Neutrophil Transmigration Research Articles

ADAM8 signaling drives neutrophil migration and ARDS severity.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8–/– mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

MiR-15p-5p Mediates the Coordination of ICAM-1 and FAK to Promote Endothelial Cell Proliferation and Migration.

Intercellular adhesion molecule-1 (ICAM-1) in endothelial cells is critical for neutrophil adhesion and transmigration across the endothelium. Focal adhesion kinase (FAK), which controls the turnover of focal adhesion to regulate cell adhesion and migration, plays a role in the resolution of inflammation. However, the coordinated involvement of ICAM-1 and FAK during endothelial inflammation has yet to be elucidated. This study reports that, as part of an inflammatory response, ICAM-1 controls FAK expression in endothelial cells via the microRNA miR-15b-5p. Induction of lung injury by lipopolysaccharide (LPS) resulted in higher levels of FAK expression in inflammatory tissues, while in ICAM-1 knockout mice, FAK expression was reduced in the lungs. FAK expression was also reduced in endothelial cells following ICAM-1 siRNA downregulation. Furthermore, ICAM-1 inhibited miR-15b-5p expression while increasing FAK mRNA and protein expression via binding of miR-15b-5p to the 3' untranslated region (UTR) of FAK. ICAM-1 inhibited miR-15b-5p promoter activity and hence reduced miR-15b-5p expression. FAK increased endothelial cell proliferation and migration, whereas miR-15b-5p inhibited cell proliferation and migration. These findings indicate that the inflammatory molecule ICAM-1 regulates FAK expression via miR-15b-5p levels, which in turn controls endothelial cell proliferation and migration.

The effect of tranexamic acid dosing regimen on trauma/hemorrhagic shock-related glycocalyx degradation and endothelial barrier permeability: An in vitro model.

Improved outcomes with early tranexamic acid (TXA) following trauma hemorrhagic shock (T/HS) may be related to its antifibrinolytic, as well as anti-inflammatory properties. Previous in vitro studies have shown that early TXA administration protects against T/HS endothelial barrier dysfunction and associated glycocalyx degradation. An intact endothelial glycocalyx may protect against subsequent neutrophil mediated tissue injury. We postulated that early TXA administration would mitigate against glycocalyx damage and resultant neutrophil adherence and transmigration through the endothelial barrier. This was studied in vitro using a microfluidic flow platform. Human umbilical vein endothelial cell monolayers were subjected to control or shock conditions (hypoxia + epinephrine) followed by administration of TXA 90 minutes or 180 minutes later. "Early" TXA administration protected against glycocalyx degradation, biomarkers of increased permeability and the development of a fibrinolytic phenotype. This was associated with decreased neutrophil endothelial adherence and transmigration. There were no differences in low versus high TXA concentrations. The protective effects were only significant with "early" TXA administration. There was a concentration and temporal effect of TXA administration on endothelial glycocalyx degradation. This was associated with "vascular leakiness" as indexed by the relative ratio of Ang-2/1 and polymorphonuclear neutrophil transmigration. Tranexamic acid if administered in patients with T/HS should be administered "early"; this includes in the prehospital setting.

Peripheral serotonin lacks effects on endothelial adhesion molecule expression in acute inflammation

BackgroundPeripheral, non‐neuronal serotonin promotes the recruitment of neutrophils to sites of acute inflammation and tissue damage. Direct effects of serotonin on neutrophil function were shown to be involved. However, the influence of serotonin on the endothelial counterpart is unknown. ObjectivesTo investigate whether serotonin alters the function of endothelial cells in leukocyte recruitment during acute inflammation. MethodsWe used two murine models of acute inflammation: intraperitoneal lipopolysaccharide (LPS) injection and mesenteric ischemia/reperfusion injury (I/R). To study effects of peripheral serotonin, leukocyte recruitment and endothelial adhesion molecule expression were compared in wild type (WT) and tryptophan hydroxylase 1 deficient (Tph1−/−) mice, which are unable to synthesize peripheral serotonin. ResultsAs expected, neutrophil transmigration into the peritoneal cavity following LPS injection was impaired in Tph1−/− mice. Abdominal blood vessels, however, showed no difference in adhesion molecule expression. In the early reperfusion phase after mesenteric I/R, the number of rolling leukocytes was significantly lower in Tph1−/− compared to WT. In line with the LPS model, endothelial adhesion molecule expression was independent of serotonin. In vitro experiments using human umbilical vein endothelial cells (HUVECs) confirmed that serotonin does not affect endothelial adhesion molecules. ConclusionsThe inflammatory release of peripheral serotonin is dispensable for the regulation of endothelial adhesion molecules.

