Transepithelial Migration Research Articles

Eosinophils in the gastrointestinal tract and their role in the pathogenesis of major colorectal disorders

Eosinophils are currently regarded as versatile mobile cells controlling and regulating multiple biological pathways and responses in health and disease. These cells store in their specific granules numerous biologically active substances (cytotoxic cationic proteins, cytokines, growth factors, chemokines, enzymes) ready for rapid release. The human gut is the main destination of eosinophils that are produced and matured in the bone marrow and then transferred to target tissues through the circulation. In health the most important functions of gut-residing eosinophils comprise their participation in the maintenance of the protective mucosal barrier and interactions with other immune cells in providing immunity to microbiota of the gut lumen. Eosinophils are closely involved in the development of inflammatory bowel disease (IBD), when their cytotoxic granule proteins cause damage to host tissues. However, their roles in Crohn’s disease and ulcerative colitis appear to follow different immune response patterns. Eosinophils in IBD are especially important in altering the structure and protective functions of the mucosal barrier and modulating massive neutrophil influx to the lamina propria followed by transepithelial migration to colorectal mucus. IBD-associated inflammatory process involving eosinophils then appears to expand to the mucus overlaying the internal gut surface. The author hypothesises that immune responses within colorectal mucus as well as ETosis exerted by both neutrophils and eosinophils on the both sides of the colonic epithelial barrier act as additional pathogenetic factors in IBD. Literature analysis also shows an association between elevated eosinophil levels and better colorectal cancer (CRC) prognosis, but mechanisms behind this effect remain to be elucidated. In conclusion, the author emphasises the importance of investigating colorectal mucus in IBD and CRC patients as a previously unexplored milieu of disease-related inflammatory responses.

Epithelial membrane protein 2 regulates transepithelial migration of neutrophils into the inflamed airspace

Abstract Neutrophil (PMN) migration from the circulation to the airway lumen is critical for pulmonary host defense but also causes bystander tissue injury that underlies a wide array of acute and chronic lung diseases. Whereas good evidence supports a role for intestinal epithelial cells in control of transmigration of extravasated PMNs into the intestinal lumen, little is known about whether and how respiratory epithelial cells regulate PMN influx into the airspace. Here, we identify epithelial membrane protein 2 (EMP2), a tetraspan membrane protein highly expressed in alveolar epithelial type 1 (AT1) cells but of unknown function in lung biology, as a master regulator of the terminal entry of PMNs into the airspace. EMP2−/− mice had reduced PMN influx into the airspace after multiple inhaled exposures and as a result exhibited attenuated lung injury and improved survival during bacterial pneumonia. Bone marrow chimeras, intravital PMN labelling, and in vitro assays suggest epithelial dysfunction in EMP2−/− lungs was responsible for deficient transepithelial PMN transit into airspace. We provide evidence that EMP2 supports epithelial surface display of multiple adhesion molecules that are required for cognate interactions with migrating PMNs and that it does so through suppression of epithelial caveolins. Moreover, EMP2 supports epithelial lipid raft mass in a manner dependent on putative cholesterol-binding motifs in its sequence. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular PMN traffic into the alveolus. Our findings identify EMP2 as a potential target for treatment of neutrophilic lung diseases.

Novel Junctional Adhesion Molecule‐Like (JAML) Regulates Neutrophil Transepithelial Migration During Intestinal Inflammation

