The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Mechanistic analysis and significance of sphingomyelinase-mediated decreases in transepithelial CFTR currents in nHBEs.

Loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). In the lungs, this manifests as immune cell infiltration and bacterial infections, leading to tissue destruction. Previous work has determined that acute bacterial sphingomyelinase (SMase) decreases CFTR function in bronchial epithelial cells from individuals without CF (nHBEs) and with CF (cfHBEs, homozygous ΔF508-CFTR mutation). This study focuses on exploring the mechanisms underlying this effect. SMase increased the abundance of dihydroceramides, a result mimicked by blockade of ceramidase enzyme using ceranib-1, which also decreased CFTR function. The SMase-mediated inhibitory mechanism did not involve the reduction of cellular CFTR abundance or removal of CFTR from the apical surface, nor did it involve the activation of 5' adenosine monophosphate-activated protein kinase. In order to determine the pathological relevance of these sphingolipid imbalances, we evaluated the sphingolipid profiles of cfHBEs and cfHNEs (nasal) as compared to non-CF controls. Sphingomyelins, ceramides, and dihydroceramides were largely increased in CF cells. Correction of ΔF508-CFTR trafficking with VX445 + VX661 decreased some sphingomyelins and all ceramides, but exacerbated increases in dihydroceramides. Additional treatment with the CFTR potentiator VX770 did not affect these changes, suggesting rescue of misfolded CFTR was sufficient. We furthermore determined that cfHBEs express more acid-SMase protein than nHBEs. Lastly, we determined that airway-like neutrophils, which are increased in the CF lung, secrete acid-SMase. Identifying the mechanism of SMase-mediated inhibition of CFTR will be important, given the imbalance of sphingolipids in CF cells and the secretion of acid-SMase from cell types relevant to CF.

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T-cell migration into tumors.

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.

Trypanosoma cruzi Exploits E- and P-Selectins To Migrate Across Endothelial Cells and Extracellular Matrix Proteins.

The Chagas disease parasite Trypanosoma cruzi must extravasate to home in on susceptible cells residing in most tissues. It remains unknown how T. cruzi undertakes this crucial step of its life cycle. We hypothesized that the pathogen exploits the endothelial cell programming leukocytes use to extravasate to sites of inflammation. Transendothelial migration (TEM) starts after inflammatory cytokines induce E-selectin expression and P-selectin translocation on endothelial cells (ECs), enabling recognition by leukocyte ligands that engender rolling cell adhesion. Here, we show that T. cruzi upregulates E- and P-selectins in cardiac ECs to which it binds in a ligand-receptor fashion, whether under static or shear flow conditions. Glycoproteins isolated from T. cruzi (TcEx) specifically recognize P-selectin in a ligand-receptor interaction. As with leukocytes, binding of P-selectin to T. cruzi or TcEx requires sialic acid and tyrosine sulfate, which are pivotal for downstream migration across ECs and extracellular matrix proteins. Additionally, soluble selectins, which bind T. cruzi, block transendothelial migration dose dependently, implying that the pathogen bears selectin-binding ligand(s) that start transmigration. Furthermore, function-blocking antibodies against E- and P-selectins, which act on endothelial cells and not T. cruzi, are exquisite in preventing TEM. Thus, our results show that selectins can function as mediators of T. cruzi transendothelial transmigration, suggesting a pathogenic mechanism that allows homing in of the parasite on targeted tissues. As selectin inhibitors are sought-after therapeutic targets for autoimmune diseases and cancer metastasis, they may similarly represent a novel strategy for Chagas disease therapy.

Platelet and Erythrocyte Extravasation across Inflamed Corneal Venules Depend on CD18, Neutrophils, and Mast Cell Degranulation.

Platelet extravasation during inflammation is under-appreciated. In wild-type (WT) mice, a central corneal epithelial abrasion initiates neutrophil (PMN) and platelet extravasation from peripheral limbal venules. The same injury in mice expressing low levels of the ß2-integrin, CD18 (CD18hypo mice) shows reduced platelet extravasation with PMN extravasation apparently unaffected. To better define the role of CD18 on platelet extravasation, we focused on two relevant cell types expressing CD18: PMNs and mast cells. Following corneal abrasion in WT mice, we observed not only extravasated PMNs and platelets but also extravasated erythrocytes (RBCs). Ultrastructural observations of engorged limbal venules showed platelets and RBCs passing through endothelial pores. In contrast, injured CD18hypo mice showed significantly less venule engorgement and markedly reduced platelet and RBC extravasation; mast cell degranulation was also reduced compared to WT mice. Corneal abrasion in mast cell-deficient (KitW-sh/W-sh) mice showed less venule engorgement, delayed PMN extravasation, reduced platelet and RBC extravasation and delayed wound healing compared to WT mice. Finally, antibody-induced depletion of circulating PMNs prior to corneal abrasion reduced mast cell degranulation, venule engorgement, and extravasation of PMNs, platelets, and RBCs. In summary, in the injured cornea, platelet and RBC extravasation depends on CD18, PMNs, and mast cell degranulation.

