Neutrophil Transepithelial Migration Research Articles

Aspergillus Enhances Eosinophil and Neutrophil Extracellular DNA Trap Formation in Chronic Rhinosinusitis.

Chronic rhinosinusitis (CRS) is characterized by inflammatory cell infiltration in the sinonasal mucosa. Eosinophil and neutrophil extracellular traps (EETs and NETs, respectively) are prominently found in CRS. This study aimed to investigate the effect of airborne fungi, Alternaria alternata and Aspergillus fumigatus, on EET and NET formation. Nasal epithelial cells, eosinophils, and neutrophils were isolated from eosinophilic CRS (ECRS), non-ECRS (NECRS), and healthy control. We determined eosinophil and neutrophil transepithelial migration after fungal treatment. We then determined the release of EETs and NETs by fungi using Sytox Green staining and determined the role of reactive oxygen species (ROS) using ROS inhibitors. We identified more abundant EETs and NETs in ECRS than in NECRS. A. alternata and A. fumigatus enhanced eosinophil and neutrophil transepithelial migration. A. fumigatus strongly induced EET and NET formation in CRS and, simultaneously, suppressed fungal metabolic activity. EET formation in CRS is associated with nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase and NET formation with NADPH-oxidase and mitochondrial ROS. A. fumigatus, but not A. alternata, induced EET and NET formation, and peripheral blood eosinophils and neutrophils exhibited different immune responses against A. fumigatus following the inflammatory status of the host. Aspergillus-fumigatus-induced EET and NET formation plays a crucial role in CRS pathogenesis.

DEL-1, as an anti-neutrophil transepithelial migration molecule, inhibits airway neutrophilic inflammation in asthma.

Neutrophil migration into the airways is a key process in neutrophilic asthma. Developmental endothelial locus-1 (DEL-1), an extracellular matrix protein, is a neutrophil adhesion inhibitor that attenuates neutrophilic inflammation. Levels of DEL-1 were measured in exhaled breath condensate (EBC) and serum in asthma patients by ELISA. DEL-1 modulation of neutrophil adhesion and transepithelial migration was examined in a co-culture model in vitro. The effects of DEL-1-adenoviral vector-mediated overexpression on ovalbumin/lipopolysaccharide (OVA/LPS)-induced neutrophilic asthma were studied in mice in vivo. DEL-1 was primarily expressed in human bronchial epithelial cells and was decreased in asthma patients. Serum DEL-1 concentrations were reduced in patients with severe asthma compared with normal subjects (567.1 ± 75.3 vs. 276.8 ± 29.36 pg/mL, p < .001) and were negatively correlated to blood neutrophils (r = -0.2881, p = .0384) and neutrophil-to-lymphocyte ratio (NLR) (r = -0.5469, p < .0001). DEL-1 concentrations in the EBC of severe asthmatic patients (113.2 ± 8.09 pg/mL) were also lower than normal subjects (193.0 ± 7.61 pg/mL, p < .001) and were positively correlated with the asthma control test (ACT) score (r = 0.3678, p = .0035) and negatively related to EBC IL-17 (r = -0.3756, p = .0131), myeloperoxidase (MPO) (r = -0.5967, p = .0055), and neutrophil elastase (NE) (r = -0.5488, p = .0009) expression in asthma patients. Neutrophil adhesion and transepithelial migration in asthma patients were associated with LFA-1 binding to ICAM-1 and inhibited by DEL-1. DEL-1 mRNA and protein expression in human bronchial epithelial cells were regulated by IL-17. Exogenous DEL-1 inhibited IL-17-enhanced neutrophil adhesion and migration. DEL-1 expression was decreased while neutrophil infiltration was increased in the airway of a murine model of neutrophilic asthma. This was prevented by DEL-1 overexpression. DEL-1 down-regulation leads to increased neutrophil migration across bronchial epithelial cells and is associated with neutrophilic airway inflammation in asthma.

Metabolomic profiling combined with network analysis of serum pharmacochemistry to reveal the therapeutic mechanism of Ardisiae Japonicae Herba against acute lung injury.

