Transepithelial Migration Research Articles

Regulation of oyster (Crassostrea virginica) hemocyte motility by the intracellular parasite Perkinsus marinus: A possible mechanism for host infection

Hemocytes associated with the mucus lining of pallial (mantle, gill) surfaces of the oyster Crassostrea virginica have been recently suggested to facilitate infection by the Alveolate parasite Perkinsus marinus by mediating the uptake and dispersion of parasite cells. These “pallial hemocytes”, which are directly exposed to microbes present in surrounding seawater, are able to migrate bi-directionally between mucosal surfaces and the circulatory system, potentially playing a sentinel role. Interestingly, P. marinus was shown to increase trans-epithelial migration of hemocytes suggesting it may regulate cell motility to favor infection establishment. The purpose of this study was to investigate the effect of P. marinus on hemocyte motility and identify specific molecular mechanisms potentially used by the parasite to regulate hemocyte migration. In a first series of experiments, various components of P. marinus (live P. marinus cells, extracellular products, fragments of P. marinus cell membrane, membrane-modified live P. marinus cells, heat-killed P. marinus) along with components of the opportunistic bacterial pathogen Vibrio alginolyticus (bacterial cells and extracellular products) were investigated for their effects on hemocyte motility. In a second series of experiments, inhibitors of specific molecular pathways involved in motility regulation (Y-27632: inhibitor of Rho-associated protein kinase, RGDS: integrin inhibitor, CK-666: Arp2/3 inhibitor) were used in conjunction with qPCR gene expression experiments to identify pathways regulated by P. marinus exposure. Results showed a specific increase in hemocyte motility following exposure to live P. marinus cells. The increase in motility induced by P. marinus was suppressed by RGDS and CK-666 implicating the involvement of integrins and Arp2/3 in cell activation. Gene expression data suggest that Arp2/3 is possibly regulated directly by an effector produced by P. marinus. The implications of increased hemocyte motility prompted by P. marinus during the early stage of the infection process are discussed.

Transepithelial migration of mucosal hemocytes in Crassostrea virginica and potential role in Perkinsus marinus pathogenesis.

We have recently described the presence of hemocytes associated with mucus covering the pallial organs (mantle, gills, and body wall) 3 of the eastern oyster Crassostrea virginica. These hemocytes, hereby designated "pallial hemocytes" share common general characteristics with circulating hemocytes but also display significant differences particularly in their cell surface epitopes. The specific location of pallial hemocytes as peripheral cells exposed directly to the marine environment confers them a putative sentinel role. The purpose of this study was to gain a better understanding of the source of these pallial hemocytes by evaluating possible exchanges between circulatory and pallial hemocyte populations and whether these exchanges are regulated by pathogen exposure. Bi-directional transepithelial migrations of hemocytes between pallial surfaces and the circulatory system were monitored using standard cell tracking approaches after staining with the vital fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in conjunction with fluorescent microscopy and flow cytometry. Results showed bi-directional migration of hemocytes between both compartments and suggest that hemocyte migration from the pallial mucus layer to the circulatory system may occur at a greater rate compared to migration from the circulatory system to the pallial mucus layer, further supporting the role of pallial hemocytes as sentinel cells. Subsequently, the effect of the obligate parasite Perkinsus marinus and the opportunistic pathogen Vibrio alginolyticus on transepithelial migration of oyster hemocytes was investigated. Results showed an increase in hemocyte migration in response to P. marinus exposure. Furthermore, P. marinus cells were acquired by pallial hemocytes before being visible in underlying tissues and the circulatory system suggesting that this parasite could use pallial hemocytes as a vehicle facilitating its access to oyster tissues. These results are discussed in light of new evidence highlighting the role of oyster pallial organs as a portal for the initiation of P. marinus infections in oysters.

