Regulation Of Leukocyte Recruitment Research Articles

Tumor necrosis factor regulates leukocyte recruitment but not bacterial persistence during Staphylococcus aureus craniotomy infection

BackgroundCraniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored.MethodsA mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity.ResultsTNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected.ConclusionThese findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection.

Recruitment of neutrophils in glomeruli in early mouse sepsis is associated with E-selectin expression and activation of endothelial NF-κB and MAPK pathways.

Sepsis is a dysregulated systemic inflammatory response to an infection, which can lead to multiple organ dysfunction syndrome that includes the kidney. Leukocyte recruitment is an important process of the host immune defense in response to sepsis. Endothelial cells (EC) actively regulate leukocyte recruitment by expressing adhesion molecules following the activation of dedicated intracellular signal transduction pathways. Previous studies reported that the expression of adhesion molecules was associated with the activation of endothelial NF-κB p65 and MAPK c-Jun pathways in vitro in response to conditions that mimic processes that occur in inflammation. This study aimed to investigate the spatiotemporal patterns of leukocyte recruitment, expression of adhesion molecules, and endothelial nuclear p65 and c-Jun localization in renal microvascular beds of septic mice. Here, we used a cecal ligation and puncture (CLP) sepsis mouse model and RT-qPCR and immunohistochemical staining. We showed that neutrophils, macrophages, and T lymphocytes were all present in the kidney, yet only neutrophils accumulated in a spatiotemporally discernible pattern, mainly in glomeruli at 4 hours after CLP-sepsis initiation. E-selectin, not VCAM-1, was expressed in glomeruli at the same time point. In a subset of mice at 72 hours after CLP-sepsis started, VCAM-1 expression was prominent in glomerular EC, which was not related to changes in mmu-microRNA(miR)-126a-3p levels, a short noncoding microRNA previously shown to inhibit the translation of VCAM-1 mRNA into protein. Nuclear localization of p65 and c-Jun occurred in EC of all microvascular segments at 4 and 7 hours after CLP-sepsis initiation. In summary, sepsis-induced recruitment of neutrophils, E-selectin expression, and NF-κB p65 and MAPK c-Jun pathway activation coincided in glomeruli at the early stage of the disease. In the other microvascular beds, sepsis led to NF-κB p65 and MAPK c-Jun pathway activation with limited expression of E-selectin and no association with VCAM-1 expression or leukocyte recruitment.

Identification of HDAC inhibitors as neutrophil recruitment modulators in zebrafish using a chemical library screen.

Tissue injury-induced neutrophil recruitment is a prerequisite for the initiation and amplification of inflammatory responses. Although multiple proteases and post-translational modification (PTM) enzymes regulate leukocyte recruitment, an unbiased functional screen of those enzymes regulating inflammatory leukocyte recruitment has yet to be undertaken. Here, using a zebrafish tail fin amputation (TFA) model to screen a chemical library consisting of 295 compounds that target proteases and PTM enzymes, we identified multiple histone deacetylase (HDAC) inhibitors that modulate inflammatory neutrophil recruitment. AR-42, a pan-HDAC inhibitor, was shown to inhibit neutrophil recruitment in three different zebrafish sterile tissue injury models: TFA, copper-induced neuromasts damage (CIND), and mechanical otic vesicle injury (MOVI), and a sterile murine peritonitis model. RNA sequencing analysis of AR-42-treated fish embryos revealed downregulation of neutrophil-associated cytokines/chemokines, and exogenous supplementation with recombinant human IL-1β and CXCL8 partially restored the defective neutrophil recruitment in AR-42-treated MOVI model fish embryos. We thus demonstrate that AR-42 non-cell autonomously modulates neutrophil recruitment by suppressing transcriptional expression of cytokines/chemokines, thereby identifying AR-42 as a promising anti-inflammatory drug for treating sterile tissue injury-associated diseases.