Introduction: Acute lung injury (ALI) is a common and devastating respiratory disease associated with uncontrolled inflammatory response and transepithelial neutrophil migration. In recent years, a growing number of studies have found that Ardisiae Japonicae Herba (AJH) has a favorable anti-inflammatory effect. However, its serum material basis and molecular mechanism are still unknown in ALI treatment. In this study, metabolomics and network analysis of serum pharmacochemistry were used to explore the therapeutic effect and molecular mechanism of AJH against lipopolysaccharide (LPS)-induced ALI. Methods: A total of 12 rats for serum pharmacochemistry analysis were randomly divided into the LPS group and LPS + AJH-treated group (treated with AJH extract 20g/kg/d), which were administered LPS (2mg/kg) by intratracheal instillation and then continuously administered for 7days. Moreover, 36 rats for metabolomic research were divided into control, LPS, LPS + AJH-treated (5, 10, and 20g/kg/d), and LPS + dexamethasone (Dex) (2.3 × 10-4g/kg/d) groups. After 1h of the seventh administration, the LPS, LPS + AJH-treated, and LPS + Dex groups were administered LPS by intratracheal instillation to induce ALI. The serum pharmacochemistry profiling was performed by UPLC-Orbitrap Fusion MS to identify serum components, which further explore the molecular mechanism of AJH against ALI by network analysis. Meanwhile, metabolomics was used to select the potential biomarkers and related metabolic pathways and to analyze the therapeutic mechanism of AJH against ALI. Results: The results showed that 71 serum components and 18 related metabolites were identified in ALI rat serum. We found that 81 overlapping targets were frequently involved in AGE-RAGE, PI3K-AKT, and JAK-STAT signaling pathways in network analysis. The LPS + AJH-treated groups exerted protective effects against ALI by reducing the infiltration of inflammatory cells and achieved anti-inflammatory efficacy by significantly regulating the interleukin (IL)-6 and IL-10 levels. Metabolomics analysis shows that the therapeutic effect of AJH on ALI involves 43 potential biomarkers and 14 metabolic pathways, especially phenylalanine, tyrosine, and tryptophan biosynthesis and linoleic acid metabolism pathways, to be influenced, which implied the potential mechanism of AJH in ALI treatment. Discussion: Our study initially elucidated the material basis and effective mechanism of AJH against ALI, which provided a solid basis for AJH application.

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 β2-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.

STAT3 mutation-associated airway epithelial defects in Job syndrome

Job syndrome is a disease of autosomal dominant hyper-IgE syndrome (AD-HIES). Patients harboring STAT3 mutation are particularly prone to airway remodeling and airway infections. Airway epithelial cells play a central role as the first line of defense against pathogenic infection and express high levels of STAT3. This study thus interrogates how AD-HIES STAT3 mutations impact the physiological functions of airway epithelial cells. This study created human airway basal cells expressing 4 common AD-HIES STAT3 mutants (R382W, V463del, V637M, and Y657S). In addition, primary airway epithelial cells were isolated from a patient with Job syndrome who was harboring a STAT3-S560del mutation and from mice harboring a STAT3-V463del mutation. Cell proliferation, differentiation, barrier function, bacterial elimination, and innate immune responses to pathogenic infection were quantitatively analyzed. STAT3 mutations reduce STAT3 protein phosphorylation, nuclear translocation, transcription activity, and protein stability in airway basal cells. As a consequence, STAT3-mutated airway basal cells give rise to airway epithelial cells with abnormal cellular composition and loss of coordinated mucociliary clearance. Notably, AD-HIES STAT3 airway epithelial cells are defective in bacterial killing and fail to initiate vigorous proinflammatory responses and neutrophil transepithelial migration in response to an experimental model of Pseudomonas aeruginosa infection. AD-HIES STAT3 mutations confer numerous abnormalities to airway epithelial cells in cell differentiation and host innate immunity, emphasizing their involvement in the pathogenesis of lung complications in Job syndrome. Therefore, therapies must address the epithelial defects as well as the previously noted immune cell defects to alleviate chronic infections in patients with Job syndrome.

Acidosis regulation of epithelial barrier function and immune signaling

A common but often underappreciated aspect of the inflammatory microenvironment is extracellular acidification. As with other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients. Colonic pH as low as 4 have been reported in IBD patients. In contrast, the pH of a healthy colon is between a pH of 7.0‐7.4. Recently, we identified an adaptive tissue response in intestinal epithelial cells (IEC) which buffered extracellular acidification during neutrophil transepithelial migration, suggesting pH homeostasis is an important aspect of acute inflammation. Extensions of these studies identified a previously undescribed CREB‐dependent pathway for sensing extracellular pH and a unique acidosis gene signature in IEC. The full impact of acidosis on IEC function remains unclear. To better understand the impact of chronic extracellular acidification on IEC function, we investigated changes in IEC immune signaling and barrier function in the setting of acute and chronic exposure to acidosis. We initially examined barrier function in T84 IEC cultured under acidic conditions and observed a significant decrease in barrier function, as determined by transepithelial electrical resistance (TER) and paracellular flux, in cells exposed to acidosis in a pH range of 6.0 – 7.4. Interestingly, following acute acidification, treatment with low pH for less than 8hr, barrier function recovered within 16hr. However, following chronic acidification, treatment with low pH beyond 16hr resulted in an appreciable barrier dysfunction for several days. We extended these studies to examine expression and localization of tight‐junction proteins in IEC exposed to acidosis and observed a time and dose‐dependent decrease in ZO‐1 and E‐cadherin expression, with almost complete loss of expression after 72hr at a pH of 6.0. Likewise, expression and localization of claudins 1, 2, and 4 decreased over time. In parallel to a loss of barrier function, IEC exposed to chronic acidosis revealed a prominent increase in the expression of inflammatory markers including several chemokines (IL‐8, CCL20, TNF) as well as anti‐microbial peptides (mucins and beta‐defensins).Thus, chronic extracellular acidosis induces the loss of IEC tight junction function, while inducing the expression of several pro‐inflammatory chemokines and the production of various anti‐microbial peptides. These results identify extracellular acidification as significant signaling mechanism which induces a loss of epithelial features and the acquisition of a more immune‐like phenotype. Furthering our understandings of the mechanisms involved in acidosis induced phenotype switching in IEC has the potential to uncover novel therapeutic drug targets.