Actin-Associated Eplin Isoforms Specifically Control Neutrophil Transmigration

Actin-Associated Eplin Isoforms Specifically Control Neutrophil Transmigration

Neutrophil DREAM Promotes Neutrophil Recruitment in Vascular Inflammation Via Nuclear Factor Kappa B-Dependent and Independent Mechanisms

The interaction between neutrophils and endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. Neutrophil recruitment to inflamed tissues is initiated by rolling on activated ECs through the interactions between P-/E-selectins and their ligands. Subsequently, activated integrins (mainly αLβ2 and αMβ2) and chemokine receptors bind to their ligands on ECs and mediate slow-rolling, adhesion, crawling, and transmigration of neutrophils. Although many neutrophil adhesion receptors have been identified, the regulation of their ligand-binding function remains not fully understood. Using real-time intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM) and their bone marrow chimeric mice, we demonstrated that hematopoietic cell DREAM contributes to neutrophil recruitment to sites of vascular inflammation induced by TNF-α- but not a G protein-coupled receptor ligand, MIP-2 or fMLP. Our studies using adoptive neutrophil transfers and flow chamber assays revealed that neutrophil DREAM positively regulates the neutrophil recruitment processes under TNF-α-induced inflammatory conditions. Using RNA-seq and biochemical and cell biological studies, we found that neutrophil DREAM upregulates numerous pro-inflammatory molecules and down-regulates anti-inflammatory molecules after TNF-α treatment. In particular, neutrophil DREAM repressed expression of A20, a negative regulator of NF-κB signaling, and enhanced phosphorylation of IκB kinase (IKK) in response to TNF-α, suggesting the role of neutrophil DREAM in NF-κB activity. Furthermore, we observed that DREAM deletion and IKK inhibition significantly diminishes the ligand-binding activity of β2 integrins in neutrophils after short-term treatment with TNF-α and that deletion of neutrophil DREAM does not affect the expression of other neutrophil adhesion receptors, such as PSGL-1, L-selectin, CD44, CXCR2, and CXCR4. As assessed by flow cytometry using conformation-specific reporter antibodies, knockdown of DREAM in neutrophil-like HL-60 cells decreased TNF-α-induced activation of β2 integrins. Neutrophil DREAM promoted degranulation through IKK-mediated SNAP-23 phosphorylation after short-term treatment with TNF-α, implying the role of neutrophil DREAM-IKK signaling in NF-κB-independent signaling. Using intravital microscopy with Berkeley mice (a mouse model of sickle cell disease) deficient in hematopoietic or nonhematopoietic DREAM, we demonstrated that hematopoietic cell DREAM is crucial for inducing intravascular cell-cell aggregation and vaso-occlusive events in microvessels following the TNF-α challenge. Furthermore, infusion of DREAM KO neutrophils, compared with WT neutrophils, significantly reduced neutrophil recruitment and vaso-occlusive events in TNF-α-challenged SCD mice. These results demonstrate that neutrophil DREAM positively regulates β2 integrin function and promotes neutrophil recruitment during sterile inflammation via NF-κB-dependent and independent mechanisms. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases. DisclosuresNo relevant conflicts of interest to declare.

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

Immunomodulation of innate immunity by brachial plexus blockade: a crossover study

The autonomic nervous system regulates innate immune function in response to afferent pain signals from injured tissue.1 During acute inflammation, vagal release of acetylcholine suppresses the expression of CD11b, a surface integrin regulating neutrophil transmigration to injured tissue. Whether regional anaesthesia modulates innate neuroimmune control has not been tested. We tested the hypothesis that brachial plexus block modulates the innate immune response to ischaemic tissue injury.

CD47 in the Brain and Neurodegeneration: An Update on the Role in Neuroinflammatory Pathways.