Proteomic Analysis Revealed the Characteristics of Key Proteins Involved in the Regulation of Inflammatory Response, Leukocyte Transendothelial Migration, Phagocytosis, and Immune Process during Early Lung Blast Injury.

Previous studies found that blast injury caused a significant increased expression of interleukin-1, IL-6, and tumor necrosis factor, a significant decrease in the expression of IL-10, an increase in Evans blue leakage, and a significant increase in inflammatory cell infiltration in the lungs. However, the molecular characteristics of lung injury at different time points after blast exposure have not yet been reported. Therefore, in this study, tandem mass spectrometry (TMT) quantitative proteomics and bioinformatics analysis were used for the first time to gain a deeper understanding of the molecular mechanism of lung blast injury at different time points. Forty-eight male C57BL/6 mice were randomly divided into six groups: control, 12 h, 24 h, 48 h, 72 h, and 1 w after low-intensity blast exposure. TMT quantitative proteomics and bioinformatics analysis were performed to analyze protein expression profiling in the lungs from control and blast-exposed mice, and differential protein expression was verified by Western blotting. The results demonstrated that blast exposure induced severe lung injury, leukocyte infiltration, and the production of inflammatory factors in mice. After analyzing the expression changes in global proteins and inflammation-related proteomes after blast exposure, the results showed that a total of 6861 global proteins and 608 differentially expressed proteins were identified, of which 215, 128, 187, 232, and 65 proteins were identified at 12 h, 24 h, 48 h, 72 h, and 1 week after blast exposure, respectively. Moreover, blast exposure-induced 177 differentially expressed proteins were associated with inflammatory responses, which were enriched in the inflammatory response regulation, leukocyte transendothelial migration, phagocytosis, and immune response. Therefore, blast exposure may induce early inflammatory response of lung tissue by regulating the expression of key proteins in the inflammatory process, suggesting that early inflammatory response may be the initiating factor of lung blast injury. These data can provide potential therapeutic candidates or approaches for the development of future treatment of lung blast injury.

Uveal melanoma cells use ameboid and mesenchymal mechanisms of cell motility crossing the endothelium.

Uveal melanomas (UMs) are malignant cancers arising from the pigmented layers of the eye. UM cells spread through the bloodstream, and circulating UM cells are detectable in patients before metastases appear. Extravasation of UM cells is necessary for formation of metastases, and transendothelial migration (TEM) is a key step in extravasation. UM cells execute TEM via a stepwise process involving the actin-based processes of ameboid blebbing and mesenchymal lamellipodial protrusion. UM cancers are driven by oncogenic mutations that activate Gαq/11, and this activates TRIO, a guanine nucleotide exchange factor for RhoA and Rac1. We found that pharmacologic inhibition of Gαq/11 in UM cells reduced TEM. Inhibition of the RhoA pathway blocked amoeboid motility but led to enhanced TEM; in contrast, inhibition of the Rac1 pathway decreased mesenchymal motility and reduced TEM. Inhibition of Arp2/3 complex allowed cells to transmigrate without intercalation, a direct mechanism similar to the one often displayed by immune cells. BAP1-deficient (+/-) UM subclones displayed motility behavior and increased levels of TEM, similar to the effects of RhoA inhibitors. We conclude that RhoA and Rac1 signaling pathways, downstream of oncogenic Gαq/11, combine with pathways regulated by BAP1 to control the motility and transmigration of UM cells.

Reversal of Pathogen-Induced Barrier Defects in Intestinal Epithelial Cells by Contra-pathogenicity Agents.

