Endothelial CLEC-1b plays a protective role against cancer hematogenous metastasis.

Metastasis, which is the spread of cancer cells into distant organs, is a critical determinant of prognosis in patients with cancer, and blood vessels are the major route for cancer cells to spread systemically. Extravasation is a critical process for the hematogenous metastasis; however, its underlying molecular mechanisms remain poorly understood. Here, we identified that senescent ECs highly express C-type lectin domain family 1 member B (CLEC-1b), and that endothelial CLEC-1b inhibits the hematogenous metastasis of a certain type of cancer. CLEC-1b expression was enhanced in ECs isolated from aged mice, senescent cultured human ECs, and ECs of aged human. CLEC-1b overexpression in ECs prevented the disruption of endothelial integrity, and inhibited the transendothelial migration of cancer cells expressing podoplanin (PDPN), a ligand for CLEC-1b. Notably, target activation of CLEC-1b in ECs decreased the hematogenous metastasis in the lungs by cancer cells expressing PDPN in mice. Our data reveal the protective role of endothelial CLEC-1b against cancer hematogenous metastasis. Considering the high CLEC-1b expression in senescent ECs, EC senescence may play a beneficial role with respect to the cancer hematogenous metastasis.

Plasma S1P Orchestrates the Reverse Transendothelial Migration of Aortic Intimal Myeloid Cells in Mice.

BACKGROUND: Myeloid cells (MCs) reside in the aortic intima at regions predisposed to atherosclerosis. Systemic inflammation triggers reverse transendothelial migration (RTM) of intimal MCs into the arterial blood, which orchestrates a protective immune response that clears intracellular pathogens from the arterial intima. Molecular pathways that regulate RTM remain poorly understood. S1P (sphingosine-1-phosphate) is a lipid mediator that regulates immune cell trafficking by signaling via 5 G-protein-coupled receptors (S1PRs [S1P receptors]). We investigated the role of S1P in the RTM of aortic intimal MCs. METHODS: Intravenous injection of lipopolysaccharide was used to model a systemic inflammatory stimulus that triggers RTM. CD11c+ intimal MCs in the lesser curvature of the ascending aortic arch were enumerated by en face confocal microscopy. Local gene expression was evaluated by transcriptomic analysis of microdissected intimal cells. RESULTS: In wild-type C57BL/6 mice, lipopolysaccharide induced intimal cell expression of S1pr1, S1pr3, and Sphk1 (a kinase responsible for S1P production). Pharmacological modulation of multiple S1PRs blocked lipopolysaccharide-induced RTM and modulation of S1PR1 and S1PR3 reduced RTM in an additive manner. Cre-mediated deletion of S1pr1 in MCs blocked lipopolysaccharide-induced RTM, confirming a role for myeloid-specific S1PR1 signaling. Global or hematopoietic deficiency of Sphk1 reduced plasma S1P levels, the abundance of CD11c+ MCs in the aortic intima, and blunted lipopolysaccharide-induced RTM. In contrast, plasma S1P levels, the abundance of intimal MCs, and lipopolysaccharide-induced RTM were rescued in Sphk1-/- mice transplanted with Sphk1+/+ or mixed Sphk1+/+ and Sphk1-/- bone marrow. Stimulation with lipopolysaccharide increased endothelial permeability and intimal MC exposure to circulating factors such as S1P. CONCLUSIONS: Functional and expression studies support a novel role for S1P signaling in the regulation of lipopolysaccharide-induced RTM and the homeostatic maintenance of aortic intimal MCs. Our data provide insight into how circulating plasma mediators help orchestrate intimal MC dynamics.

Astrocyte-Derived Extracellular Vesicular miR-143-3p Dampens Autophagic Degradation of Endothelial Adhesion Molecules and Promotes Neutrophil Transendothelial Migration after Acute Brain Injury.

Pivotal roles of extracellular vesicles (EVs) in the pathogenesis of central nervous system (CNS) disorders including acute brain injury are increasingly acknowledged. Through the analysis of EVs packaged miRNAs in plasma samples from patients with intracerebral hemorrhage (ICH), it is discovered that the level of EVs packaged miR-143-3p (EVs-miR-143-3p) correlates closely with perihematomal edema and neurological outcomes. Further study reveals that, upon ICH, EVs-miR-143-3p is robustly secreted by astrocytes and can shuttle into brain microvascular endothelial cells (BMECs). Heightened levels of miR-143-3p in BMECs induce the up-regulated expression of cell adhesion molecules (CAMs) that bind to circulating neutrophils and facilitate their transendothelial cell migration (TEM) into brain. Mechanism-wise, miR-143-3p directly targets ATP6V1A, resulting in impaired lysosomal hydrolysis ability and reduced autophagic degradation of CAMs. Importantly, a VCAM-1-targeting EVs system to selectively deliver miR-143-3p inhibitor to pathological BMECs is created, which shows satisfactory therapeutic effects in both ICH and traumatic brain injury (TBI) mouse models. In conclusion, the study highlights the causal role of EVs-miR-143-3p in BMECs' dysfunction in acute brain injury and demonstrates a proof of concept that engineered EVs can be devised as a potentially applicable nucleotide drug delivery system for the treatment of CNS disorders.