P017 CD47 REGULATES CD11B-DEPENDENT NEUTROPHIL TRANSEPITHELIAL MIGRATION DURING INTESTINAL INFLAMMATION

Neutrophil (PMN) migration across the intestinal mucosa correlates with disease flares in individuals with IBD. Despite this correlation, molecular events regulating PMN transepithelial migration (TEpM) are still elusive. However, it is clear that the leukocyte integrin CD11b/CD18 and the ubiquitously expressed membrane glycoprotein CD47 play crucial roles in this process. We hypothesized that CD47 interacts with CD11b/CD18 to regulate PMN trafficking in the gut. In vivo ileal loop assays showed reduced PMN migration into the lumen of CD47-/- mice in response to LTB4 (>60% reduction vs WT; p<0.05). This reduction is not due to the loss of CD47 on intestinal epithelial cells, as there were no differences in PMN migration between VillinCreCD47fl/fl mice and control CD47fl/fl mice. In vitro assays demonstrated a 25% reduction in LTB4 driven TEpM with CD47-/- PMN. In addition, blocking CD11b/CD18 antibody (mAb) reduced WT PMN migration to levels similar to those observed with CD47-/- PMN. However, CD11b/CD18 mAb did not reduce migration of CD47-/- PMN further, suggesting that CD47 plays a role in regulating integrin-dependent chemotaxis. In co-immunoprecipitation assays, CD47 was observed to associate with CD11b in PMN. Furthermore, we observed that upregulation of CD11b upon stimulation was reduced in CD47-/- PMN compared to WT, despite expression of similar levels at baseline. Consistent with these results, we observed reduced upregulation of CD11b upon chemoattractant stimulation in CD47 deficient human neutrophil like HL60 cells. Lastly, using antibodies specific for high affinity conformation of CD11b/CD18, we observed reduced CD11b/CD18 activation in CD47 deficient HL60 cells. These data support a role for CD47 in regulating PMN TEpM through effects on CD11b/CD18 functions. Targeting CD47 may provide a new therapeutic approach aimed at reducing pathologic intestinal inflammation in IBD.

Transmigration of Leukocytes Across Epithelial Monolayers.

Migration of leukocytes through epithelial monolayers represents an essential step in the generation of an inflammatory response and is often seen in inflammatory conditions such as Crohn's disease (Matthews et al., Toxicol Pathol 42:91-98, 2014) and asthma (Lambrecht and Hammad, Nat Med 18:684-692, 2012). Transepithelial migration involves adhesion to the basal surface of the epithelium before migration through the epithelial cell layer to the apical surface. Analyzing this process can present a technical challenge due to complications of using a coculture model and trying to recapitulate an intact monolayer. Here we describe two methods of assessing transepithelial migration based on a Transwell assay, the first of which measures the apical-basal migration of epithelial cells and the second "Inverted" transwell assay that measures basal-apical transmigration of leukocytes and therefore more closely mimics the in vivo process.

Cytosolic Phospholipase A2α Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by Streptococcus pneumoniae is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A3 (HXA3) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with S. pneumoniae As phospholipase A2 (PLA2) promotes the release of AA, we investigated the role of PLA2 in local and systemic disease during S. pneumoniae infection. The group IVA cytosolic isoform of PLA2 (cPLA2α) was activated upon S. pneumoniae infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids. Pharmacological inhibition of this enzyme blocked S. pneumoniae-induced PMN transepithelial migration in vitro Genetic ablation of the cPLA2 isoform cPLA2α dramatically reduced lung inflammation in mice upon high-dose pulmonary challenge with S. pneumoniae The cPLA2α-deficient mice also suffered no bacteremia and survived a pulmonary challenge that was lethal to wild-type mice. Our data suggest that cPLA2α plays a crucial role in eliciting pulmonary inflammation during pneumococcal infection and is required for lethal systemic infection following S. pneumoniae lung challenge.

Neutrophil-Derived Cytosolic PLA2α Contributes to Bacterial-Induced Neutrophil Transepithelial Migration.

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of Pseudomonas aeruginosa-induced neutrophil transepithelial migration has not been explored. Human and mouse neutrophil-epithelial cocultures were used to evaluate the role of neutrophil-derived cPLA2α in infection-induced transepithelial signaling by pharmacological and genetic approaches. Primary human airway basal stem cell-derived epithelial cultures and micro-optical coherence tomography, a new imaging modality that captures two- and three-dimensional real-time dynamics of neutrophil transepithelial migration, were applied. Evidence from these studies suggests that cPLA2α expressed by neutrophils, but not epithelial cells, plays a significant role in infection-induced neutrophil transepithelial migration by mediating LTB4 synthesis during migration, which serves to amplify the magnitude of neutrophil recruitment in response to epithelial infection.