BACKGROUND: Environmental enteropathy (EE) is associated with stunting, impairment of responses to oral vaccines, and other adverse health consequences in young children throughout the developing world. EE is characterized by chronic low-grade intestinal inflammation and disrupted epithelial barrier integrity, partly resulting from dysregulation of tight junction proteins, observed in other enteropathies such as celiac disease. During EE, this dysregulation of tight junction expression amplifies translocation of pathogenic bacteria across the intestinal mucosa. AIMS: The aim was to determine whether enteropathogen-mediated epithelial barrier failure can be ameliorated using contra-pathogenicity therapies. METHODS: Intestinal epithelial barrier damage was assessed in Caco-2 cells incubated with three important enteropathogens identified in EE patients: Enteropathogenic Escherichia coli (EPEC), Citrobacter rodentium (C. rodentium), and Cryptosporidium parvum (C. parvum). Potential therapeutic molecules were tested to detect effects on transepithelial resistance (TER), bacterial translocation (BT), claudin-4 expression, and regulation of the inflammatory cytokine response. RESULTS: All three enteropathogens compared to uninfected cells, reduced TER (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0007), reduced claudin-4 expression, and permitted BT (EPEC; p < 0.0001, C. rodentium; p < 0.0001, C. parvum; p < 0.0003) through the monolayer. Zinc, colostrum, epidermal growth factor, trefoil factor 3, resistin-like molecule-ß, hydrocortisone, and the myosin light chain kinase inhibitor ML7 (Hexahydro-1-[(5-iodo-1-naphthalenyl)sulfonyl]-1H-1,4-diazepine hydrochloride); ML7) improved TER (up to 70%) and decreased BT (as much as 96%). Only zinc demonstrated modest antimicrobial activity. CONCLUSION: The enteropathogens impaired intestinal-epithelial barrier integrity with dysregulation of claudin-4 and increased bacterial translocation. Enteropathogen-mediated damage was reduced using contra-pathogenicity agents which mitigated the effects of pathogens without direct antimicrobial activity.

High Endothelial Venules Accelerate Naive T Cell Recruitment by Tumor Necrosis Factor-Mediated R-Ras Upregulation.

Recruitment of naive T cells to lymph nodes is essential for the development of adaptive immunity. Upon pathogen infection, lymph nodes promptly increase the influx of naive T cells from the circulation in order to screen and prime the T cells. The precise contribution of the lymph node vasculature to the regulation of this process remains unclear. Here we show a role for the Ras GTPase, R-Ras, in the functional adaptation of high endothelial venules to increase naive T cell trafficking to the lymph nodes. R-Ras is transiently up-regulated in the endothelium of high endothelial venules by the inflammatory cytokine tumor necrosis factor (TNF) within 24 hours of pathogen inoculation. TNF induces R-Ras upregulation in endothelial cells via JNK and p38 mitogen-activated protein kinase but not NF-κB. Studies of T cell trafficking found that the loss of function of endothelial R-Ras impairs the rapid acceleration of naive T cell recruitment to the lymph nodes upon inflammation. This defect diminished the ability of naive OT-1 T cells to develop antitumor activity against ovalbumin-expressing melanoma. Proteomic analyses suggest that endothelial R-Ras facilitates TNF-dependent transendothelial migration (diapedesis) of naive T cells by modulating molecular assembly the at T cell-endothelial cell interface. These findings give new mechanistic insights into the functional adaptation of high endothelial venules to accelerate naive T cell recruitment to the lymph nodes.

Once upon a time, inflammation

Inflammation has accompanied humans since their first ancestors appeared on Earth. Aulus Cornelius Celsus (25 BC-50 AD), a Roman encyclopedist, offered a still valid statement about inflammation: "Notae vero inflammationis sunt quatuor: rubor et tumor cum calore and dolore", defining the four cardinal signs of inflammation as redness and swelling with heat and pain. While inflammation has long been considered as a morbid phenomenon, John Hunter (18th century) and Elie Metchnikoff (19th century) understood that it was a natural and beneficial event that aims to address a sterile or an infectious insult. Many other famous scientists and some forgotten ones have identified the different cellular and molecular players, and deciphered the different mechanisms of inflammation. This review pays tribute to some of the giants who made major contributions, from Hippocrates to the late 19th and first half of the 20th century. We particularly address the discoveries related to phagocytes, diapedesis, chemotactism, and fever. We also mention the findings of the various inflammatory mediators and the different approaches designed to treat inflammatory disorders.(AU)

HIF-1α is involved in blood-brain barrier dysfunction and paracellular migration of bacteria in pneumococcal meningitis.

Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae, leading to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from blood to the brain across the blood-cerebrospinal fluid barrier or the blood-brain barrier (BBB). The underlying mechanisms are still poorly understood. Current treatment strategies include adjuvant dexamethasone for inflammation and cerebral edema, followed by antibiotics. The success of dexamethasone is however inconclusive, necessitating new therapies for controlling edema, the primary reason for neurological complications. Since we have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is involved in transmigration of pathogens across the BBB. In human, murine meningitis brain samples, HIF-1α activation was observed by immunohistochemistry. S. pneumoniae infection in brain endothelial cells (EC) resulted in in vitro upregulation of HIF-1α/VEGF (Western blotting/qRT-PCR) associated with increased paracellular permeability (fluorometry, impedance measurements). This was supported by bacterial localization at cell-cell junctions in vitro and in vivo in brain ECs from mouse and humans (confocal, super-resolution, electron microscopy, live-cell imaging). Hematogenously infected mice showed increased permeability, S. pneumoniae deposition in the brain, along with upregulation of genes in the HIF-1α/VEGF pathway (RNA sequencing of brain microvessels). Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells or using primary brain ECs from HIF-1α knock-out mice revealed reduced endothelial permeability and transmigration of S. pneumoniae. Therapeutic rescue using the HIF-1α inhibitor echinomycin resulted in increased survival and improvement of BBB function in S. pneumoniae-infected mice. We thus demonstrate paracellular migration of bacteria across BBB and a critical role for HIF-1α/VEGF therein and hence propose targeting this pathway to prevent BBB dysfunction and ensuing brain damage in infections.

Cleaved endocan acts as a biologic competitor of endocan in the control of ICAM-1-dependent leukocyte diapedesis.

Dysregulated leukocyte diapedesis is a major contributor to acute severe inflammatory states like sepsis and acute respiratory distress syndrome, which are common conditions in critically ill subjects. Endocan is a circulating proteoglycan that binds to the leukocyte integrin LFA-1 and blocks its interaction with its endothelial ligand ICAM-1, subsequently leading to the inhibition of leukocyte recruitment. Recent data have highlighted the hypothetic role of p14, endocan's major catabolite found in the bloodstream of septic patients, as a potential antagonist of endocan, thus participating in the regulation of acute inflammation. We hereby characterize the role of p14 as a biologic competitor of endocan, through assessment of its molecular interactions with LFA-1, endocan, and ICAM-1, as well as its effects on human leukocyte trafficking. Using immunodetection assay, we report that p14 can bind to LFA-1, thus inhibiting the interaction between LFA-1 and endocan, which in turn leads to the restoration of the ICAM-1/LFA-1 interaction. In primary human T cells trafficking assays, we underline the absence of effect of p14 on ICAM-1-dependent adhesion and migration, as well as on transendothelial migration. However, in those models, p14 reverses the antimigratory effect of endocan. To conclude, our study supports the hypothesis of an antagonistic role of p14 versus endocan in its effect on the LFA-1/ICAM-1-dependent human leukocyte recruitment.

ß2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of ß2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of ß2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two ß2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting ß2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

The Choroid Plexus Is Permissive for a Preactivated Antigen-Experienced Memory B-Cell Subset in Multiple Sclerosis.

The role of B cells in multiple sclerosis (MS) is increasingly recognized. B cells undergo compartmentalized redistribution in blood and cerebrospinal fluid (CSF) during active MS, whereby memory B cells accumulate in the CSF. While B-cell trafficking across the blood-brain barrier has been intensely investigated, cellular diapedesis through the blood-CSF barrier (BCSFB) is incompletely understood. To investigate how B cells interact with the choroid plexus to transmigrate into the CSF we isolated circulating B cells from healthy donors (HC) and MS patients, utilized an inverted cell culture filter system of human choroid plexus papilloma (HIBCPP) cells to determine transmigration rates of B-cell subsets, immunofluorescence, and electron microscopy to analyze migration routes, and qRT-PCR to determine cytokines/chemokines mediating B-cell diapedesis. We also screened the transcriptome of intrathecal B cells from MS patients. We found, that spontaneous transmigration of HC- and MS-derived B cells was scant, yet increased significantly in response to B-cell specific chemokines CXCL-12/CXCL-13, was further boosted upon pre-activation and occurred via paracellular and transcellular pathways. Migrating cells exhibited upregulation of several genes involved in B-cell activation/migration and enhanced expression of chemokine receptors CXCR4/CXCR5, and were predominantly of isotype class switched memory phenotype. This antigen-experienced migratory subset displayed more pronounced chemotactic activities in MS than in HC and was retrieved in intrathecal B cells from patients with active MS. Trafficking of class-switched memory B cells was downscaled in a small cohort of natalizumab-exposed MS patients and the proportions of these phenotypes were reduced in peripheral blood yet were enriched intrathecally in patients who experienced recurrence of disease activity after withdrawal of natalizumab. Our findings highlight the relevance of the BCSFB as important gate for the entry of potentially harmful activated B cells into the CSF.