An Intriguing Structural Modification in Neutrophil Migration Across Blood Vessels to Inflammatory Sites: Progress in the Core Mechanisms.

The role and function of neutrophils are well known, but we still have incomplete understanding of the mechanisms by which neutrophils migrate from blood vessels to inflammatory sites. Neutrophil migration is a complex process that involves several distinct steps. To resist the blood flow and maintain their rolling, neutrophils employ tether and sling formation. They also polarize and form pseudopods and uropods, guided by hierarchical chemotactic agents that enable precise directional movement. Meanwhile, chemotactic agents secreted by neutrophils, such as CXCL1, CXCL8, LTB4, and C5a, can recruit more neutrophils and amplify their response. In the context of diapedesis neutrophils traverse the endothelial cells via two pathways: the transmigratory cup and the lateral border recycling department. These structures aid in overcoming the narrow pore size of the endothelial barrier, resulting in more efficient transmembrane migration. Interestingly, neutrophils exhibit a preference for the paracellular pathway over the transcellular pathway, likely due to the former's lower resistance. In this review, we will delve into the intricate process of neutrophil migration by focusing on critical structures that underpins this process.

BAP31-Mediated miR-206/133b Cluster Promotes Transendothelial Migration and Metastasis of Colorectal Cancer.

Dysregulated B cell receptor-associated protein 31 (BAP31) plays a crucial role in tumor progression. This study aimed to investigate the functions and molecular mechanism of BAP31 on the miR-206/133b cluster in colorectal cancer (CRC). qPCR was conducted to detect miRNA and mRNA levels in tissues and cells. Western blot assays were used to assess the levels of biomarkers and targets, as well as the levels of BAP31 and HOXD10. Wound healing, coculture and transwell assays were conducted to assess the transendothelial migration abilities of CRC cells. A luciferase assay was employed to assess miRNA binding effects on targets, as well as the initiating transcription effect of genomic fragments. Tumor growth and lung metastatic models were established through an in vivo animal study. BAP31 overexpression in CRC cells led to a reduction in the expression of the miR-206/133b cluster. The expression of the miR-206/133b cluster was correlated with the transendothelial migration capability of CRC cells. The miR-206/133b cluster was found to directly regulate cell division cycle 42 (CDC42) and actin-related protein 2/3 complex subunit 5 (ARPC5) in the tight junction pathway (hsa04530). Moreover, a potential transcription regulator of the miR-206/133b cluster was also found to be Homeobox D10 (HOXD10). We further elucidated the molecular mechanisms and functional mechanisms of BAP31's regulatory role in the expression levels of the miR-206/133b cluster by inhibiting HOXD10 translocation from the cytoplasm to the nucleus. In conclusion, this study provides valuable insights into how BAP31 regulates the transcription of the miR-206/133b cluster and how BAP31-related lung metastases arise in CRC.

PECAM-1 mediates temsirolimus-induced increase in neutrophil transendothelial migration that leads to lung injury.

Temsirolimus is a first-generation mTOR inhibitor commonly used in the clinical treatment of cancers that is associated with lung injury. However, the mechanism underlying this adverse effect remains elusive. Endothelial barrier dysfunction plays a pivotal role in the infiltration of neutrophils into the pulmonary alveoli, which eventually induces lung injury. The present study demonstrates that temsirolimus induces the aberrant expression of adhesion molecules in endothelial cells, leading to enhanced neutrophil infiltration and subsequent lung injury. Results of a mouse model revealed that temsirolimus disrupted capillary-alveolar barrier function and facilitated neutrophil transmigration across the endothelium within the alveolar space. Consistent with our in vivo observations, temsirolimus impaired intercellular barrier function within monolayers of human lung endothelial cells, resulting in increased neutrophil infiltration. Furthermore, we demonstrated that temsirolimus-induced neutrophil transendothelial migration was mediated by platelet endothelial cell adhesion molecule-1 (PECAM-1) in both in vitro and in vivo experiments. Collectively, these findings highlight that temsirolimus induces endothelial barrier dysfunction via PECAM-1-dependent pathway both in vitro and in vivo, ultimately leading to neutrophil infiltration and subsequent pulmonary injury.

Mechanotransduction via endothelial adhesion molecule CD31 initiates transmigration and reveals a role for VEGFR2 in diapedesis.