Role of G protein-coupled receptor kinase-6 in Escherichia coli lung infection model in mice.

G protein-coupled receptor kinase-6 (GRK6) is a serine/threonine kinase that is important in inflammatory processes. In this study, we examined the role of GRK6 in Escherichia coli-induced lung infection and inflammation using GRK6 knockout (KO) and wild-type (WT) mice. Intratracheal instillation of E. coli significantly enhanced bacterial load in the bronchoalveolar lavage (BAL) of KO compared with WT mice. Reduced bacterial clearance in the KO mice was not due to an intrinsic defect in neutrophil phagocytosis or killing but as a result of reduced neutrophil numbers in the KO BAL. Interestingly, neutrophil numbers in the lung were increased in the KO compared with WT mice, suggesting a potential dysfunction in transepithelial migration of neutrophils from the lungs to the bronchoalveolar space. This effect was selective for lung tissue because peritoneal neutrophil numbers were similar between the two genotypes following peritoneal infection. Although neutrophil expression of CXCR2/CXCR3 was similar between WT and KO, IL-17A expression was higher in the KO compared with WT mice. These results suggest that enhanced neutrophil count in the KO lungs but reduced numbers in BAL are likely due to transepithelial migration defect and/or altered chemokines/cytokines. Together, our studies suggest a previously unrecognized and novel role for GRK6 in neutrophil migration specific to pulmonary tissue during bacterial infection.

Potential roles of neutrophils in regulating intestinal mucosal inflammation of inflammatory bowel disease.

Inflammatory bowel diseases (IBD), comprising of ulcerative colitis and Crohn's disease, are inflammatory disorders of the gastrointestinal tract characterized by chronically relapsing mucosal inflammation. Neutrophils, as the effector cells of acute inflammation, have long been reported to play a role in the maintenance of intestinal homeostasis and pathogenesis of IBD. At the early stage of mucosal inflammation in patients with IBD, neutrophils flood into intestinal mucosa, phagocytose pathogenic microbes, and promote mucosal healing and resolution of inflammation. However, large numbers of neutrophils infiltrating in the inflamed mucosa and accumulating in the epithelia cause damage of mucosal architecture, compromised epithelial barrier and production of inflammatory mediators. In this review we discuss the critical roles of neutrophils in modulating innate and adaptive immune responses in intestinal mucosa, and, importantly, clarify the potential roles of neutrophils related to their production of inflammatory mediators, transenthothelial and transepithelial migration into intestinal mucosa, and the underlying mechanisms in regulating mucosal inflammation of IBD. Moreover, we also describe a new subset of neutrophils (i.e., CD177+ neutrophils) and illustrate its protective role in modulating intestinal mucosal immune responses in IBD.

Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface.

Pseudomonas aeruginosa ExoU augments neutrophil transepithelial migration.

Excessive neutrophil infiltration of the lungs is a common contributor to immune-related pathology in many pulmonary disease states. In response to pathogenic infection, airway epithelial cells produce hepoxilin A3 (HXA3), initiating neutrophil transepithelial migration. Migrated neutrophils amplify this recruitment by producing a secondary gradient of leukotriene B4 (LTB4). We sought to determine whether this two-step eicosanoid chemoattractant mechanism could be exploited by the pathogen Pseudomonas aeruginosa. ExoU, a P. aeruginosa cytotoxin, exhibits phospholipase A2 (PLA2) activity in eukaryotic hosts, an enzyme critical for generation of certain eicosanoids. Using in vitro and in vivo models of neutrophil transepithelial migration, we evaluated the impact of ExoU expression on eicosanoid generation and function. We conclude that ExoU, by virtue of its PLA2 activity, augments and compensates for endogenous host neutrophil cPLA2α function, leading to enhanced transepithelial migration. This suggests that ExoU expression in P. aeruginosa can circumvent immune regulation at key signaling checkpoints in the neutrophil, resulting in exacerbated neutrophil recruitment.