Neutrophil (PMN) migration in the intestine is an essential component of host defense and wound repair. However, dysregulated PMN recruitment is a hallmark of inflammatory bowel disease (IBD). Disease flares in IBD are associated with robust PMN migrating across the intestinal mucosa resulting in crypt abscess formation and mucosal ulceration. Despite strong association of PMN infiltration into the gut with patient symptoms in IBD, the molecular basis of PMN transepithelial migration (TEpM) represents a significantly understudied area. It is now appreciated that PMN migration into intestinal mucosa requires multiple adhesive interactions between migrating PMN and intestinal epithelial cells (IECs), that represents a multi‐step process facilitated by complex interactions between PMN and IEC expressed proteins including members of the Junctional Adhesion Molecules (JAMs) family. We have reported that the novel JAM‐like protein termed JAML expressed on PMN, associates with the coxsackie and adenovirus receptor (CAR) expressed on epithelial cells. Binding of soluble JAML ectodomains to CAR has been shown to disrupt JAML‐CAR interactions and reduce PMN TEpM in‐vitro. To define the molecular mechanism of JAML regulation of PMN TEpM during intestinal inflammation in‐vivo, we generated JAML knockout mice (JAML−/−). PMN TEpM was evaluated using a small intestinal loop model, and PMN migration in response to the chemoattractant LTB4 (1nM) quantified by flow cytometry. While WT and JAML−/− mice had similar numbers of circulating leukocytes, we observed reduced PMN TEpM in‐vivo in JAML−/− mice compared to WT animals. This decrease in PMN traffic into the lumen was associated with increased retention of PMN within the intestinal epithelium in JAML−/− mice. In addition, epithelial accumulation of PMN in JAML−/− mice paralleled an increase in intestinal permeability to a 4KDa FITC‐dextran as detected by fluorescence intensity in serum. Finally, we studied the role of JAML on susceptibility to DSS induced colitis. Loss of JAML markedly increased disease activity index, inflammation and mucosal ulceration compared to WT mice. Overall, these observations suggest that JAML is an important adhesion molecule that regulates PMN TEpM in the intestinal mucosa. JAML thus represents a new therapeutic target to dampen excessive PMN migration during intestinal inflammation as seen in IBD.Support or Funding InformationCCFA CDA454814 and NIH 3R01 DK072564‐23S1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting Epithelial Expressed Sialyl Lewis A Improves Intestinal Mucosal Wound Healing and Protects Against Colitis

Dysregulated healing of mucosal wounds is a hallmark of many pathological conditions including inflammatory bowel disease (IBD). Mucosal wound healing involves a complex interplay between epithelial cells and infiltrating immune cells that trigger cell signaling events and epithelial cell migration, proliferation and differentiation that are necessary for mucosal homeostasis. Specific alterations in surface glycosylation have been reported in intestinal epithelial cells following injury, and we have shown that targeting the inflammation induced epithelial glycan sialyl Lewis A (sLea) with a specific mAb termed GM35 results in inhibition of neutrophil (PMN) trafficking in the inflamed intestine. Despite this observation, the role of glycans in intestinal wound healing has not been examined. Here we report that, in addition to inhibiting PMN transepithelial migration, targeting epithelial expressed sLea increases intestinal epithelial wound healing and recovery from barrier disruption in vitro. We report that these protective effects are mediated through deactivation of Akt (a kinase previously shown to destabilize epithelial adherens junctions) downstream of sLea engagement. Furthermore, wound bed injection of GM35 directly into biopsy‐induced colonic wounds resulted in a significant increase in mucosal wound healing in vivo. Finally, administration of GM35 to C57BL/6J mice followed by induction of colitis with dextran sodium sulfate resulted in reduced disease activity as assessed clinically and histologically, compared to mice treated with an isotype control mAb. Given our observations of robust upregulation of epithelial sLea during intestinal inflammation in mice and humans, these data suggest that targeting sLea may represent a promising new target for regulating intestinal trafficking of PMNs. In addition, targeting of this glycan may also provide an additional therapeutic strategy for improved recovery of epithelial barrier function and restitution of intestinal homeostasis following inflammation or injury.Support or Funding InformationThis work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, 410855404), a senior Research Award from the Crohn's and Colitis Foundation (CCF) and R01 awards from the NIH (DK072564 and DK079392).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protease Activity of Campylobacter jejuni HtrA Modulates Distinct Intestinal and Systemic Immune Responses in Infected Secondary Abiotic IL-10 Deficient Mice.

