Neutrophil Mobility Research Articles

Heterozygous Variants in the Clpbgene Associated with Neutropenia Affect Neutrophil Function

Introduction The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation. Methods Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden's chamber model was used to determine the level of the neutrophil's mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%). Results Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p .Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p .Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1). Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P<0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P<0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients' plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P<0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups. Conclusion Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Rutin hydrate relieves neuroinflammation in zebrafish models: Involvement of NF-κB pathway as a central network

Neuroinflammation is prevalent in multiple brain diseases and may also lead to dementia, cognitive impairment, and impaired spatial memory function associated with neurodegenerative diseases. A neuroprotective and antioxidant flavonoid, rutin hydrate (RH), was evaluated for the anti-neuroinflammatory activity mediated by copper sulfate (CuSO4) solution and lipopolysaccharide (LPS) in zebrafish. The results showed that 100 mg/L RH significantly reduced the ratio of neutrophil mobility in caudal hematopoietic tissue (CHT) region caused by CuSO4 and the number of neutrophils co-localized with facial peripheral nerves. In the LPS model, RH co-injection significantly diminished neutrophil and macrophage migration. Therefore, RH exhibited a significant rescue effect on both models. In addition, RH treatment remarkably reduced the effects of neuroinflammation on the locomotor ability, expression levels of genes associated with behavioral disorders, and acetylcholinesterase (AChE) activity. Furthermore, network pharmacology techniques were employed to investigate the potential mechanisms, and the associated genes and enzyme activities were validated in order to elucidate the underlying mechanisms. Network pharmacological analysis and zebrafish model indicated that RH regulated the expressions of NF-κB pathway-related targets (Toll-like receptor 9 (tlr9), nuclear factor kappa B subunit 1 (nfkb1), RELA proto-oncogene (RelA), nitric oxide synthase 2a, inducible (nos2a), tumour necrosis factor alpha-like (tnfα), interleukin 6 (il6), interleukin 1β (il1β), chemokine 8 (cxcl8), and macrophage migration inhibitory factor (mif)) as well as six key factors (arachidonic acid 4 alpha-lipoxygenase (alox4a), arachidonate 5-lipoxygenase a (alox5), prion protein a (prnpa), integrin, beta 2 (itgb2), catalase (CAT), and alkaline phosphatase (ALP) enzymes). Through this study, a thorough understanding of the mechanism underlying the therapeutic effects of RH in neuroinflammation has been achieved, thereby establishing a solid foundation for further research on the potential therapeutic applications of RH in neuroinflammatory disorders.

β-hydroxybutyrate impairs the directionality of migrating neutrophils through inhibiting the autophagy-dependent degradation of Cdc42 and Rac1 in ketotic cows

Dairy cows have high incidence of ketosis during perinatal. According to our previous studies, elevated ketone bodies (mainly β-hydroxybutyrate, BHB) in the peripheral blood are believed to contribute to the impairment of neutrophils mobility and directionality thereby contributing to the immunosuppression and further infectious disease secondary to ketosis. However, the specific effect of BHB on the directionality of bovine neutrophil need further study and the underlying molecular mechanisms are still unclear. According to the concentration of serum BHB, 40 multiparous cows (within 3 wk postpartum) were selected and divided into the control (n = 20, BHB <0.6 mM) or clinical ketosis (n = 20, BHB >3.0 mM) group. Blood samples were collected for baseline serum characteristics analysis and neutrophil mobility and directionality detection. Platelet activation factor was used as a chemoattractant in cell migration experiments. Our ex-vivo data showed ketotic cows, compared with control cows, were in a negative energy balance state, and their neutrophils had shorter migration distance, lower migration speed, and impaired migration directionality. Neutrophils from control cows were incubated with 3.0 mM BHB for 6 h in vitro. Similarly, BHB stimulation resulted in impaired mobility and directionality of bovine neutrophils. We further specifically studied the underlying molecular mechanism of BHB regulating neutrophil migration directionality in the present study. Cell division control protein 42 homolog (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), 2 key markers in the regulation of migration directionality, were found increased after BHB treatment in their total and activated protein levels while decreasing in their transcription level, suggesting that an imbalance of the protein degradation system may be involved. Interestingly, transmission electron microscopy data revealed a decrease in autophagosome number in neutrophils from ketotic cows. Western blotting data showed the accumulation of sequestosome-1 (p62) protein and a decrease in microtubule-associated protein 1 light chain 3-II (LC3-II) protein abundance after BHB treatment, further confirming that the autophagy flux was inhibited in neutrophils from ketotic cows. Additionally, rapamycin (RAPA), a specific autophagy activator, was used with or without BHB treatment in vitro. Accordingly, the BHB-induced impairment of migration directionality but not mobility was relieved by RAPA. Furthermore, as verified by in vivo experiments, compared with the control cows, the protein abundance of total and activated Cdc42 and Rac1 increased and their mRNA abundance decreased in neutrophils from ketotic cows. Overall, the present study revealed that pathological concentration of BHB impairs neutrophil migration directionality through inhibiting the autophagy-mediated degradation of Cdc42 and Rac1. These findings help explain the immunosuppression caused by ketosis.