VITAMIN E BOOSTS NEUTROPHIL ELASTASE ACTIVITY AND THEIR ABILITY TO KILL STREPTOCOCCUS PNEUMONIAE

Streptococcus pneumoniae (pneumococcus) remain a leading cause of life-threatening infections such as pneumonia, bacteremia and meningitis in the elderly. Neutrophils are innate immune cells that are key determinants of disease following infection as their presence is initially required to control bacterial numbers, but their persistence in the lungs can lead to tissue destruction and bacterial spread. We previously found that vitamin E (VE) supplementation reverses the age-associated increase in susceptibility to S. pneumoniae in mice by modulating pulmonary recruitment of neutrophils. The objective of this study was to test the effect of VE on the ability of neutrophils isolated from young (22–35 years) or elderly (65–69 years) volunteers to migrate across lung epithelial cell in response to S. pneumoniae and to kill complement-opsonized bacteria in vitro. We found no distinguishable differences in neutrophil migration from young and elderly donors and that VE diminished transepithelial migration across all ages. Surprisingly, unlike previous studies showing defective killing of antibody opsonized bacteria with aging, we found that when compared to young donors, neutrophils of older donors were better at killing complement opsonized pneumococci ex vivo. This increased antibacterial response in neutrophils from elderly individuals correlated with elevated activity of the antimicrobial serine protease neutrophil elastase. Exposure of neutrophils to VE elevated enzymatic activity of neutrophil elastase in young donors and increased pneumococcal killing by these cells. These findings demonstrate that VE is an effective modulator of innate immune cell function with potential to fight bacterial pneumonia.

The metalloproteinase ADAM8 promotes leukocyte recruitment in vitro and in acute lung inflammation.

Alveolar leukocyte recruitment is a hallmark of acute lung inflammation and involves transmigration of leukocytes through endothelial and epithelial layers. The disintegrin and metalloproteinase (ADAM) 8 is expressed on human isolated leukocytic cells and can be further upregulated on cultured endothelial and epithelial cells by proinflammatory cytokines. By shRNA-mediated knockdown we show that leukocytic ADAM8 is required on monocytic THP-1 cells for chemokine-induced chemotaxis as well as transendothelial and transepithelial migration. Furthermore, ADAM8 promotes αL-integrin upregulation and THP-1 cell adhesion to endothelial cells. On endothelial cells ADAM8 enhances transendothelial migration and increases cytokine-induced permeability. On epithelial cells the protease facilitates migration in a wound closure assay but does not affect transepithelial leukocyte migration. Blood leukocytes and bone marrow-derived macrophages (BMDM) from ADAM8-deficient mice show suppressed chemotactic response. Intranasal application of LPS to mice is accompanied with ADAM8 upregulation in the lung. In this model of acute lung inflammation ADAM8-deficient mice are protected against leukocyte infiltration. Finally, transfer experiments of BMDM in mice indicate that ADAM8 exerts a promigratory function predominantly on leukocytes. Our study provides in vitro and in vivo evidence that ADAM8 on leukocytes holds a proinflammatory function in acute lung inflammation by promoting alveolar leukocyte recruitment.

The Alpha-Tocopherol Form of Vitamin E Boosts Elastase Activity of Human PMNs and Their Ability to Kill Streptococcus pneumoniae.

Despite the availability of vaccines, Streptococcus pneumoniae remains a leading cause of life-threatening infections, such as pneumonia, bacteremia and meningitis. Polymorphonuclear leukocytes (PMNs) are a key determinant of disease course, because optimal host defense requires an initial robust pulmonary PMN response to control bacterial numbers followed by modulation of this response later in infection. The elderly, who manifest a general decline in immune function and higher basal levels of inflammation, are at increased risk of developing pneumococcal pneumonia. Using an aged mouse infection model, we previously showed that oral supplementation with the alpha-tocopherol form of vitamin E (α-Toc) decreases pulmonary inflammation, in part by modulating neutrophil migration across lung epithelium into alveolar spaces, and reverses the age-associated decline in resistance to pneumococcal pneumonia. The objective of this study was to test the effect of α-Toc on the ability of neutrophils isolated from young (22–35 years) or elderly (65–69 years) individuals to migrate across epithelial cell monolayers in response to S. pneumoniae and to kill complement-opsonized pneumococci. We found that basal levels of pneumococcal-induced transepithelial migration by PMNs from young or elderly donors were indistinguishable, suggesting that the age-associated exacerbation of pulmonary inflammation is not due to intrinsic properties of PMNs of elderly individuals but rather may reflect the inflammatory milieu of the aged lung. Consistent with its anti-inflammatory activity, α-Toc treatment diminished PMN migration regardless of donor age. Unexpectedly, unlike previous studies showing poor killing of antibody-opsonized bacteria, we found that PMNs of elderly donors were more efficient at killing complement-opsonized bacteria ex vivo than their younger counterparts. We also found that the heightened antimicrobial activity in PMNs from older donors correlated with increased activity of neutrophil elastase, a serine protease that is required to kill pneumococci. Notably, incubation with α-Toc increased PMN elastase activity from young donors and boosted their ability to kill complement-opsonized pneumococci. These findings demonstrate that α-Toc is a potent modulator of PMN responses and is a potential nutritional intervention to combat pneumococcal infection.