Optimized cultivation of porcine choroid plexus epithelial cells, a blood-cerebrospinal fluid barrier model, for studying granulocyte transmigration.

The blood-cerebrospinal fluid barrier (BCSFB) plays important roles during the transport of substances into the brain, the pathogenesis of central nervous system (CNS) diseases, and neuro-immunological processes. Along these lines, transmigration of granulocytes across the blood-cerebrospinal fluid (CSF) barrier (BCSFB) is a hallmark of inflammatory events in the CNS. Choroid plexus (CP) epithelial cells are an important tool to generate in vitro models of the BCSFB. A porcine CP epithelial cell line (PCP-R) has been shown to present properties of the BCSFB, including a strong barrier function, when cultivated on cell culture filter inserts containing a membrane with 0.4 µm pore size. For optimal analysis of pathogen and host immune cell interactions with the basolateral side of the CP epithelium, which presents the physiologically relevant "blood side", the CP epithelial cells need to be grown on the lower face of the filter in an inverted cell culture insert model, with the supporting membrane possessing a pore size of at least 3.0 µm. Here, we demonstrate that PCP-R cells cultivated in the inverted model on filter support membranes with a pore size of 3.0 µm following a "conventional" protocol grow through the pores and cross the membrane, forming a second layer on the upper face. Therefore, we developed a cell cultivation protocol, which strongly reduces crossing of the membrane by the cells. Under these conditions, PCP-R cells retain important properties of a BCSFB model, as was observed by the formation of continuous tight junctions and a strong barrier function demonstrated by a high transepithelial electrical resistance and a low permeability for macromolecules. Importantly, compared with the conventional cultivation conditions, our optimized model allows improved investigations of porcine granulocyte transmigration across the PCP-R cell layer.

Simultaneous Study of the Recruitment of Monocyte Subpopulations Under Flow In Vitro.

The recruitment of monocytes from the blood to targeted peripheral tissues is critical to the inflammatory process during tissue injury, tumor development and autoimmune diseases. This is facilitated through a process of capture from free flow onto the luminal surface of activated endothelial cells, followed by their adhesion and transendothelial migration (transmigration) into the underlying affected tissue. However, the mechanisms that support the preferential and context-dependent recruitment of monocyte subpopulations are still not fully understood. Therefore, we have developed a method that allows the recruitment of different monocyte subpopulations to be simultaneously visualized and measured under flow. This method, based on time-lapse confocal imaging, allows for the unambiguous distinction between adherent and transmigrated monocytes. Here, we describe how this method can be used to simultaneously study the recruitment cascade of pro-angiogenic and non-angiogenic monocytes in vitro. Furthermore, this method can be extended to study the different steps of recruitment of up to three monocyte populations.

Image-Assisted Microvessel-on-a-Chip Platform for Studying Cancer Cell Transendothelial Migration Dynamics.

With the push to reduce in vivo approaches, the demand for microphysiological models that recapitulate the in vivo settings in vitro is dramatically increasing. Here, we present an extracellular matrix-integrated microfluidic chip with a rounded microvessel of ~100 µm in diameter. Our system displays favorable characteristics for broad user adaptation: simplified procedure for vessel creation, minimised use of reagents and cells, and the ability to couple live-cell imaging and image analysis to study dynamics of cell-microenvironment interactions in 3D. Using this platform, the dynamic process of single breast cancer cells (LM2-4175) exiting the vessel lumen into the surrounding extracellular matrix was tracked. Here, we show that the presence of endothelial lining significantly reduced the cancer exit events over the 15-hour imaging period: there were either no cancer cells exiting, or the fraction of spontaneous exits was positively correlated with the number of cancer cells in proximity to the endothelial barrier. The capability to map the z-position of individual cancer cells within a 3D vessel lumen enabled us to observe cancer cell transmigration 'hot spot' dynamically. We also suggest the variations in the microvessel qualities may lead to the two distinct types of cancer transmigration behaviour. Our findings provide a tractable in vitro model applicable to other areas of microvascular research.

A differentiating neural stem cell-derived astrocytic population mitigates the inflammatory effects of TNF-α and IL-6 in an iPSC-based blood-brain barrier model.