Autophagy Induced by Palmitic Acid Regulates Neutrophil Adhesion Through the Granule-Dependent Degradation of αMβ2 Integrin in Dairy Cows With Fatty Liver.

β2 integrins are critical for neutrophil firm adhesion, trans-endothelial migration, and the recruitment to the inflamed tissue. Autophagy is implicated in cell migration and tumor metastasis through facilitating the turnover of β1 integrins; however, whether autophagy is able to control neutrophil migration by promoting the degradation of β2 integrins is unexplored. Here, we show that high blood levels of palmitic acid (PA) strongly triggered neutrophil autophagy activation, leading to adhesion deficiency in dairy cows with fatty liver. The three neutrophil granule subtypes, namely, azurophil granules (AGs), specific granules (SGs), and gelatinase granules (GGs), were engulfed by the autophagosomes for degradation, resulting in an increased vacuolation in fatty liver dairy cow neutrophils. Importantly, the adhesion-associated molecules CD11b and CD18 distributed on AGs, SGs, and GGs were degraded with the three granule subtypes by autophagy. Moreover, FGA, Hsc70, and TRIM21 mediated the degradation of cytosolic oxidized–ubiquitinated CD11b and CD18. Collectively, our results demonstrate that high blood PA triggers neutrophil autophagy-dependent vacuolation and granule-dependent adhesion deficiency, decreasing neutrophil mobility, and impairing the innate immune system of dairy cow with fatty liver. This theory extends the category of autophagy in maintaining granule homeostasis and provides a novel strategy to improve the immune of dairy cows with metabolic disease.

Characteristics of neutrophil extracellular traps in patients with periodontitis and gingivitis.

We sought to compare the characteristics and clinical significance of neutrophil extracellular traps in gingival samples from patients with periodontitis and those with gingivitis. The clinical indexes of gingival samples from patients with periodontitis and gingivitis were measured; the expression of TNF-alpha and IL-8 was measured by real-time fluorescent quantitative PCR; and the expression of TLR-8 and MMP-9 was measured by western blotting assays. Chemotaxis, phagocytosis and phagocytic activity of neutrophils were measured. Compared with the healthy group, the expression of TNF-α and IL-8 in the periodontitis group and the gingivitis group increased significantly (p < 0.05), and TNF-α in the gingivitis group was significantly lower than that in the healthy group (p < 0.05). The expression of IL-8 in the periodontitis group was significantly higher than that in the periodontitis group (p < 0.05). Furthermore, the expression of TLR-8 and MMP-9 in the periodontitis group was different from that in the gingivitis group and the healthy group, and the expression of TLR-8 and MMP-9 in the gingivitis group was significantly different from that in the healthy group (p < 0.05). In addition, the neutrophil mobility index in healthy people was 3.02 ± 0.53, that in the periodontitis group was 2.21 ± 0.13, and that in the gingivitis group was 2.31 ± 0.12. In conclusion, the chemotaxis of neutrophils in gingival samples of patients with periodontitis and gingivitis was decreased, the phagocytotic ability and activity of neutrophils were reduced, and the release of the extracellular trap-releasing inducible factors TNF-alpha and IL-8 also declined.