Even though human Campylobacter jejuni infections are progressively increasing worldwide, the underlying molecular mechanisms of pathogen-host-interactions are still not fully understood. We have recently shown that the secreted serine protease HtrA plays a key role in C. jejuni cellular invasion and transepithelial migration in vitro, and is involved in the onset of intestinal pathology in murine infection models in vivo. In the present study, we investigated whether the protease activity of HtrA had an impact in C. jejuni induced acute enterocolitis. For this purpose, we perorally infected secondary abiotic IL-10−/− mice with wildtype C. jejuni strain NCTC11168 (11168WT) or isogenic bacteria carrying protease-inactive HtrA with a single point mutation at S197A in the active center (11168HtrA−S197A). Irrespective of the applied pathogenic strain, mice harbored similar C. jejuni loads in their feces and exhibited comparably severe macroscopic signs of acute enterocolitis at day 6 postinfection (p.i.). Interestingly, the 11168HtrA−S197A infected mice displayed less pronounced colonic apoptosis and immune cell responses, but enhanced epithelial proliferation as compared to the 11168WT strain infected controls. Furthermore, less distinct microscopic sequelae in 11168HtrA−S197A as compared to parental strain infected mice were accompanied by less distinct colonic secretion of pro-inflammatory cytokines such as MCP-1, IL-6, TNF, and IFN-γ in the former as compared to the latter. Strikingly, the S197A point mutation was additionally associated with less pronounced systemic pro-inflammatory immune responses as assessed in serum samples. In conclusion, HtrA is a remarkable novel virulence determinant of C. jejuni, whose protease activity is not required for intestinal colonization and establishment of disease, but aggravates campylobacteriosis by triggering apoptosis and pro-inflammatory immune responses.

How Adhesion Molecule Patterns Change While Neutrophils Traffic through the Lung during Inflammation.

In acute pulmonary inflammation, polymorphonuclear cells (PMNs) pass a transendothelial barrier from the circulation into the lung interstitium followed by a transepithelial migration into the alveolar space. These migration steps are regulated differentially by a concept of adhesion molecules and remain—despite decades of research—incompletely understood. Current knowledge of changes in the expression pattern of adhesion molecules mainly derives from in vitro studies or from studies in extrapulmonary organ systems, where regulation of adhesion molecules differs significantly. In a murine model of lung inflammation, we determined the expression pattern of nine relevant neutrophilic adhesion molecules on their way through the different compartments of the lung. We used a flow cytometry-based technique that allowed describing spatial distribution of the adhesion molecules expressed on PMNs during their migration through the lung in detail. For example, the highest expression of CD29 was found in the intravascular compartment, highlighting its impact on the initial adhesion to the endothelium. CD47 showed its peak of expression on the later phase of transendothelial migration, whereas CD11b and CD54 expression peaked interstitial. A pivotal role for transepithelial migration was found for the adhesion molecule CD172a. Thereby, expression may correlate with functional impact for specific migration steps. In vitro studies further confirmed our in vivo findings. In conclusion, we are the first to determine the changes in expression patterns of relevant adhesion molecules on their migration through the different compartments of the lung. These findings may help to further understand the regulation of neutrophil trafficking in the lung.

Neutrophil Dysfunction in the Airways of Children with Acute Respiratory Failure Due to Lower Respiratory Tract Viral and Bacterial Coinfections

Neutrophils are recruited to the airways of patients with acute respiratory distress syndrome (ARDS) where they acquire an activated pro-survival phenotype with an enhanced respiratory burst thought to contribute to ARDS pathophysiology. Our in vitro model enables blood neutrophil transepithelial migration into cell-free tracheal aspirate fluid from patients to recapitulate the primary airway neutrophil phenotype observed in vivo. Neutrophils transmigrated through our model toward airway fluid from children with lower respiratory viral infections coinfected with bacteria had elevated levels of neutrophil activation markers but paradoxically exhibited an inability to kill bacteria and a defective respiratory burst compared with children without bacterial coinfection. The airway fluid from children with bacterial coinfections had higher levels of neutrophil elastase activity, as well as myeloperoxidase levels compared to children without bacterial coinfection. Neutrophils transmigrated into the aspirate fluid from children with bacterial coinfection showed decreased respiratory burst and killing activity against H. influenzae and S. aureus compared to those transmigrated into the aspirate fluid from children without bacterial coinfection. Use of a novel transmigration model recapitulates this pathological phenotype in vitro that would otherwise be impossible in a patient, opening avenues for future mechanistic and therapeutic research.

Estradiol impairs epithelial CXCL1 gradient in the cervix to delay neutrophil transepithelial migration during insemination

Female reproductive mucosa must allow allogenic sperm survival whereas at the same time, avoid pathogen infection. To preserve sperm from neutrophil attack, neutrophils disappear from the vagina during the ovulatory phase (high estradiol); although the mechanisms that regulate neutrophil influx to the vagina during insemination remain controversial.We investigated the sex hormone regulation of the neutrophil migration through the cervix during insemination and revealed that ovulatory estradiol dose fades the CXCL1 epithelial expression in the ectocervix and fornix; hence, retarding neutrophil migration and retaining them in the epithelium. These mechanisms spare sperm from neutrophil attack to preserve reproduction, but might compromise immunity. However, luteal progesterone dose promotes the CXCL1 gradient expression to restore neutrophil migration, to eliminate sperm and prevent sperm associated pathogen dissemination. Surprisingly, these mechanisms are hormone dependent and independent of the insemination. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil transepithelial migration in the fornix and ectocervix.