Inhibition of SDF-1 receptors CXCR4 and CXCR7 attenuates acute pulmonary inflammation via the adenosine A2B-receptor on blood cells

Acute pulmonary inflammation is characterized by migration of polymorphonuclear neutrophils into the different compartments of the lung. Recent studies showed evidence that the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 influence migration of immune cells and their activity was linked to adenosine concentrations. We investigated the particular role of CXCR4- and CXCR7-inhibition and the potential link to the adenosine A2B-receptor, which plays an important anti-inflammatory role in the lung. After LPS-inhalation for 45 minutes, administration of the CXCR4-inhibitor (AMD3100) decreased transendothelial and transepithelial migration, whereas CXCR7-antagonism influenced epithelial migration exclusively. In A2B−/− mice, no anti-inflammatory effects were detectible through either one of the agents. Using chimeric mice, we identified A2B on hematopoietic cells to be crucial for these anti-inflammatory effects of CXCR4/7-inhibition. Both inhibitors decreased TNFα, IL6, CXCL1 and CXCL2/3 levels in the bronchoalveolar lavage of wild type mice, while not influencing the chemokine release in A2B−/− mice. Inflammation augmented the expression of both receptors and their inhibition increased A2B-levels upon inflammation. In vitro assays with human epithelium/endothelium confirmed our in vivo findings. During inflammation, inhibition of CXCR4- and CXCR7-receptors prevented microvascular permeability in wild type but not in A2B−/− mice, highlighting the pivotal role of an active A2B-receptor in this setting. The combination of both inhibitors had a synergistic effect in preventing capillary leakage. In conclusion, we determined the pivotal role of CXCR4- and CXCR7-inhibition in acute pulmonary inflammation, which depended on A2B-receptor signalling.

Neuralized regulates Crumbs endocytosis and epithelium morphogenesis via specific Stardust isoforms.

Crumbs (Crb) is a conserved determinant of apical membrane identity that regulates epithelial morphogenesis in many developmental contexts. In this study, we identify the Crb complex protein Stardust (Sdt) as a target of the E3 ubiquitin ligase Neuralized (Neur) in Drosophila melanogaster Neur interacts with and down-regulates specific Sdt isoforms containing a Neur binding motif (NBM). Using a CRISPR (clustered regularly interspaced short palindromic repeats)-induced deletion of the NBM-encoding exon, we found that Sdt is a key Neur target and that Neur acts via Sdt to down-regulate Crb. We further show that Neur promotes the endocytosis of Crb via the NBM-containing isoforms of Sdt. Although the regulation of Crb by Neur is not strictly essential, it contributes to epithelium remodeling in the posterior midgut and thereby facilitates the trans-epithelial migration of the primordial germ cells in early embryos. Thus, our study uncovers a novel regulatory mechanism for the developmental control of Crb-mediated morphogenesis.