Megakaryocyte volume modulates bone marrow niche properties and cell migration dynamics.

All hematopoietic cells that develop in the bone marrow must cross the endothelial barrier to enter the blood circulation. Blood platelets, however, are released by bigger protrusions of huge progenitor cells, named megakaryocytes, and enter the blood stream as so-called proplatelets before fragmenting into mature platelets. Recently, a second function of megakaryocytes has been identified, as they modulate the quiescence of hematopoietic stem cells, mostly via different soluble factors. We know from light sheet fluorescence microscopy images that megakaryocytes are distributed throughout the bone marrow facing a dense vascular network. Here, we used such three-dimensional images to provide a realistic simulation template reflecting the in vivo cell-vessel distributions resulting in reliable whole-bone analysis in silico. Combining this approach with an automated image analysis pipeline, we found that megakaryocytes influence migration of neutrophils and hematopoietic stem cells, and thus act as biomechanical restrainers modulating cell mobility and extravasation. Indeed, as a consequence of increased megakaryocyte volumes in platelet-depleted mice neutrophil mobility was reduced in these animals.

The 17,18-epoxyeicosatetraenoic acid–G protein–coupled receptor 40 axis ameliorates contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques

Metabolites of eicosapentaenoic acid exert various physiologic actions. 17,18-Epoxyeicosatetraenoic acid (17,18-EpETE) is a recently identified new class of antiallergic and anti-inflammatory lipid metabolite of eicosapentaenoic acid, but its effects on skin inflammation and the underlying mechanisms remain to be investigated. We evaluated the effectiveness of 17,18-EpETE for control of contact hypersensitivity in mice and cynomolgus macaques. We further sought to reveal underlying mechanisms by identifying the responsible receptor and cellular target of 17,18-EpETE. Contact hypersensitivity was induced by topical application of 2,4-dinitrofluorobenzene. Skin inflammation and immune cell populations were analyzed by using flow cytometric, immunohistologic, and quantitative RT-PCR analyses. Neutrophil mobility was examined by means of imaging analysis invivo and neutrophil culture invitro. The receptor for 17,18-EpETE was identified by using the TGF-α shedding assay, and the receptor's involvement in the anti-inflammatory effects of 17,18-EpETE was examined by using KO mice and specific inhibitor treatment. We found that preventive or therapeutic treatment with 17,18-EpETE ameliorated contact hypersensitivity by inhibiting neutrophil mobility in mice and cynomolgus macaques. 17,18-EpETE was recognized by G protein-coupled receptor (GPR) 40 (also known as free fatty acid receptor 1) and inhibited chemoattractant-induced Rac activation and pseudopod formation in neutrophils. Indeed, the antiallergic inflammatory effect of 17,18-EpETE was abolished in the absence or inhibition of GPR40. 17,18-EpETE inhibits neutrophil mobility through GPR40 activation, which is a potential therapeutic target to control allergic inflammatory diseases.

SiMA: A simplified migration assay for analyzing neutrophil migration.

In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB4 )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS® FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB4 , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and three non-asthmatic patients gives a first hint to the capability of SiMA assay in the context of migration based diagnostics. Using SiMA we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB4 , the effect of the matrices FN versus HEM as well as the response to different medications, that is, Prednisolone induced a change of direction of migrating neutrophils in FN but no such effect was observed in human placental matrix. In addition, neutrophils of asthmatic individuals showed an increased proportion of cells migrating toward the vehicle. With the SiMA platform we presented a simplified but yet flexible platform for cost-effective tracking and quantification of neutrophil migration. The introduced method is based on a simple microscopic video stage, standardized, commercially available, µ-fluidic migration chambers and automated image analysis, and track validation software. © 2017 International Society for Advancement of Cytometry.

GRPR antagonist protects from drug-induced liver injury by impairing neutrophil chemotaxis and motility.

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL-8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life-threatening tissue damage that ensues. A GRPR (gastrin-releasing peptide receptor) antagonist impairs IL-8-induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen-induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen-overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2-induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8-driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.