Engagement of platelet endothelial cell adhesion molecule 1 (PECAM, PECAM-1, CD31) on the leukocyte pseudopod with PECAM at the endothelial cell border initiates transendothelial migration (TEM, diapedesis). We show, using fluorescence lifetime imaging microscopy (FLIM), that physical traction on endothelial PECAM during TEM initiated the endothelial signaling pathway. In this role, endothelial PECAM acted as part of a mechanotransduction complex with VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2), and this predicted that VEGFR2 was required for efficient TEM. We show that TEM required both VEGFR2 and the ability of its Y1175 to be phosphorylated, but not VEGF or VEGFR2 endogenous kinase activity. Using inducible endothelial-specific VEGFR2-deficient mice, we show in three mouse models of inflammation that the absence of endothelial VEGFR2 significantly (by ≥75%) reduced neutrophil extravasation by selectively blocking diapedesis. These findings provide a more complete understanding of the process of transmigration and identify several potential anti-inflammatory targets.

Transendothelial Migration of Human B Cells: Chemokine versus Antigen.

B cells, like T cells, can infiltrate sites of inflammation, but the processes and B cell subsets involved are poorly understood. Using human cells and in vitro assays, we find only a very small number of B cells will adhere to TNF-activated (but not to resting) human microvascular endothelial cells (ECs) under conditions of venular flow and do so by binding to ICAM-1 and VCAM-1. CXCL13 and, to a lesser extent, CXCL10 bound to the ECs can increase adhesion and induce transendothelial migration (TEM) of adherent naive and memory B cells in 10-15 min through a process involving cell spreading, translocation of the microtubule organizing center (MTOC) into a trailing uropod, and interacting with EC activated leukocyte cell adhesion molecule. Engagement of the BCR by EC-bound anti-κ L chain Ab also increases adhesion and TEM of κ+ but not λ+ B cells. BCR-induced TEM takes 30-60 min, requires Syk activation, is initiated by B cell rounding up and translocation of the microtubule organizing center to the region of the B cell adjacent to the EC, and also uses EC activated leukocyte cell adhesion molecule for TEM. BCR engagement reduces the number of B cells responding to chemokines and preferentially stimulates TEM of CD27+ B cells that coexpress IgD, with or without IgM, as well as CD43. RNA-sequencing analysis suggests that peripheral blood CD19+CD27+CD43+IgD+ cells have increased expression of genes that support BCR activation as well as innate immune properties in comparison with total peripheral blood CD19+ cells.

Exploring the Process of Neutrophil Transendothelial Migration Using Scanning Ion-Conductance Microscopy.

The dynamics of neutrophil transendothelial migration was investigated in a model of experimental septicopyemia. Scanning ion-conductance microscopy allowed us to determine changes in morphometric characteristics of endothelial cells during this process. In the presence of a pyogenic lesion simulated by Staphylococcus aureus, such migration was accompanied by both compensatory reactions and alteration of both neutrophils and endothelial cells. Neutrophils demonstrated crawling along the contact sites between endothelial cells, swarming phenomenon, as well as anergy and formation of neutrophil extracellular traps (NETs) as a normergic state. Neutrophil swarming was accompanied by an increase in the intercellular spaces between endothelial cells. Endothelial cells decreased the area of adhesion to the substrate, which was determined by a decrease in the cell projection area, and the cell membrane was smoothed. However, endothelial cell rigidity was paradoxically unchanged compared to the control. Over time, neutrophil migration led to a more significant alteration of endothelial cells: first, shallow perforations in the membrane were formed, which were repaired rather quickly, then stress fibrils were formed, and finally, endothelial cells died and multiple perforations were formed on their membrane.

Porphyromonas gingivalis Gingipains Destroy the Vascular Barrier and Reduce CD99 and CD99L2 Expression To Regulate Transendothelial Migration.

Porphyromonas gingivalis is an important periodontal pathogen that can cause vascular injury and invade local tissues through the blood circulation, and its ability to evade leukocyte killing is critical to its distal colonization and survival. Transendothelial migration (TEM) is a series of that enable leukocytes to squeeze through endothelial barriers and migrate into local tissues to perform immune functions. Several studies have shown that P. gingivalis-mediated endothelial damage initiates a series of proinflammatory signals that promote leukocyte adhesion. However, whether P. gingivalis is involved in TEM and thus influences immune cell recruitment remains unknown. In our study, we found that P. gingivalis gingipains could increase vascular permeability and promote Escherichia coli penetration by downregulating platelet/endothelial cell adhesion molecule 1 (PECAM-1) expression in vitro. Furthermore, we demonstrated that although P. gingivalis infection promoted monocyte adhesion, the TEM capacity of monocytes was substantially impaired, which might be due to the reduced CD99 and CD99L2 expression on gingipain-stimulated endothelial cells and leukocytes. Mechanistically, gingipains mediate CD99 and CD99L2 downregulation, possibly through the inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, our in vivo model confirmed the role of P. gingivalis in promoting vascular permeability and bacterial colonization in the liver, kidney, spleen, and lung and in downregulating PECAM-1, CD99, and CD99L2 expression in endothelial cells and leukocytes. IMPORTANCE P. gingivalis is associated with a variety of systemic diseases and colonizes in distal locations in the body. Here, we found that P. gingivalis gingipains degrade PECAM-1 to promote bacterial penetration while simultaneously reducing leukocyte TEM capacity. A similar phenomenon was also observed in a mouse model. These findings established P. gingivalis gingipains as the key virulence factor in modulating the permeability of the vascular barrier and TEM processes, which may provide a new rationale for the distal colonization of P. gingivalis and its associated systemic diseases.