P147 NOVEL JUNCTIONAL ADHESION MOLECULE-LIKE (JAML) REGULATES NEUTROPHIL TRANSEPITHELIAL MIGRATION DURING INTESTINAL INFLAMMATION

Neutrophil (PMN) migration in the intestine is an essential component of host defense and wound repair. However, dysregulated PMN recruitment is a hallmark of chronic active inflammatory bowel disease (IBD) characterized by persistent inflammation. Disease flares in IBD are associated with marked recruitment of PMN into the intestinal mucosa that results in crypt abscess formation and mucosal ulcerations. Despite the strong association with IBD, the process of PMN transepithelial migration (TEpM) represents a significantly understudied area. PMN migration into the intestinal mucosa requires multiple adhesive interactions between migrating PMN and intestinal epithelial cells (IECs). PMN TEpM is a multi-step process facilitated by complex interactions between PMN and IEC expressed proteins including members of the Juntional Adhesion Molecules (JAMs) family. We have reported that the novel JAM-like protein termed JAML expressed on PMN, associates with the coxsackie and adenovirus receptor (CAR) on epithelial cells. Binding of recombinant JAML ectodomains disrupts JAML-CAR interactions and reduces PMN TEpM. To define the molecular mechanism of JAML regulation of PMN TEpM during intestinal inflammation in-vivo, we generated JAML knockout mice (JAML-/-). PMN TEpM was evaluated using a small intestinal loop model, and PMN migration into the intestinal lumen in response to the chemoattractant LTB4(1nM) for 1h was quantified by flow cytometry. While WT and JAML-/- mice had similar numbers of circulating leukocytes, we observed reduced PMN migration into the intestinal lumen in response to LTB4 in JAML-/- mice compared to WT animals. In addition, there were increased PMN in the epithelial cell fractions derived from intestines of JAML-/- mice suggesting that JAML regulates PMN TEpM in vivo. We also assessed the role of JAML on susceptibility to DSS induced colitis and recovery. Markedly increased disease activity index, inflammation and mucosal ulceration were observed in JAML-/- mice compared to controls. These observations suggest that JAML represents an important adhesion molecule that regulates PMN TEpM. Furthermore, loss of JAML increases susceptibility to colitis. JAML may thus represent a new therapeutic target to dampen excessive PMN migration during intestinal inflammation as seen in IBD.

Mitochondrial damage-associated molecular patterns released by lung transplants are associated with primary graft dysfunction.

Primary graft dysfunction (PGD) is a major limitation in short- and long-term lung transplant survival. Recent work has shown that mitochondrial damage-associated molecular patterns (mtDAMPs) can promote solid organ injury, but whether they contribute to PGD severity remains unclear. We quantitated circulating plasma mitochondrial DNA (mtDNA) in 62 patients, before lung transplantation and shortly after arrival to the intensive care unit. Although all recipients released mtDNA, high levels were associated with severe PGD development. In a mouse orthotopic lung transplant model of PGD, we detected airway cell-free damaged mitochondria and mtDNA in the peripheral circulation. Pharmacologic inhibition or genetic deletion of formylated peptide receptor 1 (FPR1), a chemotaxis sensor for N-formylated peptides released by damaged mitochondria, inhibited graft injury. An analysis of intragraft neutrophil-trafficking patterns reveals that FPR1 enhances neutrophil transepithelial migration and retention within airways but does not control extravasation. Using donor lungs that express a mitochondria-targeted reporter protein, we also show that FPR1-mediated neutrophil trafficking is coupled with the engulfment of damaged mitochondria, which in turn triggers reactive oxygen species (ROS)-induced pulmonary edema. Therefore, our data demonstrate an association between mtDAMP release and PGD development and suggest that neutrophil trafficking and effector responses to damaged mitochondria are drivers of graft damage.