Structural basis of PDZ-mediated chemokine receptor CXCR2 scaffolding by guanine nucleotide exchange factor PDZ-RhoGEF

The CXC chemokine receptor 2 (CXCR2) is a G protein coupled receptor mediating interleukin-8 chemotactic signaling and plays an important role in neutrophil mobility and tumor migration. However, efficient CXCR2 signaling requires PDZ domain-mediated scaffolding of signaling complexes at the plasma membrane and functional coupling of the signaling to specific downstream signaling pathways, in which only one PDZ protein has been characterized to interact with CXCR2. Here, we identified five novel CXCR2-binding PDZ-containing proteins, among which PDZ-RhoGEF is of particular interest because this PDZ and RGS-containing guanine nucleotide exchange factor (GEF) is also involved in cell signaling and mobility. To reveal the molecular basis of the interaction, we solved the crystal structure of PDZ-RhoGEF PDZ domain in complex with the CXCR2 C-terminal PDZ binding motif. The structure reveals that the PDZ–CXCR2 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four CXCR2 residues contributing to specific interactions. Structural comparison of CXCR2-binding PDZ domains and PDZ-RhoGEF PDZ bound with different ligands reveals PDZ- and ligand-specific interactions that may underlie the ability of promiscuous CXCR2 binding by different PDZ domains and PDZ binding promiscuity. The structure also reveals an unexpected asymmetric disulfide bond-linked PDZ dimer that allows simultaneous parallel binding of CXCR2 to two PDZ domains. This study provides not only the structural basis for PDZ-mediated CXCR2–PDZ-RhoGEF interaction, but also a new mode of PDZ dimerization, which both could prove valuable in understanding signaling complex scaffolding in CXCR2 signaling and coupling to specific signaling pathways.

Neutrophil-Mediated Delivery of Therapeutic Nanoparticles across Blood Vessel Barrier for Treatment of Inflammation and Infection.

Endothelial cells form a monolayer in lumen of blood vessels presenting a great barrier for delivery of therapeutic nanoparticles (NPs) into extravascular tissues where most diseases occur, such as inflammation disorders and infection. Here, we report a strategy for delivering therapeutic NPs across this blood vessel barrier by nanoparticle in situ hitchhiking activated neutrophils. Using intravital microscopy of TNF-α-induced inflammation of mouse cremaster venules and a mouse model of acute lung inflammation, we demonstrated that intravenously (iv) infused NPs made from denatured bovine serum albumin (BSA) were specifically internalized by activated neutrophils, and subsequently, the neutrophils containing NPs migrated across blood vessels into inflammatory tissues. When neutrophils were depleted using anti-Gr-1 in a mouse, the transport of albumin NPs across blood vessel walls was robustly abolished. Furthermore, it was found that albumin nanoparticle internalization did not affect neutrophil mobility and functions. Administration of drug-loaded albumin NPs markedly mitigated the lung inflammation induced by LPS (lipopolysaccharide) or infection by Pseudomonas aeruginosa. These results demonstrate the use of an albumin nanoparticle platform for in situ targeting of activated neutrophils for delivery of therapeutics across the blood vessel barriers into diseased sites. This study demonstrates our ability to hijack neutrophils to deliver nanoparticles to targeted diseased sites.

Current insights into inherited bone marrow failure syndromes.

Inherited bone marrow failure syndrome (IBMFS) encompasses a heterogeneous and complex group of genetic disorders characterized by physical malformations, insufficient blood cell production, and increased risk of malignancies. They often have substantial phenotype overlap, and therefore, genotyping is often a critical means of establishing a diagnosis. Current advances in the field of IBMFSs have identified multiple genes associated with IBMFSs and their pathways: genes involved in ribosome biogenesis, such as those associated with Diamond-Blackfan anemia and Shwachman-Diamond syndrome; genes involved in telomere maintenance, such as dyskeratosis congenita genes; genes encoding neutrophil elastase or neutrophil adhesion and mobility associated with severe congenital neutropenia; and genes involved in DNA recombination repair, such as those associated with Fanconi anemia. Early and adequate genetic diagnosis is required for proper management and follow-up in clinical practice. Recent advances using new molecular technologies, including next generation sequencing (NGS), have helped identify new candidate genes associated with the development of bone marrow failure. Targeted NGS using panels of large numbers of genes is rapidly gaining potential for use as a cost-effective diagnostic tool for the identification of mutations in newly diagnosed patients. In this review, we have described recent insights into IBMFS and how they are advancing our understanding of the disease's pathophysiology; we have also discussed the possible implications they will have in clinical practice for Korean patients.

