494, and in AIS patients, elevated miR-494 expression in neutrophils was a predictor of worse neurological outcomes. MiR-494 correlates with the upregulation of adhesion molecules in neutrophils of AIS patients. Systemically administered antagomiR-494 partly shifts neutrophils into the N2 phenotype in MCAO mice. AntagomiR-494-treated neutrophils exert a neuroprotective role in vitro. ChIP-seq revealed that HDAC2 targets multiple MMP genes in neutrophils of AIS patients. Further in vitro and in vivo experiments showed that antagomiR-494 repressed expression of MMP genes, including MMP7, MMP10, MMP13, and MMP16, to reduce the number of brain-infiltrating neutrophils by regulating HDAC2. Conclusion: MiR-494 may serve as an alternative predictive biomarker of the outcome of AIS patients, and antagomiR-494 treatment decreased the expression of multiple MMPs and the infiltration of neutrophils partly by targeting HDAC2.

scholarly journals Gα13-Mediated β2 Integrin Outside-in Signaling in Neutrophil Migration

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 436-436
Author(s):  
Claire W Chang ◽  
Ni Cheng ◽  
Randal Skidgel ◽  
Yanyan Bai ◽  
Xiaoping Du
Keyword(s):  
Neutrophil Migration ◽  
Neutrophil Infiltration ◽  
Transendothelial Migration ◽  
Wild Type ◽  
Infiltration Model ◽  
Β2 Integrin ◽  
And Migration ◽  
Integrin Ligand

Abstract Transendothelial migration of neutrophils requires chemoattractant signals and also integrin family of adhesion receptors, particularly the β 2 family of integrins, including Mac-1 and LFA-1. Signals transmitted by G protein-coupled receptors (GPCR) for chemoattractants and cytokines induces inside-out signaling activating the ligand binding function of integrins. Conversely ligand binding to integrins stimulates outside-in signaling, leading to cell spreading, retraction and migration. The heterotrimeric G protein subunit, Gα13, is important for GPCR signaling leading to RhoA activation but also binds to integrins, including β2 integrins to stimulate outside-in signaling. To study the roles of Gα13 in neutrophil migration, we tested the effect of Gα13 knockout on transendothelial migration of neutrophils stimulated by chemoattractant fMIVIL. We demonstrate that transendothelial migration of Gα13 knockout neutrophils was significantly but partially reduced as compared with wild type mice. Transendothelial migration of Gα13 knockout neutrophils is similar to wild type neutrophil migration neutralized with an anti-Mac1 (anti-αm) antibody, and was not further inhibited by the anti-Mac1 antibody, suggesting that transendothelial migration mediated by integrin αmβ2 was predominantly Gα13-dependent. Interestingly, either anti-β2 antibody or anti-LFA1 (anti-αL) antibody appeared to inhibit transendothelial migration of not only wild type neutrophils, but also to a degree, Gα13-knockout neutrophils, suggesting a minor LFA1-dependent but Gα13-independent component of transendothelial migration in addition to the Gα13-dependent transendothlial migration. Furthermore, even though the fibrinogen and ICAM-1 are both β2 ligands, we show that more neutrophils migrated through ICAM-1-coated transwells than fibrinogen-coated transwells, and only ICAM-1-mediated neutrophil migration is Gα13 dependent, suggesting that Gα13-dependent neutrophil migration is selective for certain β2 integrin ligand (ICAM-1). Importantly, Gα13 knockout selectively inhibited the velocity of neutrophil migration on integrin ligand ICAM-1, but had no effect on the directionality of neutrophil migration which requires GPCR-dependent chemoattactant signaling. To understand whether and how Gα13 regulate integrin signaling, we show that Gα13 knockout did not affect the static adhesion of neutrophils to ICAM1, but significantly inhibited neutrophil spreading on ICAM-1. Furthermore, Gα13 bound to β2 integrins in neutrophils adherent on ICAM-1, and this binding was inhibited by the ExE motif peptide MB2mP6 derived from the Gα13 binding site of β2 integrin cytoplasmic domain. MB2mP6 also inhibited transendothelial cell migration similarly as Gα13 knockout. These data suggest that Gα13 plays an important role in promoting β2-integrin dependent neutrophil transendothelial migration mainly by mediating integrin outside-in signaling. Consistent with previous findings of the role of Gα13-dependent outside-in signaling in negative regulation of RhoA in other integrin subtypes, both Gα13 knockout and MB2mP6 abolished the transient inhibition in RhoA during adhesion of neutrophils on ICAM-1. These data suggest that Gα13 mediates outside-in signaling and transient inhibition of RhoA, and thus promotes neutrophil spreading and migration on integrin ligands. To test the role of Gα13 in neutrophil migration in vivo, we showed that neutrophil infiltration in vivo was reduced in leukocyte-selective Gα13 knockout mice using both thioglycolate-induced peritoneal neutrophil infiltration model and LPS-induced neutrophil lung infiltration model in vivo. Furthermore, MB2mP6 inhibited neutrophil infiltration in cardiac tissues in the cardiac ischemia-reperfusion injury model in mice. These data suggest that Gα13-integrin interaction plays an essential role in the integrin-dependent transendothelial migration and is likely to be important in neutrophils' immune and inflammatory functions. Disclosures No relevant conflicts of interest to declare.

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