ROR2 regulates leukocyte recruitment and atherosclerotic plaque formation in disturbed flow vascular regions

Endothelial cells (ECs) sense and transduce mechanical forces created by blood flow into intracellular signals, and are highly sensitive to shear stress (SS). In regions of curvature or bifurcation, blood flow exerts low or disturbed SS promoting vascular inflammation and atherosclerosis whereas physiologically high SS is protective. Recent studies showed that ROR2, a tyrosine kinase receptor, by regulating cholesterol homeostasis and inflammatory responses in vascular smooth muscle cells may be involved in the pathophysiology of atherosclerosis. However, its role in ECs has been poorly studied. Here, we wondered if ROR2 in ECs, could control the inflammatory response and/or atherosclerotic plaque formation. The impact of ROR2 deletion on gene expression was investigated in vitro on HUVEc submitted to distrubed flow (DF). Leukocytes recrutment and atherosclerotic plaque were quantified on mice aorta from ROR2 mutant mice and in ApoE-/- mice deleted for ROR2 in EC. First, we showed in vivo, in the mouse aorta that ROR2 was highly expressed in ECs of DF areas. By in vitro studies, using an orbital flow model, we showed that ROR2 loss of function decreased the expression of pro-inflammatory cytokines and adhesion molecules in ECs exposed to DF, suggesting a pro-inflammatory effect of ROR2. In vivo, deletion of Ror2 in ECs, decreased leukocyte recruitment in the aortic arch of mice under physiological conditions. Finally, deletion of Ror2 in ApoE-/- mice showed a decrease of leukocytes in the aortic arch associated with a decreased of intimal thickening in the aortic sinus. This study shows that ROR2 is a new actor in endothelial cell responses to disturbed flow under both physiological and pathological conditions by promoting leukocyte recruitment probably via its effect on the expression of pro-inflammatory cytokines and/or adhesion molecules.

CD47 Binding on Vascular Endothelial Cells Inhibits IL-17-Mediated Leukocyte Adhesion.

To address the conflicting role of thrombospondin (TSP)-1 reported in acute and chronic pathologies, this study investigated the role of TSP-1 in regulating leukocyte recruitment and regulation of VCAM-1 expression using mouse models of uveitis. The spontaneously increased VCAM-1 expression and leukocyte adhesion in retinas of TSP-1-deficient mice suggested a TSP-1-mediated regulation of VCAM-1 expression. In a chronic uveitis model, induced by immunizing wild-type mice with specific interphotoreceptor retinoid-binding protein (IRBP) peptide, topically applied TSP-1-derived CD47-binding peptide significantly reduced the clinical disease course and retinal leukocyte adhesion as compared to the control peptide-treated group. In contrast, in LPS-mediated acute uveitis, TSP-1 deficiency significantly reduced the retinal leukocyte adhesion. The results of our in vitro study, using vascular endothelial cell (EC) cultures, demonstrate that unlike TNF-α, VCAM-1 expression induced by IL-17 is associated with a reduced expression of endogenous TSP-1. Such reduced endogenous TSP-1 expression in IL-17-stimulated ECs helps limit the CD36-mediated increased VCAM-1 expression, while favoring CD47-mediated inhibition of VCAM-1 expression and leukocyte adhesion. Thus, our study identifies TSP-1:CD47 interaction as a molecular pathway that modulates IL-17-mediated VCAM-1 expression, contributing to its anti-inflammatory effect in chronic inflammatory conditions.

Role of CXCL5 in Regulating Chemotaxis of Innate and Adaptive Leukocytes in Infected Lungs Upon Pulmonary Influenza Infection.