Endothelial Cell Peroxisome Proliferator–Activated Receptor γ Reduces Endotoxemic Pulmonary Inflammation and Injury

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARγ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARγ. Endothelial cell PPARγ (ePPARγ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARγ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARγ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARγ ligands. Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARγ, but not in its absence, and also inhibited neutrophil mobility in a PPARγ-dependent manner. Our results demonstrate a key protective role of ePPARγ against endotoxemic injury and a potential ePPARγ-mediated anti-inflammatory role for NFAs.

Abstract 5054: Bone Marrow MyD88 Contributes to Ischemic Myocardial Infarction and Modulates Neutrophil Function and Chemokine Receptor Expression

INTRODUCTION: MyD88 is an adaptor protein critical for innate immune signaling and plays a pivotal role in host defense against infection, but its role in non-infectious myocardial inflammation and injury after ischemia-reperfusion (I/R) is unclear. Our previous studies in MyD88 −/− (KO) mice have demonstrated that MyD88 is essential for I/R-induced myocardial injury. The current study was to delineate the specific contribution of cardiac and bone marrow (BM)-derived MyD88 in mediating ischemic cardiac injury and to explore the underlying mechanisms. METHODS: Chimeric models were generated by lethal irradiation and BM reconstruction. For I/R model, the coronary artery was ligated for 60 min and followed by reperfusion. At the end of reperfusion, the hearts were examined for ischemic and infarct sizes. To assess neutrophil mobility function, thioglycollate was injected intra-peritoneally. Six hours later, peritoneal cells were counted. Neutrophils were labeled with anti-Ly-6C and counted by flow cytometry. CXCR2 expression, which is critical for neutrophil mobility function, was measured by flow cytometry. RESULTS: There was a significant decrease in infarct sizes in KO compared to WT (22±1% vs. 37±2%, p &lt;0.001). Compared to WT→WT control (Donor→Recipient), KO→WT mice, which lacked MyD88 in BM cells but maintained intact MyD88 signaling in the heart, had smaller MI sizes ( p &lt; 0.05). Surprisingly, infarct sizes of WT→WT mice were also significantly reduced compared to WT ( p &lt; 0.05). To determine if MyD88 deficiency inhibits neutrophil function that contributes the observed cardiac benefit, we examined the neutrophil mobility and CXCR2 expression. Indeed, neutrophil mobility was dramatically decreased in KO mice compared to WT mice ( p &lt; 0.01). Neutrophil mobilization also decreased in WT→WT and KO→WT chimeric mice ( p &lt; 0.01). Moreover, there was a significant decrease in CXCR2 expression on the peritoneal neutrophils of KO and KO→WT mice ( p &lt; 0.01), but not in WT→WT mice. CONCLUSIONS: Taken together, these findings suggest that MyD88 signaling, most likely that of BM-derived neutrophils plays a critical role in mediating myocardial I/R injury. MyD88 may mediate I/R injury partly by modulating CXCR2 expression and thus neutrophil function. This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).

Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis

Clostridium perfringens gas gangrene is a fulminant necrotizing infection in which inflammatory cells are notably absent from infected tissues but are often massed within adjacent vessels. It has been shown that C. perfringens phospholipase C (PLC) stimulates formation of large intravascular platelet/leukocyte complexes and that PLC-induced activation of platelet gpIIbIIIa plays a major role. In vivo, such aggregates contribute to microvascular thrombosis and ischaemic necrosis of tissue. However, the effects of adherent platelets on neutrophil diapedesis have not been established. The present work investigated (1) the contribution of platelet P-selectin (CD62P) to PLC-induced cellular complex formation and (2) the effects of platelet adhesion on neutrophil diapedesis. The effects of anti-gpIIbIIIa and anti-CD62P strategies on PLC-induced complex formation were measured by flow cytometry and followed by light microscopy. Both platelet gpIIbIIIa and CD62P contributed to the formation of platelet/leukocyte complexes. Specifically, gpIIbIIIa mediated the formation of large platelet/platelet aggregates that were tethered to the leukocyte principally via CD62P. Neutrophil diapedesis, quantified by a transendothelial cell migration assay and visualized by electron microscopy, was significantly reduced (>60%) by the adherence of large platelet aggregates. It was concluded that the absence of a tissue inflammatory response in C. perfringens gas gangrene is due, in part, to impaired neutrophil mobility caused by large aggregates of adherent platelets induced by PLC. Further, an adjunctive immunotherapeutic strategy targeting both gpIIbIIIa and CD62P may improve the tissue inflammatory response, prevent vascular occlusion, maintain tissue viability, and reduce the need for radical amputation in patients with clostridial gas gangrene.

Interleukin-17 regulates expression of the CXC chemokine LIX/CXCL5 in osteoblasts: implications for inflammation and neutrophil recruitment.

Interleukin (IL)-17 is the founding member of an emerging family of inflammatory cytokines whose functions remain poorly defined. IL-17 has been linked to the pathogenesis of rheumatoid arthritis, and numerous studies implicate this cytokine in inflammation-induced bone loss. It is clear that a major function of IL-17 is to amplify the immune response by triggering production of chemokines, cytokines, and cell-surface markers, ultimately leading to neutrophil chemotaxis and inflammation. As an IL-17 signaling deficiency in mice causes a dramatic reduction in neutrophil chemotaxis and a consequent increased susceptibility to bacterial infection, it is important to define gene targets involved in IL-17-mediated neutrophil trafficking. Here, we demonstrate that IL-17 and tumor necrosis factor alpha (TNF-alpha) cooperatively induce the lipopolysaccharide-inducible CXC chemokine (LIX; a.k.a., CXC chemokine ligand 5, Scya5, or murine granulocyte chemotactic protein-2) in the preosteoblast cell line MC3T3. LIX is induced rapidly at the mRNA and protein levels, likely through the activation of new gene transcription. Conditioned media from MC3T3 cells treated with IL-17 and/or TNF-alpha stimulates neutrophil mobility potently, and LIX is a significant contributing factor to this process. In addition, IL-17 cooperates with bacterial components involved in periodontal disease to up-regulate LIX expression. This study is the first demonstration of LIX expression in bone cells and has implications for inflammatory bone diseases such as arthritis and periodontal disease.

Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

We examined the in vitro fungicidal activity of human neutrophils against conidia and yeast cells of Penicillium marneffei. Neutrophils showed a small but significant anti-fungal effect against the yeast form of P. marneffei. Treatment of neutrophils with GM-CSF significantly augmented their anti-fungal activity. In contrast, the conidia form resisted killing even by stimulated neutrophils. Neutrophil fungicidal effect was not inhibited by superoxide dismutase (SOD), while the same treatment significantly suppressed the killing of Candida albicans by GM-CSF-stimulated neutrophils. For effective killing of P. marneffei yeast cells by GM-CSF-stimulated neutrophils, direct contact between the two was essential; interference in such interaction by separation using a 0. 45-microm-pored membrane prevented such an effect. Addition of colchicine attenuated GM-CSF-stimulated neutrophil fungicidal activity in a dose-dependent manner. This effect did not appear to be mediated by interference with neutrophil mobility toward yeast cells, because similar results were obtained when the cultures were set in round-bottomed wells which facilitate their direct contact. Finally, granular extracts derived from unstimulated neutrophils significantly suppressed the growth of microorganisms. Pretreatment of neutrophils with GM-CSF markedly enhanced this effect. The fungicidal activity of granular lysates was strongly, but not completely, reduced by heat treatment. Considered together, our results indicate that GM-CSF-stimulated neutrophils killed the yeast form of P. marneffei present in close proximity, probably in a superoxide anion-independent mechanism, but through exocytosis of granular enzymes which were largely heat-labile.