Plasma membrane profiling during enterohemorrhagic E. coli infection reveals that the metalloprotease StcE cleaves CD55 from host epithelial surfaces

Enterohemorrhagic Escherichia coli (EHEC) is one of several E. coli pathotypes that infect the intestinal tract and cause disease. Formation of the characteristic attaching and effacing lesion on the surface of infected cells causes significant remodeling of the host cell surface; however, limited information is available about changes at the protein level. Here we employed plasma membrane profiling, a quantitative cell-surface proteomics technique, to identify host proteins whose cell-surface levels are altered during infection. Using this method, we quantified more than 1100 proteins, 280 of which showed altered cell-surface levels after exposure to EHEC. 22 host proteins were significantly reduced on the surface of infected epithelial cells. These included both known and unknown targets of EHEC infection. The complement decay–accelerating factor cluster of differentiation 55 (CD55) exhibited the greatest reduction in cell-surface levels during infection. We showed by flow cytometry and Western blot analysis that CD55 is cleaved from the cell surface by the EHEC-specific protease StcE and found that StcE-mediated CD55 cleavage results in increased neutrophil adhesion to the apical surface of intestinal epithelial cells. This suggests that StcE alters host epithelial surfaces to depress neutrophil transepithelial migration during infection. This work is the first report of the global manipulation of the epithelial cell surface by a bacterial pathogen and illustrates the power of quantitative cell-surface proteomics in uncovering critical aspects of bacterial infection biology.

Resistance of uterine radial artery blood flow was correlated with peripheral blood NK cell fraction and improved with low molecular weight heparin therapy in women with unexplained recurrent pregnancy loss

Female reproductive mucosa must allow allogenic sperm survival whereas at the same time, avoid pathogen infection. To preserve sperm from neutrophil attack, neutrophils disappear from the vagina during the ovulatory phase (high estradiol); although the mechanisms that regulate neutrophil influx to the vagina during insemination remain controversial.We investigated the sex hormone regulation of the neutrophil migration through the cervix during insemination and revealed that ovulatory estradiol dose fades the CXCL1 epithelial expression in the ectocervix and fornix; hence, retarding neutrophil migration and retaining them in the epithelium. These mechanisms spare sperm from neutrophil attack to preserve reproduction, but might compromise immunity. However, luteal progesterone dose promotes the CXCL1 gradient expression to restore neutrophil migration, to eliminate sperm and prevent sperm associated pathogen dissemination. Surprisingly, these mechanisms are hormone dependent and independent of the insemination. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil transepithelial migration in the fornix and ectocervix.

Analysis of leukocyte transepithelial migration using an in vivo murine colonic loop model.

Molecular mechanisms that control leukocyte migration across the vascular endothelium (transendothelial migration; TEndoM) have been extensively characterized in vivo, but details of leukocyte transepithelial migration (TEpM) and its dysregulation (a pathologic feature of many mucosal diseases) are missing due to the lack of suitable animal models. Here, we describe a murine model that utilizes a vascularized proximal colonic segment (pcLoop) and enables quantitative studies of leukocyte trafficking across colonic epithelium. Consistent with previous in vitro studies, intraluminal injection of antibodies against integrin CD11b/CD18 reduced recruitment of polymorphonuclear neutrophils (PMN) into the lumen of pcLoops, and it increased subepithelial accumulation of PMN. We extended studies using the pcLoop to determine contributions of Junctional Adhesion Molecule-A (JAM-A, or F11R) in PMN TEpM and confirmed that mice with total loss of JAM-A or mice with intestinal epithelial selective loss of JAM-A had increased colonic permeability. Furthermore, there was reduced PMN migration into the colonic lumen that paralleled subepithelial accumulation of PMN in global-KO mice, as well as in intestinal epithelial-targeted JAM-A-deficient mice. These findings highlight a potentially novel role for JAM-A in regulating PMN TEpM in vivo and demonstrate utility of this model for identifying receptors that may be targeted in vivo to reduce pathologic intestinal inflammation.

Transporters MRP1 and MRP2 Regulate Opposing Inflammatory Signals To Control Transepithelial Neutrophil Migration during Streptococcus pneumoniae Lung Infection.