Respirovirus such as influenza virus infection induces pulmonary anti-viral immune response, orchestration of innate and adaptive immunity restrain viral infection, otherwise causes severe diseases such as pneumonia. Chemokines regulate leukocyte recruitment to the inflammation site. One chemokine CXCL5, plays a scavenging role to regulate pulmonary host defense against bacterial infection, but its role in pulmonary influenza virus infection is underdetermined. Here, using an influenza (H1N1) infected CXCL5-/- mouse model, we found that CXCL5 not only responds to neutrophil infiltration into infected lungs at the innate immunity stage, but also affects B lymphocyte accumulation in the lungs by regulating the expression of the B cell chemokine CXCL13. Inhibition of CXCL5-CXCR2 axis markedly induces CXCL13 expression in CD64+CD44hiCD274hi macrophages/monocytes in infected lungs, and in vitro administration of CXCL5 to CD64+ alveolar macrophages suppresses CXCL13 expression via the CXCL5-CXCR2 axis upon influenza challenge. CXCL5 deficiency leads to increased B lymphocyte accumulation in infected lungs, contributing to an enhanced B cell immune response and facilitating induced bronchus-associated lymphoid tissue formation in the infected lungs during the late infection and recovery stages. These data highlight multiple regulatory roles of CXCL5 in leukocyte chemotaxis during pulmonary influenza infection.

Macrophage-mediated RON signaling supports breast cancer growth and progression through modulation of IL-35.

Tumor associated macrophages (TAMs) play a major role in regulating mammary tumor growth and in directing the responses of tumor infiltrating leukocytes in the microenvironment. However, macrophage-specific mechanisms regulating the interactions of macrophages with tumor cells and other leukocytes that support tumor progression have not been extensively studied. In this study, we show that the activation of the RON receptor tyrosine kinase signaling pathway specifically in macrophages supports breast cancer growth and metastasis. Using clinically relevant murine models of breast cancer, we demonstrate that loss of macrophage RON expression results in decreases in mammary tumor cell proliferation, survival, cancer stem cell self-renewal, and metastasis. Macrophage RON signaling modulates these phenotypes via direct effects on the tumor proper and indirectly by regulating leukocyte recruitment including macrophages, T-cells, and B-cells in the mammary tumor microenvironment. We further show that macrophage RON expression regulates the macrophage secretome including IL-35 and other immunosuppressive factors. Overall, our studies implicate activation of RON signaling in macrophages as a key player in supporting a thriving mammary pro-tumor microenvironment through novel mechanisms including the augmentation of tumor cell properties through IL-35.

Immunological Role and Prognostic Value of APBB1IP in Pan-Cancer Analysis.

Objective: APBB1IP is a Rap1-binding protein that mainly acts as a regulator of leukocyte recruitment and pathogen clearance through complement-mediated phagocytosis. However, the role of APBB1IP in tumor immunity remains unclear. This study was carried out to evaluate the prognostic landscape of APBB1IP in pan-cancer analysis and investigate the relationship between APBB1IP expression and immune infiltration.Methods: We explored the expression pattern and prognostic value of APBB1IP in pan-cancer analysis through Kaplan-Meier Plotter and multiple databases, including TCGA, Oncomine. We then assessed the correlation between APBB1IP expression and immune cell infiltration using the TIMER database. Furthermore, we identified the proteins that interact with APBB1IP and performed epigenetic and transcriptional analyses. Multivariate Cox regression analyses were applied to construct a prognostic model, which consisted of APBB1IP and its interacting proteins, based on the lung cancer cohorts from the Gene Expression Omnibus (GEO) database.Results: The expression of APBB1IP was correlated with the prognosis of several types of cancer. APBB1IP upregulation was found to be associated with increased immune cell infiltration, especially for CD8+ T cells, natural killer (NK) cells, and immune regulators. A link was found between APBB1IP and immune-related proteins including RAP1A/B, TLN1/2 and VCL in the interaction network.Conclusion: APBB1IP can serve as a prognostic biomarker in pan-cancer analysis. APBB1IP upregulation was correlated with increased immune-cell infiltration, and the expression APBB1IP in different tumors might be related to the tumor immune microenvironment.