Cyclosporin A protects lung function from hyperoxic damage.

Cyclosporin A (CsA), an inhibitor of protein phosphatase 2B (calcineurin), has been shown to play a role in exocytosis and neutrophil mobility. Hyperoxia (>95% oxygen for 72 h) causes lung injury and reduces lung compliance. This model is indicative of deficiencies in surfactant and elicits a vigorous immune response leading to further damage. We examined the effects of CsA on surfactant-secreting lung alveolar type II cells. CsA enhances ATP-stimulated increases in whole cell capacitance in the presence of 2 mM extracellular Ca2+. This measurement corresponds with increases in exocytosis. Because of its effect on the immune system and exocytosis from type II cells, CsA was examined for its protective effects against hyperoxia-induced lung damage in mice. We found that CsA (50 mg. kg-1. day-1) attenuated hyperoxia-induced reductions in lung compliance when administered before or during 72 h of >95% oxygen (P < 0.05). CsA (10 mg. kg-1. day-1) also had a protective effect against hyperoxia-induced changes in neutrophil infiltration, capillary congestion, edema, and hyaline membrane formation. Wet lung weight-to-dry lung weight ratios did not show any significant changes after hyperoxia or hyperoxia plus CsA (P < 0. 05). CsA may be useful to treat patients undergoing prolonged high-oxygen therapy and possibly other lung injuries.

Effect of activators on neutrophil mobility

Effect of various activators on chaotic movement of neutrophls on the glass is studied using a Magiscan 2A automatic image analysis system. All tested agents in concentration inducing neutrophil activation suppress their mobility, but no one completely inhibits neutrophil motion. The cells retain the ability to change their shape and partially to move.

CD18-Dependent and L-Selectin-Dependent Neutrophil Emigration Is Diminished in Neonatal Rabbits

Human neonatal neutrophils manifest decreases in mobility, adherence, and emigration compared with adult neutrophils that may contribute to the increased susceptibility of neonates to infection. In a developmental rabbit model, we show a reduced ability of neutrophils from 1-day-old rabbit pups to emigrate to inflamed peritoneium (3.7 +/- 0.35 x 10(6) neutrophils/mL peritoneal exudate) compared with 14-day-old (8.5 +/- 0.7 x 10(6)/mL) and adult rabbits (9.4 +/- 1.4 x 10(6) mL, P < .05) despite significantly increased blood neutrophil counts. Because the reductions in functional Mac-1 (CD11b/CD18) as well as the amount of surface L-selectin are hypothesized to be primarily responsible for the differences in human neonatal neutrophil mobility, we examined CD11b/CD18 and L-selectin in our model. Using flow cytometric analysis we found that similar to human neonates, neutrophils from 1-day-old rabbit pups had 57% of adult rabbit levels of L-selectin and, in contrast with adults, failed to show significant decreases in L-selectin after chemotactic stimulation. In addition, neutrophils from 1-day-old pups compared with adults showed a significantly diminished capacity to upregulate CD11b/CD18 after chemotactic stimulation in vitro, or after emigration to the inflamed peritoneum. Systemic administration of anti-L-selectin monoclonal antibody (MoAb) resulted in significant reduction in peritoneal neutrophils in adult (47%, P < .05) and 14-day-old rabbits (47%, P < .05), but was without effect in 1-day-old rabbits. Administration of anti-CD18 MoAb resulted in significant reduction in peritoneal neutrophil accumulation in all age groups though less in 1 day and 14 day (58% and 65%, respectively) than in adults (91%, P < .05). Only in the 14-day-old rabbits was there an additive effect of anti-L-selectin and anti-CD18 MoAbs compared with anti-CD18 alone (84% v 65%, P < .05). The findings in this in vivo rabbit model support the hypothesis that the previously described in vitro defects in human neonatal L-selectin and CD11b/CD18 may be major contributors to human neonatal inflammatory deficits.