Illuminating dynamic neutrophil trans-epithelial migration with micro-optical coherence tomography

A model of neutrophil migration across epithelia is desirable to interrogate the underlying mechanisms of neutrophilic breach of mucosal barriers. A co-culture system consisting of a polarized mucosal epithelium and human neutrophils can provide a versatile model of trans-epithelial migration in vitro, but observations are typically limited to quantification of migrated neutrophils by myeloperoxidase correlation, a destructive assay that precludes direct longitudinal study. Our laboratory has recently developed a new isotropic 1-μm resolution optical imaging technique termed micro-optical coherence tomography (μOCT) that enables 4D (x,y,z,t) visualization of neutrophils in the co-culture environment. By applying μOCT to the trans-epithelial migration model, we can robustly monitor the spatial distribution as well as the quantity of neutrophils chemotactically crossing the epithelial boundary over time. Here, we demonstrate the imaging and quantitative migration results of our system as applied to neutrophils migrating across intestinal epithelia in response to a chemoattractant. We also demonstrate that perturbation of a key molecular event known to be critical for effective neutrophil trans-epithelial migration (CD18 engagement) substantially impacts this process both qualitatively and quantitatively.

CD47 regulates CD11b‐dependent neutrophil transepithelial migration during intestinal inflammation

Inflammatory bowel disease (IBD), including Crohn's disease and Ulcerative colitis, is a chronic, relapsing disorder of the gastrointestinal tract characterized by neutrophil (PMN) associated bystander tissue damage, crypt abscess formation and mucosal ulceration. It is well appreciated that neutrophil transepithelial migration (TEpM) in the intestine correlates with disease flares in individuals with IBD. While the molecular events regulating PMN TEpM are far from understood, it is now clear that the integrin CD11b/CD18 and CD47 play crucial roles in this process. CD47 is a highly glycosylated protein that associates in cis with β1, β2 and β3 integrins in different cell types while also serving as a receptor for Signal Regulatory Proteins (SIRP‐α and ‐γ) and thrombospondin (TSP). CD47 is involved in regulation of innate and adaptive immune responses. For example, it has been reported that PMN recruitment in CD47−/− mice is reduced in models of peritonitis, lung injury and dermal air pouch inflammation, but the exact mechanism(s) behind CD47 regulation of PMN infiltration into inflamed mucosal tissues is unclear. We have previously reported that CD47 expressed in T‐cells regulates transendothelial migration through its physical and functional association with the β2 integrin CD11a/CD18. In the gut, it has been shown that PMN transmigration across intestinal epithelium is dependent on CD11b/CD18 but not CD11a/CD18. Thus, we hypothesized that CD47 interacts with CD11b/CD18 to regulate PMN chemotaxis and TEpM in the intestine. Here we report that CD47 co‐associates with CD11b/CD18 in PMN as determined by co‐immunoprecipitation and proximal ligation assay (PLA). Flow cytometry analyses of murine PMN deficient in CD47 showed that, despite expressing similar levels of CD11b/CD18 at baseline, there was defective upregulation of CD11b/CD18 upon stimulation with the chemoattractants fMLF or LTB4 compared with WT PMN. Additionally, loss of CD47 in murine PMN resulted in a 25% reduction in LTB4 driven chemotaxis. Furthermore, antibody mediated inhibition of CD11b/CD18 reduced WT PMN migration to similar levels as CD47−/− PMN. In contrast, blocking CD11b on CD47−/− PMN did not further reduce migration, suggesting a functional regulation of integrin‐dependent chemotaxis by CD47. In vivo studies modeling PMN TEpM into the intestinal lumen in response to LTB4 revealed decreased migration in CD47−/− mice (&gt;60% reduction vs. WT; p&lt;0.05). Taken together, our data suggest that CD47 plays an important role in PMN intestinal recruitment through regulation of CD11b/CD18 adhesive function. We suggest that CD47 represents a promising new candidate for therapeutic approaches aimed at dampening pro‐inflammatory signals, reducing PMN influx and improving mucosal wound healing in IBD.Support or Funding InformationSupported by NIH 5R01DK079392‐12 and R01HL125780

The Pivotal Role of CXCR7 in Stabilization of the Pulmonary Epithelial Barrier in Acute Pulmonary Inflammation.