Characterisation of endogenous Galectin-1 and -9 expression in monocyte and macrophage subsets under resting and inflammatory conditions

Macrophages are key cells in both acute and chronic inflammatory settings. Their activation and function highly depends on the cytokines, chemokines and adhesion molecules that direct monocytes to infiltrate tissues, differentiate into macrophages, and finally lead to the clearance of such inflammatory signals. Galectins, β‐galactoside‐binding lectins, are differentially expressed by various immune cells, and some members of this family have been identified as regulators of leukocyte recruitment and activation. Galectin-1 (Gal-1) and galectin-9 (Gal-9) expression has been described in immune cells, but the specific molecular mechanisms by which they modulate the inflammatory response in macrophages/monocytes are not completely understood. In this study we sought to comprehensively characterise the expression profile of endogenous Gal-1 and Gal-9 in different murine and human monocyte/macrophage populations in response to different inflammatory stimuli. All subsets of murine and human macrophages expressed significant levels of Gal-1 and -9. Interestingly, murine bone marrow derived macrophages stimulated with M2 (pro-resolution) polarising agents preferentially upregulated Gal-1, while Gal-9 expression was upregulated by M1/pro-inflammatory stimulation. However, we observed differing results in human monocyte derived macrophages. Collectively, our findings report a differential expression pattern of endogenous Gal-1 and -9 in macrophage and monocyte subsets in response to a range of inflammatory stimuli. Future studies will endeavour to elucidate whether the galectins make attractive therapeutic targets or agents for regulating the inflammatory response.

Lack of CD34 produces defects in platelets, microparticles, and lung inflammation.

Lung innate immune activation results in acute lung inflammation, which is characterized by alveolar barrier disruption and accumulation of cellular lung aggregates comprising neutrophils, platelets, mononuclear cells, and microparticles. CD34 is a sialomucin, with pan-selectin affinity and recently shown to protect the endothelial barrier in a bleomycin-induced lung injury model. However, there is very little information about the fundamental role of CD34 in regulation of the lung innate immune response. We hypothesized that CD34 regulates leukocyte recruitment by promoting optimal platelet activation (aggregation and spread) during bacterial lipopolysaccharide (LPS)-induced acute lung injury. Therefore, we utilized CD34 knock-out (KO) and wild-type (WT) mice to analyze and compare the morphology and expression of leukocyte subsets from the pulmonary and systemic compartments. We utilized the chemotactic N-formylated tri-peptide, fMLP, to understand platelet aggregation in vitro, and the fundamental immune stimulant, LPS, to induce lung injury and understand platelet activation ex vivo. Our data reveal that under steady-state conditions, KO mice possess large aggregates of integrin β3 (CD61)-positive microparticles in peripheral blood. Moreover, the KO mice recruit a large number of neutrophils to lungs, which are not cleared even at 36-h post-LPS exposure. The KO mice display an increased platelet CD61 expression, which aggregates, but does not spread normally in response to in vitro fMLP treatment. The KO platelets display similar deficits in their spreading ability even after ex vivo LPS exposure. Thus, our data demonstrate that CD34 modulates platelet biology, microparticle aggregation, and neutrophil recruitment during murine lung inflammation.

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of β2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

ICAM-1: A master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis.

ICAM-1 is a cell surface glycoprotein and an adhesion receptor that is best known for regulating leukocyte recruitment from circulation to sites of inflammation. However, in addition to vascular endothelial cells, ICAM-1 expression is also robustly induced on epithelial and immune cells in response to inflammatory stimulation. Importantly, ICAM-1 serves as a biosensor to transduce outside-in-signaling via association of its cytoplasmic domain with the actin cytoskeleton following ligand engagement of the extracellular domain. Thus, ICAM-1 has emerged as a master regulator of many essential cellular functions both at the onset and at the resolution of pathologic conditions. Because the role of ICAM-1 in driving inflammatory responses is well recognized, this review will mainly focus on newly emerging roles of ICAM-1 in epithelial injury-resolution responses, as well as immune cell effector function in inflammation and tumorigenesis. ICAM-1 has been of clinical and therapeutic interest for some time now; however, several attempts at inhibiting its function to improve injury resolution have failed. Perhaps, better understanding of its beneficial roles in resolution of inflammation or its emerging function in tumorigenesis will spark new interest in revisiting the clinical value of ICAM-1 as a potential therapeutic target.

