Search

Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Results: Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Conclusion: Our results suggest that SKAP2 has a dual role. It may restrict CD11b/ CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils. [ABSTRACT FROM AUTHOR]

BackgroundThe importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood.MethodWe performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC).ResultsAmong the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells.ConclusionOur results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of β2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of β2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two β2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting β2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of β 2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of β 2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two β 2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting β 2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease. [ABSTRACT FROM AUTHOR]

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD.

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the αMβ2 (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic target cells. These properties have been exploited to engineer the CyaA toxin as a potent non-replicating vector able to deliver antigens into antigen presenting cells and elicit specific cell-mediated immune responses. Antigens of interest can be inserted into the CyaA protein to yield recombinant molecules that are targeted in vivo to dendritic cells, where the antigens are processed and presented by the major class I and class II histocompatibility complexes (MHC-I and II). CyaA turned out to be a remarkably effective and versatile vaccine vector capable of inducing all the components of the immune response (T-CD4, T-CD8, and antibody). In this chapter, we summarize the basic knowledge on the adenylate cyclase toxin and then describe the application of CyaA in vaccinology, including some recent results of clinical trials of immunotherapy using a recombinant CyaA vaccine.

Abstract: The beta 2 integrin CR3 is a leukocyte adhesion heterodimeric glycoprotein which functions both as receptor for iC3b and in several cell–cell and cell–substrate adhesion interactions. In order to elucidate the molecular evolution of the CR3 receptor, here we report the cloning and characterization of its β2 (CD18) and aM (CD11b) subunits in rainbow trout (Oncorhynchus mykiss). The predicted polypeptide sequences of trout CD18 and CD11b-like exhibit 50, 49, or 61% and 25, 25, or 30% identity with human, mouse, and zebrafish orthologs, respectively. The ‘domain’ architecture of trout CD18 and CD11b-like subunits retains several characteristics of the mammalian ortholog proteins, such as cysteine-rich regions, N-linked glycosylation sites and several proposed domains and signal sequences (von Willebrand factor type A, Integrin alpha, Integrin B tail, EGF, and Transmembrane domain). The tissue expression profiles of trout CR3 subunits diverge from those of mammalian counterparts, showing the kidney as the main source of the trout CD18 and CD11b-like mRNA transcripts. This is the first report of cloning and characterization of the CR3 receptor in low vertebrates. [Copyright &y& Elsevier]

Depression is an independent risk factor for cardiovascular disease (CVD). We have previously shown that repeated social defeat (RSD) exaggerates atherosclerosis development by enhancing neutrophil extracellular trap (NET) formation. In this study, we investigated the impact of RSD on arterial thrombosis. Eight-week-old male wild-type mice (C57BL/6J) were exposed to RSD by housing with larger CD-1 mice in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. After confirming depression-like behaviors, mice underwent FeCl3-induced carotid arterial injury and were analyzed after 3 h. Although the volume of thrombi was comparable between the two groups, fibrin(ogen)-positive areas were significantly increased in defeated mice, in which Ly-6G-positive cells were appreciably co-localized with Cit-H3-positive staining. Treatment with DNase I completely diminished exaggerated fibrin-rich clot formation in defeated mice. Flow cytometric analysis showed that neutrophil CD11b expression before FeCl3 application was significantly higher in defeated mice than in control mice. In vitro NET formation induced by activated platelets was significantly augmented in defeated mice, which was substantially inhibited by anti-CD11b antibody treatment. Our findings demonstrate that RSD enhances fibrin-rich clot formation after arterial injury by enhancing NET formation, suggesting that NET can be a new therapeutic target in depression-related CVD. [ABSTRACT FROM AUTHOR]

Dysregulated chronic inflammation plays a crucial role in the pathophysiology of atherosclerosis and may be a result of impaired resolution. Thus, restoring levels of specialized pro-resolving mediators (SPMs) to promote the resolution of inflammation has been proposed as a therapeutic strategy for patients with atherosclerosis, in addition to standard clinical care. Herein, we evaluated the effects of the SPM lipids, lipoxin A 4 (LXA 4 ) and lipoxin B 4 (LXB 4 ), on neutrophils isolated from patients with atherosclerosis compared with healthy controls. Patients displayed altered endogenous SPM production, and we demonstrated that lipoxin treatment in whole blood from atherosclerosis patients attenuates neutrophil oxidative burst, a key contributor to atherosclerotic development. We found the opposite effect in neutrophils from healthy controls, indicating a potential mechanism whereby lipoxins aid the endogenous neutrophil function in health but reduce its excessive activation in disease. We also demonstrated that lipoxins attenuated upregulation of the high-affinity conformation of the CD11b/CD18 integrin, which plays a central role in clot activation and atherosclerosis. Finally, LXB 4 enhanced lymphatic transmigration of human neutrophils isolated from patients with atherosclerosis. This finding is noteworthy, as impaired lymphatic function is now recognized as an important contributor to atherosclerosis. Although both lipoxins modulated neutrophil function, LXB 4 displayed more potent effects than LXA 4 in humans. This study highlights the therapeutic potential of lipoxins in atherosclerotic disease and demonstrates that the effect of these SPMs may be specifically tailored to the need of the individual., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

The adenylate cyclase toxin (CyaA) is one of the major virulence factors of Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells where, upon activation by endogenous calmodulin, it synthesizes massive amounts of cAMP that alters cellular physiology. The CyaA toxin is a 1706 residues-long bifunctional protein: the catalytic domain is located in the 400 amino-proximal residues, whereas the carboxy-terminal 1306 residues are implicated in toxin binding to the cellular receptor, the αMβ2 (CD11b/CD18) integrin, and subsequently in the translocation of the catalytic domain across the cytoplasmic membrane of the target cells. Indeed, this protein is endowed with the unique capability of delivering its N-terminal catalytic domain directly across the plasma membrane of eukaryotic target cells. These properties have been exploited to engineer the CyaA toxin as a potent non-replicating vector able to deliver antigens into antigen presenting cells and elicit specific cell-mediated immune responses. Antigens of interest can be inserted into the CyaA protein to yield recombinant molecules that are targeted in vivo to dendritic cells, where the antigens are processed and presented by the major class I and class II histocompatibility complexes (MHC-I and II). CyaA turned out to be a remarkably effective and versatile vaccine vector capable of inducing all the components of the immune response (T-CD4, T-CD8, and antibody). In this chapter, we summarize the basic knowledge on the adenylate cyclase toxin and then describe the application of CyaA in vaccinology, including some recent results of clinical trials of immunotherapy using a recombinant CyaA vaccine. [ABSTRACT FROM AUTHOR]