Methods for Imaging Inflammation and Transendothelial Migration in Vivo and ex Vivo.

Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.

CD31 as a probable responding and gate-keeping protein of the blood-brain barrier and the risk of Alzheimer's disease.

Several studies have shown that an abnormal vascular-immunity link could increase Alzheimer's disease (AD) risk; however, the mechanism is unclear. CD31, also named platelet endothelial cell adhesion molecule (PECAM), is a surface membrane protein of both endothelial and immune cells and plays important roles in the interaction between the vascular and immune systems. In this review, we focus on research regarding CD31 biological actions in the pathological process that may contribute to AD based on the following rationales. First, endothelial, leukocyte and soluble forms of CD31 play multi-roles in regulating transendothelial migration, increasing blood-brain barrier (BBB) permeability and resulting in neuroinflammation. Second, CD31 expressed by endothelial and immune cells dynamically modulates numbers of signaling pathways, including Src family kinases, selected G proteins, and ß-catenin which in turn affect cell-matrix and cell-cell attachment, activation, permeability, survival, and ultimately neuronal cell injury. In endothelia and immune cells, these diverse CD31-mediated pathways act as a critical regulator in the immunity-endothelia-brain axis, thereby mediating AD pathogenesis in ApoE4 carriers, which is the major genetic risk factor for AD. This evidence suggests a novel mechanism and potential drug target for CD31 in the background of genetic vulnerabilities and peripheral inflammation for AD development and progression.

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration.

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.

Pseudomonas aeruginosa LecB suppresses immune responses by inhibiting transendothelial migration.

Pseudomonas aeruginosa is a Gram-negative bacterium causing morbidity and mortality in immuno-compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen-specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE-cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P. aeruginosa infection.

Nectins and Nectin-like molecules drive vascular development and barrier function.

Angiogenesis, barriergenesis, and immune cell migration are all key physiological events that are dependent on the functional characteristics of the vascular endothelium. The protein family of Nectins and Nectin-like molecules (Necls) is a group of cell adhesion molecules that are widely expressed by different endothelial cell types. The family includes four Nectins (Nectin-1 to -4) and five Necls (Necl-1 to -5) that either interact with each other by forming homo- and heterotypical interactions or bind to ligands expressed within the immune system. Nectin and Necl proteins are mainly described to play a role in cancer immunology and in the development of the nervous system. However, Nectins and Necls are underestimated players in the formation of blood vessels, their barrier properties, and in guiding transendothelial migration of leukocytes. This review summarizes their role in supporting the endothelial barrier through their function in angiogenesis, cell-cell junction formation, and immune cell migration. In addition, this review provides a detailed overview of the expression patterns of Nectins and Necls in the vascular endothelium.

Sialylation regulates neutrophil transepithelial migration, CD11b/CD18 activation, and intestinal mucosal inflammatory function.

Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The ß2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3-linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide-mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.

Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM-1.

Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration "hotspot" regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photoconvertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, the loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in the loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, the loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4th Ig-like dimerization domain are not involved, indicating that intracellular signaling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A.

Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.

LEUKOCYTE-EPITHELIAL INTERACTIONS: A DOUBLE-EDGED SWORD THAT PROTECTS AND INJURES DURING HEALTH AND DISEASE.

Neutrophils (PMNs) play a critical role in innate immunity, yet many pathologic conditions are associated with dysregulated infiltration of PMNs into tissues. In the gut, robust PMN accumulation and migration across the intestinal epithelium closely correlates with clinical symptoms in conditions such as ulcerative colitis. While much is known about how PMNs migrate out of blood vessels, far less is understood about how PMNs traverse epithelial barriers. Until fairly recently, in vitro models of PMN transepithelial migration (TEpM) across cultured intestinal epithelial cell lines provided many of the insights into the molecular basis of TEpM. However, innovative animal models have provided new avenues for investigating in vivo mechanisms regulating PMN TEpM. This report will highlight molecular insights gained from studies on PMN TEpM and provide a rationale for developing tissue targeted strategies directed at reducing pathologic consequences of dysregulated PMN trafficking in the gut.