Streptococcus pneumoniae remains a source of morbidity and mortality in both developed and underdeveloped nations of the world. Disease can manifest as pneumonia, bacteremia, and meningitis, depending on the localization of infection. Interestingly, there is a correlation in experimental murine infections between the development of bacteremia and influx of neutrophils into the pulmonary lumen. Reduction of this neutrophil influx has been shown to improve survivability during infection. In this study, we use in vitro biotinylation and neutrophil transmigration and in vivo murine infection to identify a system in which two epithelium-localized ATP-binding cassette transporters, MRP1 and MRP2, have inverse activities dictating neutrophil transmigration into the lumen of infected mouse lungs. MRP1 effluxes an anti-inflammatory molecule that maintains homeostasis in uninfected contexts, thus reducing neutrophil infiltration. During inflammatory events, however, MRP1 decreases and MRP2 both increases and effluxes the proinflammatory eicosanoid hepoxilin A3. If we then decrease MRP2 activity during experimental murine infection with S. pneumoniae, we reduce both neutrophil infiltration and bacteremia, showing that MRP2 coordinates this activity in the lung. We conclude that MRP1 assists in depression of polymorphonuclear cell (PMN) migration by effluxing a molecule that inhibits the proinflammatory effects of MRP2 activity.IMPORTANCEStreptococcus pneumoniae is a Gram-positive bacterium that normally inhabits the human nasopharynx asymptomatically. However, it is also a major cause of pneumonia, bacteremia, and meningitis. The transition from pneumonia to bacteremia is critical, as patients that develop septicemia have ~20% mortality rates. Previous studies have shown that while neutrophils, a major bacterium-induced leukocyte, aid in S.pneumoniae elimination, they also contribute to pathology and may mediate the lung-to-blood passage of the bacteria. Herein, we show that epithelium-derived MRP1 and MRP2 efflux immunomodulatory agents that assist in controlling passage of neutrophils during infection and that limiting neutrophil infiltration produced less bacteremia and better survival during murine infection. The importance of our work is twofold: ours is the first to identify an MRP1/MRP2 axis of neutrophil control in the lung. The second is to provide possible therapeutic targets to reduce excess inflammation, thus reducing the chances of developing bacteremia during pneumococcal pneumonia.

Drosophila immune cells extravasate from vessels to wounds using Tre1 GPCR and Rho signaling.

Inflammation is pivotal to fight infection, clear debris, and orchestrate repair of injured tissues. Although Drosophila melanogaster have proven invaluable for studying extravascular recruitment of innate immune cells (hemocytes) to wounds, they have been somewhat neglected as viable models to investigate a key rate-limiting component of inflammation-that of immune cell extravasation across vessel walls-due to their open circulation. We have now identified a period during pupal development when wing hearts pulse hemolymph, including circulating hemocytes, through developing wing veins. Wounding near these vessels triggers local immune cell extravasation, enabling live imaging and correlative light-electron microscopy of these events in vivo. We show that RNAi knockdown of immune cell integrin blocks diapedesis, just as in vertebrates, and we uncover a novel role for Rho-like signaling through the GPCR Tre1, a gene previously implicated in the trans-epithelial migration of germ cells. We believe this new Drosophila model complements current murine models and provides new mechanistic insight into immune cell extravasation.

Endocervical and Neutrophil Lipoxygenases Coordinate Neutrophil Transepithelial Migration to Neisseria gonorrhoeae.

Infection with Neisseria gonorrhoeae (GC) is characterized by robust neutrophil influx that is insufficient to clear the bacteria. Sustained neutrophilic inflammation contributes to serious clinical sequelae that particularly affect women, including pelvic inflammatory disease and infertility. We established a 3-component system using GC, End1 polarized human endocervical cells, and primary human neutrophils to investigate neutrophil transepithelial migration following infection. Neutrophil migration across endocervical monolayers increased with the infectious dose and required GC-epithelial cell contact. Epithelial protein kinase C, cytosolic phospholipase A2, 12R-lipoxygenase (LOX), and eLOX3 hepoxilin synthase were required for neutrophil transmigration to GC, and migration was abrogated by blocking the MRP2 efflux pump and by adding recombinant soluble epoxide hydrolase. These results are all consistent with epithelial cell production of the neutrophil chemoattractant hepoxilin A3 (HXA3). Neutrophil transmigration was also accompanied by increasing apical concentrations of leukotriene B4 (LTB4). Neutrophil 5-lipoxygenase and active BLT1 receptor were required for apical LTB4 and neutrophil migration. Our data support a model in which GC-endocervical cell contact infection stimulates HXA3 production, driving neutrophil migration that is amplified by neutrophil-derived LTB4. Therapeutic targeting of these pathways could limit inflammation and deleterious clinical sequelae in women with gonorrhea.