Inflammation can be a risk factor for neurodegenerative diseases such as Alzheimer's disease (AD) and may also contribute to the progression of AD. Here, we sought to understand how inflammation affects the properties of the brain microvascular endothelial cells (BMECs) that compose the blood-brain barrier (BBB), which is impaired in AD. A fully human in vitro BBB model with brain microvascular endothelial cells derived from induced pluripotent stem cells and differentiating neural stem cell (NSC)-derived astrocytic cells was used to investigate the effects of neuroinflammation on barrier function. The cytokines TNF-α and IL-6 directly cause BBB dysfunction measured by a decrease in transendothelial electrical resistance, an increase in sodium fluorescein permeability, and a decrease in cell polarity, providing a link between neuroinflammation and specific aspects of BBB breakdown. An NSC-derived astrocytic cell population was added to the model and secreted cytokines and chemokines were quantified in monoculture and coculture both in the presence and absence of TNF-α and IL-6. Increased concentrations of pro-inflammatory cytokines known to be secreted by astrocytes or endothelial cells such as MCP-1, IL-8, IP-10, MIP-1ß, IL-1 ß, MIG, and RANTES peaked in inflammatory conditions when NSC-astrocytic cells were present. Despite the presence of several pro-inflammatory cytokines, the NSC-derived astrocytic cells mitigated the effects of inflammation measured by a restoration of transendothelial electrical resistance and IgG permeability. These results also suggest a breakdown in transcellular transport that precedes any increase in paracellular permeability in neuroinflammation. This model has the potential to resolve questions about neurodegenerative disease progression and delivery of therapeutics to the brain.

Interaction with an endothelial lumen increases neutrophil lifetime and motility in response to P aeruginosa.

Neutrophil infiltration into tissues is essential for host defense and pathogen clearance. Although many of the signaling pathways involved in the transendothelial migration of neutrophils are known, the role of the endothelium in regulating neutrophil behavior in response to infection within interstitial tissues remains unclear. Here we developed a microscale 3-dimensional (3D) model that incorporates an endothelial lumen, a 3D extracellular matrix, and an intact bacterial source to model the host microenvironment. Using this system, we show that an endothelial lumen significantly increased neutrophil migration toward a source of Pseudomonas aeruginosa Surprisingly, we found neutrophils, which were thought to be short-lived cells in vitro, migrate for up to 24 hours in 3D in the presence of an endothelial lumen and bacteria. In addition, we found that endothelial cells secrete inflammatory mediators induced by the presence of P aeruginosa, including granulocyte-macrophage colony-stimulating factor (GM-CSF), a known promoter of neutrophil survival, and interleukin (IL)-6, a proinflammatory cytokine. We found that pretreatment of neutrophils with a blocking antibody against the IL-6 receptor significantly reduced neutrophil migration to P aeruginosa but did not alter neutrophil lifetime, indicating that secreted IL-6 is an important signal between endothelial cells and neutrophils that mediates migration. Taken together, these findings demonstrate an important role for endothelial paracrine signaling in neutrophil migration and survival.

Pediatric acute lymphoblastic leukemia-Conquering the CNS across the choroid plexus.

Despite the high prevalence of central nervous system (CNS) involvement in relapsing pediatric acute lymphoblastic leukemia (ALL), our understanding of CNS invasion is still vague. As lymphoblasts have to overcome the physiological blood-CNS barriers to enter the CNS, we investigated the cellular interactions of lymphoblasts with the choroid plexus (CP) epithelium of the blood-cerebrospinal fluid barrier (BCSFB). Both a precurser B cell ALL (pB-ALL) cell line (SD-1) and a T cell ALL (T-ALL) cell line (P12-Ishikawa) were able to actively cross the CP epithelium in a human in vitro model. We could illustrate a transcellular and (supposedly) paracellular transmigration by 3-dimensional immunofluorescence microscopy as well as electron microscopy. Chemotactic stimulation with CXCL12 during this process led to a significantly increased transmigration and blocking CXCL12/CXCR4-signaling by the CXCR4-inhibitor AMD3100 inhibited this effect. However, CXCR4 expression in primary ALL samples did not correlate to CNS disease, indicating that CXCR4-driven CNS invasion across the BCSFB might be a general property of pediatric ALL. Notably, we present a unique in vitro BCSFB model suitable to study CNS invasion of lymphoblasts in a human setting, providing the opportunity to investigate experimental variables, which may determine CNS disease childhood ALL.