The Role of Protein SUMOylation in the Pathogenesis of Atherosclerosis.

Atherosclerosis is a progressive, inflammatory cardiovascular disorder characterized by the development of lipid-filled plaques within arteries. Endothelial cell dysfunction in the walls of blood vessels results in an increase in vascular permeability, alteration of the components of the extracellular matrix, and retention of LDL in the sub-endothelial space, thereby accelerating plaque formation. Epigenetic modification by SUMOylation can influence the surface interactions of target proteins and affect cellular functionality, thereby regulating multiple cellular processes. Small ubiquitin-like modifier (SUMO) can modulate NFκB and other proteins such as p53, KLF, and ERK5, which have critical roles in atherogenesis. Furthermore, SUMO regulates leukocyte recruitment and cytokine release and the expression of adherence molecules. In this review, we discuss the regulation by SUMO and SUMOylation modifications of proteins and pathways involved in atherosclerosis.

Retinopathy severity correlates with RANTES concentrations and CCR 5-positive microvesicles in diabetes.

RANTES regulates leukocyte recruitment to areas affected by the inflammatory process. Microvesicles (MVs) belong to a subpopulation of extracellular vesicles and show proangiogenic potential by transferring bioactive molecules to target cells. the aim of this study was to determine the relationship between circulating proangiogenic factors (MVs and RANTES) and diabetes complications in patients with different severities of diabetic retinopathy (DR). CCR5 (CD195) receptors transported by annexin V-labeled MVs were also investigated. Diabetic patients (n = 61), among whom 35 had confirmed DR classified according to guidelines, and controls (n = 25) were included. MVs were isolated by centrifugation and analyzed using flow cytometry, RANTES was assessed by ELISA. the study group differed from the control group with respect to BMI, age, heart rate and systolic blood pressure. Additionally, glucose and creatinine concentrations were significantly increased: 5.30 [5.09-5.62] vs. 9.38 [7.48-11.55] (p <0.0001) mmol/l and 74.59 [64-84] vs. 89.00 [77.11-105.44] μmol/l (p = 0.0005), respectively. RANTES concentrations were significantly increased in diabetic patients compared to those of controls (15.5 (9.7-18.1) vs. 8.9 (0.9-14.6) μg/ml (p = 0.011)), and RANTES concentration significantly increased with respect to nonproliferative DR progression. Moreover, the number of CCR5-positive MVs was significantly increased in patients with heavy nonproliferative diabetic retinopathy (HNPDR) compared to those with so nonproliferative DR (SNPDR): 1178 [836-2254] vs. 394 [275-799] counts/μl. Correlation of RANTES concentrations with the stage of nonproliferative DR and the statistically significant dependence of CCR5-positive MVs with disease progression suggest that MVs and RANTES can be considered new biomarkers.