CD47 is a receptor belonging to the immunoglobulin (Ig) superfamily and broadly expressed on cell membranes. Through interactions with ligands such as SIRPα, TSP-1, integrins, and SH2-domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1), CD47 regulates numerous functions like cell adhesion, proliferation, apoptosis, migration, homeostasis, and the immune system. In this aspect, previous research has shown that CD47 modulates phagocytosis via macrophages, the transmigration of neutrophils, and the activation of T-cells, dendritic cells, and B-cells. Moreover, several studies have reported the increased expression of the CD47 receptor in a variety of diseases, including acute lymphoblastic leukemia (ALL), chronic myeloid leukemia, non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM), bladder cancer, acute myeloid leukemia (AML), Gaucher disease, Multiple Sclerosis and stroke among others. The ubiquitous expression of the CD47 cell receptor on most resident cells of the CNS has previously been established through different methodologies. However, there is little information concerning its precise functions in the development of different neurodegenerative pathologies in the CNS. Consequently, further research pertaining to the specific functions and roles of CD47 and SIRP is required prior to its exploitation as a druggable approach for the targeting of various neurodegenerative diseases that affect the human population. The present review attempts to summarize the role of both CD47 and SIRP and their therapeutic potential in neurodegenerative disorders.

P259 Safety, tolerability and pharmacokinetics of BT051, an oral inhibitor of neutrophil migration and activation in clinical development for Inflammatory Bowel Disease

Abstract Background BT051 is an oral, locally acting inhibitor of neutrophil trafficking and activation in clinical development for the treatment of patients with Inflammatory Bowel Disease (IBD). Herein we report the results of this first-in-human study of BT051 in healthy subjects. Methods This was a phase 1, randomised, double-blind, placebo-controlled, single-centre, single ascending dose (SAD) study. Healthy subjects (n=50) were randomised into 5 SAD cohorts to receive BT051 (100, 300, 700, 1500 or 3500 mg) or placebo (8 active:2 placebo in each cohort). A safety review committee evaluated any dose-limiting adverse events (AEs) through Day 3 before proceeding to the next cohort. Samples for pharmacokinetic (PK) analysis were collected from blood, stool and urine predose and up to 48 hours postdose; stool samples for PK analysis were also collected on Day 7. An exploratory endpoint of systemic immunosuppression was performed by assessing cytokine secretion induced from T-cells in subjects’ peripheral blood mononuclear cells (PBMC) at 4 and 24 hours postdose. Results Fifty healthy subjects were dosed with BT051 (n=40) or placebo (n=10). No dose-limiting toxicities, serious AEs or study discontinuations due to AEs were observed. Nine (22.5%) BT051 subjects and 2 (20%) placebo subjects experienced at least 1 mild or moderate AE with only vessel puncture site pain and constipation reported in more than 1 subject (n=2). Mean % of dose excreted in stool from partial collection ranged between 11.3–26.3%. At all doses tested, concentrations of BT051 in the large intestine were estimated to exceed >20x the threshold for inhibition of neutrophil transmigration and activation in vitro (1 μM) and continued to be detected out to 7 days postdose. Generally, blood concentrations of BT051 were undetectable except in two subjects at one time point each: one subject at 24 hours postdose who received 700 mg BT051 (70 ng/mL) and another subject at 12 hours postdose who received 1500 mg BT051 (8 ng/mL). Mean % of dose excreted in urine through 48 hours postdose ranged between 0.01–0.03%. No evidence of systemic T-cell immunosuppression was observed at any dose level. Conclusion Single ascending doses up to 3500 mg of BT051 were safe and well-tolerated in healthy subjects. Stool concentrations out to 7 days postdose that exceed the expected efficacious threshold, minimal to no systemic exposure and the lack of systemic immunosuppression support the continued development of BT051 as a gut-targeted therapy for patients with IBD.

Transmigration of Neutrophils From Patients With Familial Mediterranean Fever Causes Increased Cell Activation.

Familial Mediterranean fever (FMF) is caused by pyrin-encoding MEFV gene mutations and characterized by the self-limiting periods of intense inflammation, which are mainly mediated by a massive influx of polymorphonuclear neutrophils (PMNs) into the inflamed sites. Perturbation of actin polymerization by different pathogens was shown to activate the pyrin inflammasome. Our aim was to test whether cytoskeletal dynamics in the absence of pathogens may cause abnormal activation of PMNs from FMF patients. We also aimed to characterize immunophenotypes of circulating neutrophils and their functional activity. Circulating PMNs displayed heterogeneity in terms of cell size, granularity and immunophenotypes. Particularly, PMNs from the patients in acute flares (FMF-A) exhibited a characteristic of aged/activated cells (small cell size and granularity, up-regulated CXCR4), while PMNs form the patients in remission period (FMF-R) displayed mixed fresh/aged cell characteristics (normal cell size and granularity, up-regulated CD11b, CD49d, CXCR4, and CD62L). The findings may suggest that sterile tissue-infiltrated PMNs undergo reverse migration back to bone marrow and may explain why these PMNs do not cause immune-mediated tissue damage. A multidirectional expression of FcγRs on neutrophils during acute flares was also noteworthy: up-regulation of FcγRI and down-regulation of FcγRII/FcγRIII. We also observed spontaneous and fMPL-induced activation of PMNs from the patients after transmigration through inserts as seen by the increased expression of CD11b and intracellular expression of IL-1β. Our study suggests heightened sensitivity of mutated pyrin inflammasome towards cytoskeletal modifications in the absence of pathogens.

Reconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics.

Alveolar-capillary basement membrane (BM) is ultra-thin (<2 µm) extracellular matrix that maintains integral epithelial-endothelial cell layers. In vitro reconstructions of alveolar-capillary barrier supported on synthetic scaffolds closely resembling the fibrous and ultra-thin natural BM are essential in mimicking the lung pathophysiology. Although BM topology and dimensions are well known to significantly influence cellular behavior, conventionally used BM mimics fail to recreate this natural niche. To overcome this, electrospun ultra-thin 2 µm poly(caprolactone) (PCL) nanofibrous mesh is used to establish an alveolar-capillary barrier model of lung endothelial/epithelial cells. Transepithelial electrical resistance (TEER) and permeability studies reveal integral tight junctions and improved mass transport through the highly porous PCL meshes compared to conventional dense membranes with etched pores. The chemotaxis of neutrophils is shown across the barrier in presence of inflammatory response that is naturally impeded in confined regions. Conventional requirement of 3 µm or larger pore size can lead to barrier disruption due to epithelial/endothelial cell invasion. Despite high porosity, the interconnected BM mimic prevents barrier disruption and allows neutrophil transmigration, thereby demonstrating the physiological relevance of the thin nanofibrous meshes. It is envisioned that these bipolar cultured barriers would contribute to an organ-level in vitro model for pathological disease, environmental pollutants, and nanotoxicology.

Impact of resolvin mediators in the immunopathology of diabetes and wound healing

ABSTRACT Introduction: Wound healing in diabetes may be delayed by persistent wound infection due to deficient immune and cellular response to tissue injury. Hyperglycemia due to decreased insulin availability and increased insulin resistance affects the immune response of the body. Accumulation of inflammatory immune cells and pro-inflammatory cytokines results in chronic inflammation and an altered resolution and remodeling phase of wound healing. Areas covered: Pro-resolving mediators called ‘resolvins’ target the resolution phase of wound healing and are becoming an area of increased interest. Resolvins stimulate self-limited innate immune responses and enhance innate microbial killing and clearance. Resolvins resolve inflammation by decreasing neutrophil infiltration and transmigration, increasing the phagocytic activity of macrophages, decreasing adipose tissue macrophages, downregulating platelet activation, suppressing nuclear factor-kappa beta activation, promoting the apoptosis of polymorphonuclear leukocytes, and improving insulin sensitivity. This review discusses the role of resolvins in diabetic wound healing and potential therapeutic strategies. The review is based on a literature search of PubMed and the Web of Science restricted to publications between January 2001 and October 2020. Expert opinion: There is increasing support for the use of resolvins in clinical applications related to diabetes and wound healing. Further research will help clarify this potential.

E-selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity.

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.

β2 Integrin Regulation of Neutrophil Functional Plasticity and Fate in the Resolution of Inflammation.

Neutrophils act as the first line of cellular defense against invading pathogens or tissue injury. Their rapid recruitment into inflamed tissues is critical for the elimination of invading microorganisms and tissue repair, but is also capable of inflicting damage to neighboring tissues. The β2 integrins and Mac-1 (CD11b/CD18, αMβ2 or complement receptor 3) in particular, are best known for mediating neutrophil adhesion and transmigration across the endothelium and phagocytosis of microbes. However, Mac-1 has a broad ligand recognition property that contributes to the functional versatility of the neutrophil population far beyond their antimicrobial function. Accumulating evidence over the past decade has demonstrated roles for Mac-1 ligands in regulating reverse neutrophil transmigration, lifespan, phagocytosis-induced cell death, release of neutrophil extracellular traps and efferocytosis, hence extending the traditional β2 integrin repertoire in shaping innate and adaptive immune responses. Understanding the functions of β2 integrins may partly explain neutrophil heterogeneity and may be instrumental to develop novel therapies specifically targeting Mac-1-mediated pro-resolution actions without compromising immunity. Thus, this review details novel insights on outside-in signaling through β2 integrins and neutrophil functional heterogeneity pertinent to the resolution of inflammation.