Acute pulmonary inflammation is still a frightening complication in intensive care units and has a high mortality. Specific treatment is not available, and many details of the pathomechanism remain unclear. The recently discovered chemokine receptor CXCR7 and its ligand stromal cell-derived factor (SDF)-1 are known to be involved in inflammation. We chose to investigate the detailed role of CXCR7 in a murine model of LPS inhalation. Inflammation increased pulmonary expression of CXCR7, and the receptor was predominantly expressed on pulmonary epithelium and on polymorphonuclear neutrophil (PMNs) after transepithelial migration into the alveolar space. Specific inhibition of CXCR7 reduced transepithelial PMN migration by affecting the expression of adhesion molecules. CXCR7 antagonism reduced the most potent PMN chemoattractants CXCL1 and CXCL2/3. After inhibiting CXCR7, NF-κB phosphorylation was reduced in lungs of mice, tight junction formation increased, and protein concentration in the bronchoalveolar lavage diminished, showing the impact of CXCR7 on stabilizing microvascular permeability. In vitro studies with human cells confirmed the pivotal role of CXCR7 in pulmonary epithelium. Immunofluorescence of human lungs confirmed our in vivo data and showed an increase of the expression of CXCR7 in pulmonary epithelium. Highlighting the clinical potential of CXCR7 antagonism, nebulization of the agent before and after the inflammation showed impressive anti-inflammatory effects. Additional CXCR7 inhibition potentiated the effect of SDF-1 antagonism, most probably by downregulating SDF-1 and the second receptor of the chemokine (CXCR4) expression. In conclusion, our data identified the pivotal role of the receptor CXCR7 in pulmonary inflammation with a predominant effect on the pulmonary epithelium and PMNs.

Upregulated expression of substance P (SP) and NK1R in eczema and SP-induced mast cell accumulation

Substance P (SP) was reported to be associated with eczema and acts as a potent skin mast cell secretagogue. However, little is known of its expression in inflammatory cells in eczema and its ability in induction of mast cell accumulation. In the present study, we investigated expression of SP and neurokinin-1 receptor (NK1R) on peripheral blood leukocytes and mast cells from patients with eczema and influence of SP on mast cell accumulation by using flow cytometry analysis, trans-epithelial cell migration assay and mouse peritoneal model. The results showed that plasma SP and IL-17A levels in eczema patients were higher than that in healthy control subject. The percentages of SP+ and NK1R+ expression populations of monocytes, helper T cells, natural killer T cells and basophils in peripheral blood of eczema patients were markedly elevated. It was observed that not only absolute number of mast cells but also SP+ and NK1R+ mast cells are enhanced in the lesion skin of eczema. SP showed a potent chemoattractant action on mast cells as assessed by a mouse peritoneal model and a trans-endothelium cell migration assay. SP-induced mast cell accumulation appears a CD18/CD11a complex, L-selectin and ICAM-1-dependent event which can be blocked by a NK-1R antagonist RP67580. In conclusion, elevated expression of SP in patients with eczema and the ability of SP in induction of mast cell accumulation indicate strongly that SP is a potent proinflammatory mediator, which contributes to the pathogenesis of eczema. Inhibitors of SP and blockers of NK1R are likely useful agents for treatment of eczema.

Pseudomonas aeruginosa sabotages the generation of host proresolving lipid mediators

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A4 (15-epi LXA4), plays a critical role in limiting neutrophil activation and tissue inflammation, thus promoting the return to tissue homeostasis. Here, we show that a secreted P. aeruginosa epoxide hydrolase, cystic fibrosis transmembrane conductance regulator inhibitory factor (Cif), can disrupt 15-epi LXA4 transcellular biosynthesis and function. In the airway, 15-epi LXA4 production is stimulated by the epithelial-derived eicosanoid 14,15-epoxyeicosatrienoic acid (14,15-EET). Cif sabotages the production of 15-epi LXA4 by rapidly hydrolyzing 14,15-EET into its cognate diol, eliminating a proresolving signal that potently suppresses IL-8-driven neutrophil transepithelial migration in vitro. Retrospective analyses of samples from patients with CF supported the translational relevance of these preclinical findings. Elevated levels of Cif in bronchoalveolar lavage fluid were correlated with lower levels of 15-epi LXA4, increased IL-8 concentrations, and impaired lung function. Together, these findings provide structural, biochemical, and immunological evidence that the bacterial epoxide hydrolase Cif disrupts resolution pathways during bacterial lung infections. The data also suggest that Cif contributes to sustained pulmonary inflammation and associated loss of lung function in patients with CF.