Cathepsin B‐mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels

Cathepsin B (CtsB) contributes to atherosclerosis and cancer progression by processing the extracellular matrix and promoting angiogenesis. Although CtsB was reported to promote and reduce angiogenesis, there is no mechanistic explanation that reconciles this apparent discrepancy. CtsB cleaves CD18 from the surface of immune cells, but its contribution to angiogenesis has not been studied. We developed an in vivo technique for visualization of immune cell transmigration from corneal vessels toward implanted cytokines. Wild-type (WT) leukocytes extravasated from limbal vessels, angiogenic stalks, and growing tip vessels and migrated toward the cytokines, indicating immune competence of angiogenic vessels. Compared to WT leukocytes, CtsB-/- leukocytes accumulated in a higher number in angiogenic vessels, but extravasated less toward the implanted cytokine. The accumulated CtsB-/- leukocytes in angiogenic vessels expressed more CD18. CD18-/- leukocytes extravasated later than WT leukocytes. However, once extravasated, CD18-/- leukocytes transmigrated more rapidly than their WT counterparts. These results suggest that, although CD18 facilitates efficient extravasation, outside of the vessel CD18 interaction with the extracellular matrix, it reduced transmigration velocity. Our results reveal an unexpected role for CtsB in leukocyte extravasation and transmigration, which advances our understanding of the complex contribution of CtsB to angiogenesis.-Nakao, S., Zandi, S., Sun, D., Hafezi-Moghadam, A. Cathepsin B-mediated CD18 shedding regulates leukocyte recruitment from angiogenic vessels.

Interleukin-18-induced cell adhesion molecule expression is associated with feedback regulation by PPAR-γ and NF-κB in Apo E-/- mice.

Focal recruitment of monocytes and lymphocytes is one of the earliest detectable cellular responses in atherosclerotic lesion formation. Endothelium may regulate leukocyte recruitment by expressing specific adhesion molecules. Interleukin-18 is a proinflammatory cytokine that plays an important role in vascular pathologies. The present study highlights the modulation of adhesion molecules and PPAR-γ by IL-18 and proposes a novel feedback mechanism by which PPAR-γ may regulate IL-18 expression. Three groups of normal chow diet-fed, male Apo E-/- mice, aged 12weeks (n = 6/group) were employed: Gp I, phosphate-buffered saline (PBS) (2 mo): Gp II, recombinant IL-18 (rIL-18) (1 mo) followed by PBS (1 mo); Gp III, rIL-18 (1 mo) followed by pyrrolidine dithiocarbamate (PDTC) (1 mo). Significantly augmented mRNA expression of ICAM-1 (~5.7-fold), VCAM-1 (~3.6-fold), and NF-κB (~7-fold) was observed in Gp II mice as compared to Gp I, whereas PPAR-γ expression was not altered. PDTC treatment caused a significant downregulation of ICAM-1 (~4.2-fold), VCAM-1(~2-fold), and NF-κB (~4.5-fold) and upregulation of PPAR-γ expression (~5-fold) in Gp III mice. A similar trend was observed in protein expression. In vivo imaging results demonstrated a marked increase in probe (CF750 dye conjugated to VCAM-1 antibody) fluorescence intensity for VCAM-1 expression in Gp II mice, whereas it was moderately decreased in Gp III. PPAR-γ was found to significantly downregulate both IL-18 levels and IL-18-induced adhesion molecules. The underlying mechanism was found to be via inhibition of NF-κB activity by PDTC, thereby leading to decreased adherence of monocytes to the activated endothelial cells and a step to halt the progression and development of atherosclerotic lesions.

β2-Adrenergic receptor-dependent chemokine receptor 2 expression regulates leukocyte recruitment to the heart following acute injury