Estrogen Receptor-Alpha (ESR1) Governs the Lower Female Reproductive Tract Vulnerability to Candida albicans.

Estradiol-based therapies predispose women to vaginal infections. Moreover, it has long been known that neutrophils are absent from the vaginal lumen during the ovulatory phase (high estradiol). However, the mechanisms that regulate neutrophil influx to the vagina remain unknown. We investigated the neutrophil transepithelial migration (TEM) into the vaginal lumen. We revealed that estradiol reduces the CD44 and CD47 epithelial expression in the vaginal ectocervix and fornix, which retain neutrophils at the apical epithelium through the estradiol receptor-alpha. In contrast, luteal progesterone increases epithelial expression of CD44 and CD47 to promote neutrophil migration into the vaginal lumen and Candida albicans destruction. Distinctive to vaginal mucosa, neutrophil infiltration is contingent to sex hormones to prevent sperm from neutrophil attack; although it may compromise immunity during ovulation. Thus, sex hormones orchestrate tolerance and immunity in the vaginal lumen by regulating neutrophil TEM.

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

Pseudomonas aeruginosa-induced lung epithelial production of IL-8 contributes to PMN sub-mucosa recruitment but not trans-epithelial migration in vitro.

Abstract Background Numerous lung associated diseased states such as acute pneumonia and cystic fibrosis (CF) are associated with increased concentrations of IL8 and infiltration of neutrophils (PMNs) in the airspace. IL8 production by lung epithelial cells (ECs) is stimulated by Pseudomonas aeruginosa infection, an opportunistic pathogen involved in acute pneumonia, and lung infection of CF patients. The exact role IL8 plays in migration of circulating PMNs into the infected lung and airspace is still uncertain. Objective Assess the role of IL8 in mediating PMN migration in response to P. aeruginosa infected lung epithelium. Methods CRISPR/Cas9 was used to delete the IL8 gene from human lung ECs, which were grown as polarized monolayers in a co-culture transwell model. Basolateral to apical PMN migration to PAO1 infection was assessed. A novel double transwell model was developed to observe PMN submucosa migration in response to PAO1 infection in the presence and absence of IL8 lung EC production. Results PAO1 induced PMN trans-epithelial (TE) migration was not impacted in the absence of IL8 expression. When PMNs were placed in a separate transwell suspended 5 mm above the basolateral side of the infected epithelium, the IL8 gradient produced was sufficient to cause relevant migration of PMNs to the basolateral side of the infected epithelium. The level of migrated PMNs in the double transwell system with tissues devoid of the IL8 gene was equivalent to the presence of PMNs which responded to un-infected epithelial tissues. Conclusion These findings further support that IL8 is integral in PMN recruitment, and suggests that IL8 is most important towards recruiting PMNs to the submucosa during P. aeruginosa PAO1 infection of lung epithelium.

An in vitro transepithelial migration assay to evaluate the role of neutrophils in Respiratory Syncytial Virus (RSV) induced epithelial damage

Large numbers of neutrophils migrate into the lungs of children with severe Respiratory Syncytial Virus (RSV) disease. It is unclear how these cells contribute to viral clearance and recovery from infection or whether they contribute to disease pathology. We have developed a novel in vitro model to study neutrophil migration through airway epithelial cells (AECs), the main cellular target of RSV infection. Our model reproduces a physiologically relevant cell polarity and directionality of neutrophil migration. Using this model, we found that RSV infected AECs induced rapid neutrophil transepithelial migration. We also detected increased AEC damage associated with RSV infection, with a further increase in epithelial cells shedding from the Transwell membrane following neutrophil migration. This was not observed in the mock infected controls. Neutrophils that migrated through the RSV infected AECs showed increased cell surface expression of CD11B and MPO compared to neutrophils that had not migrated. In conclusion, our in vitro co-culture assay can be used to identify critical mechanisms that mediate epithelial cell damage and promote inflammation in children with severe RSV disease.