Acinetobacter baumannii LOS Regulate the Expression of Inflammatory Cytokine Genes and Proteins in Human Mast Cells.

Herein, we investigated the effect of bacterial lipooligosaccharides (LOS), from Acinetobacter baumannii, on the expression of pro-inflammatory genes that play an essential role in bacterial clearance. LAD2 human mast cells were stimulated with LOS derived from two strains of A. baumannii—ATCC 19606 and MDRA T14. LOS exposure induced the expression of genes for pro-inflammatory mediators, including TNF-α, IL-8, LTC4S, CCL4, and TLR4. The mRNA expression levels of a majority of the pro-inflammatory genes, except TLR4, in A. baumannii-LOS stimulated mast cells were increased. Moreover, co-culture of neutrophils with the supernatant obtained from LOS (ATCC 19606 and MDRA T14)-induced LAD2 cells increased the transmigration of neutrophils, which plays a critical role in the early protection against bacterial infections. The results of the present study suggest that LOS could be involved in the pathogenicity of A. baumannii by inducing inflammatory responses via mast cells and that IL-8 is involved in recruiting neutrophils in response to bacterial invasion.

Neutrophil-Induced Liver Injury and Interactions Between Neutrophils and Liver Sinusoidal Endothelial Cells.

Neutrophils are the most abundant type of leukocytes with diverse functions in immune defense including production of reactive oxygen species, bacteriocidal proteins, neutrophil extracellular traps, and pro-inflammatory mediators. However, aberrant accumulation of neutrophils in host tissues and excessive release of bacteriocidal compounds can lead to unexpected injury to host organs. Neutrophil-mediated liver injury has been reported in various types of liver diseases including liver ischemia/reperfusion injury, nonalcoholic fatty liver disease, endotoxin-induced liver injury, alcoholic liver disease, and drug-induced liver injury. Yet the mechanisms of neutrophil-induced hepatotoxicity in different liver diseases are complicated. Current knowledge of these mechanisms are summarized in this review. In addition, a substantial body of evidence has emerged showing that liver sinusoidal endothelial cells (LSECs) participate in several key steps of neutrophil-mediated liver injury including neutrophil recruitment, adhesion, transmigration, and activation. This review also highlights the current understanding of the interactions between LSECs and neutrophils in liver injury. The future challenge is to explore new targets for selectively interfering neutrophil-induced liver injury without impairing host defense function against microbial infection. Further understanding the role of LSECs in neutrophil-induced hepatotoxicity would aid in developing more selective therapeutic approaches for liver disease.

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

During inflammation, neutrophils are one of the first responding cells of innate immunity, contributing to a fast clearance of infection and return to homeostasis. However, excessive neutrophil infiltration accelerates unsolicited disproportionate inflammation for instance in autoimmune diseases such as rheumatoid arthritis. The transient-receptor-potential channel-kinase TRPM7 is an essential regulator of immune system homeostasis. Naïve murine T cells with genetic inactivation of the TRPM7 enzyme, due to a point mutation at the active site, are unable to differentiate into pro-inflammatory T cells, whereas regulatory T cells develop normally. Moreover, TRPM7 is vital for lipopolysaccharides (LPS)-induced activation of murine macrophages. Within this study, we show that the channel-kinase TRPM7 is functionally expressed in neutrophils and has an important impact on neutrophil recruitment during inflammation. We find that human neutrophils cannot transmigrate along a CXCL8 chemokine gradient or produce reactive oxygen species in response to gram-negative bacterial lipopolysaccharide LPS, if TRPM7 channel or kinase activity are blocked. Using a recently identified TRPM7 kinase inhibitor, TG100-115, as well as murine neutrophils with genetic ablation of the kinase activity, we confirm the importance of both TRPM7 channel and kinase function in murine neutrophil transmigration and unravel that TRPM7 kinase affects Akt1/mTOR signaling thereby regulating neutrophil transmigration and effector function. Hence, TRPM7 represents an interesting potential target to treat unwanted excessive neutrophil invasion.

Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria.IMPORTANCE Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite Plasmodium falciparum The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.

Triple co-culture and perfusion bioreactor for studying the interaction between Neisseria gonorrhoeae and neutrophils: A novel 3D tissue model for bacterial infection and immunity

Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.