Following cardiac injury, early immune cell responses are essential for initiating cardiac remodeling and tissue repair. We previously demonstrated the importance of β2-adrenergic receptors (β2ARs) in the regulation of immune cell localization following acute cardiac injury, with deficient leukocyte infiltration into the damaged heart. The purpose of this study was to investigate the mechanism by which immune cell-expressed β2ARs regulate leukocyte recruitment to the heart following acute cardiac injury. Chemokine receptor 2 (CCR2) expression and responsiveness to C-C motif chemokine ligand 2 (CCL2)-mediated migration were abolished in β2AR knockout (KO) bone marrow (BM), both of which were rescued by β2AR reexpression. Chimeric mice lacking immune cell-specific CCR2 expression, as well as wild-type mice administered a CCR2 antagonist, recapitulated the loss of monocyte/macrophage and neutrophil recruitment to the heart following myocardial infarction (MI) observed in mice with immune cell-specific β2AR deletion. Converse to β2AR ablation, β2AR stimulation increased CCR2 expression and migratory responsiveness to CCL2 in BM. Mechanistically, G protein-dependent β2AR signaling was dispensable for these effects, whereas β-arrestin2-biased β2AR signaling was required for the regulation of CCR2 expression. Additionally, activator protein 1 (AP-1) was shown to be essential in mediating CCR2 expression in response to β2AR stimulation in both murine BM and human monocytes. Finally, reconstitution of β2ARKO BM with rescued expression of a β-arrestin-biased β2AR in vivo restored BM CCR2 expression as well as cardiac leukocyte infiltration following MI. These results demonstrate the critical role of β-arrestin2/AP-1-dependent β2AR signaling in the regulation of CCR2 expression and recruitment of leukocytes to the heart following injury.

Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency.

Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. We found that chemoattractant-induced activation of β1 and β2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of β1 and β2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type-selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis.

Recruitment of CD16+ monocytes to endothelial cells in response to LPS-treatment and concomitant TNF release is regulated by CX3CR1 and interfered by soluble fractalkine

Fractalkine (FKN, CX3CL1) is a regulator of leukocyte recruitment and adhesion, and controls leukocyte migration on endothelial cells (ECs). We show that FKN triggers different effects in CD16+ and CD16− monocytes, the two major subsets of human monocytes. In the presence of ECs a lipopolysaccharide (LPS)-stimulus led to a significant increase in tumor necrosis factor (TNF)-secretion by CD16+ monocytes, which depends on the interaction of CX3CR1 expressed on CD16+ monocytes with endothelial FKN. Soluble FKN that was efficiently shed from the surface of LPS-activated ECs in response to binding of CD16+ monocytes to ECs, diminished monocyte adhesion in down-regulating CX3CR1 expression on the surface of CD16+ monocytes resulting in decreased TNF-secretion. In this process the TNF-converting enzyme (TACE) acts as a central player regulating FKN-shedding and TNFα-release through CD16+ monocytes interacting with ECs. Thus, the release and local accumulation of sFKN represents a mechanism that limits the inflammatory potential of CD16+ monocytes by impairing their interaction with ECs during the initial phase of an immune response to LPS. This regulatory process represents a potential target for therapeutic approaches to modulate the inflammatory response to bacterial components.

Regulation of allergic lung inflammation by endothelial cell transglutaminase 2.

Tissue transglutaminase 2 (TG2) is an enzyme with multiple functions, including catalysis of serotonin conjugation to proteins (serotonylation). Previous research indicates that TG2 expression is upregulated in human asthma and in the lung endothelium of ovalbumin (OVA)-challenged mice. It is not known whether endothelial cell TG2 is required for allergic inflammation. Therefore, to determine whether endothelial cell TG2 regulates allergic inflammation, mice with an endothelial cell-specific deletion of TG2 were generated, and these mice were sensitized and challenged in the airways with OVA. Deletion of TG2 in endothelial cells blocked OVA-induced serotonylation in lung endothelial cells, but not lung epithelial cells. Interestingly, deletion of endothelial TG2 reduced allergen-induced increases in respiratory system resistance, number of eosinophils in the bronchoalveolar lavage, and number of eosinophils in the lung tissue. Endothelial cell deletion of TG2 did not alter expression of adhesion molecules, cytokines, or chemokines that regulate leukocyte recruitment, consistent with other studies, demonstrating that deletion of endothelial cell signals does not alter lung cytokines and chemokines during allergic inflammation. Taken together, the data indicate that endothelial cell TG2 is required for allergic inflammation by regulating the recruitment of eosinophils into OVA-challenged lungs. In summary, TG2 functions as a critical signal for allergic lung responses. These data identify potential novel targets for intervention in allergy/asthma.