Neutrophil-Epithelial Crosstalk During Intestinal Inflammation.

Neutrophils are the most abundant leukocyte population in the human circulatory system and are rapidly recruited to sites of inflammation. Neutrophils play a multifaceted role in intestinal inflammation, as they contribute to the elimination of invading pathogens. Recently, their role in epithelial restitution has been widely recognized; however, they are also associated with bystander tissue damage. The intestinal epithelium provides a physical barrier to prevent direct contact of luminal contents with subepithelial tissues, which is extremely important for the maintenance of intestinal homeostasis. Numerous studies have demonstrated that transepithelial migration of neutrophils is closely related to disease symptoms and disruption of crypt architecture in inflammatory bowel disease and experimental colitis. There has been growing interest in how neutrophils interact with the epithelium under inflammatory conditions. Most studies focus on the effects of neutrophils on intestinal epithelial cells; however, the effects of intestinal epithelial cells on neutrophils during intestinal inflammation need to be well-established. Based on these data, we have summarized recent articles on the role of neutrophil–epithelial interactions in intestinal inflammation, particularly highlighting the epithelium-derived molecular regulators that mediate neutrophil recruitment, transepithelial migration, and detachment from the epithelium, as well as the functional consequences of their crosstalk. A better understanding of these molecular events may help develop novel therapeutic targets for mitigating the deleterious effects of neutrophils in inflammatory bowel disease.

ICAM-1 and ICAM-2 Are Differentially Expressed and Up-Regulated on Inflamed Pulmonary Epithelium, but Neither ICAM-2 nor LFA-1: ICAM-1 Are Required for Neutrophil Migration Into the Airways In Vivo.

Neutrophil migration into the airways is an important process to fight infection and is mediated by cell adhesion molecules. The intercellular adhesion molecules, ICAM-1 (CD54) and ICAM-2 (CD102) are known ligands for the neutrophil integrins, lymphocyte function associated antigen (LFA)-1 (αLβ2; CD11a/CD18), and macrophage-1 antigen (Mac-1;αMβ2;CD11b/CD18) and are implicated in leukocyte migration into the lung. However, it is ill-defined how neutrophils exit the lung and the role for ICAMs in trans-epithelial migration (TEpM) across the bronchial or alveolar epithelium. We found that human and murine alveolar epithelium expressed ICAM-1, whilst the bronchial epithelium expressed ICAM-2, and both were up-regulated during inflammatory stimulation in vitro and in inflammatory lung diseases such as cystic fibrosis. Although β2 integrins interacting with ICAM-1 and -2 mediated neutrophil migration across human bronchial epithelium in vitro, neither ICAM-2 nor LFA-1 binding of ICAM-1 mediated murine neutrophil migration into the lung or broncho-alveolar space during LPS-induced inflammation in vivo. Furthermore, TEpM of neutrophils themselves resulted in increased epithelial junctional permeability and reduced barrier function in vitro. This suggests that although β2 integrins interacting with ICAMs may regulate low levels of neutrophil traffic in healthy lung or early in inflammation when the epithelial barrier is intact; these interactions may be redundant later in inflammation when epithelial junctions are disrupted and no longer limit TEpM.

Type I interferon enhances γδ intraepithelial lymphocyte migratory behavior via CD47 upregulation

Abstract γδ intraepithelial lymphocytes (IEL) migrate along the basement membrane and into the lateral intracellular space (LIS) between adjacent intestinal epithelial cells (IEC) and is critical to limit microbial translocation across the barrier. Although activated γδ IELs produce interferon (IFN)-α, whether IFNα directly influences γδ IEL migratory behavior remains unknown. To test this, intravital microscopy was performed on GFP γδ T cell reporter mice treated with PBS or IFNα (1 μg, i.v., 3h). We found that γδ IEL track speed was increased 30% and migration into the LIS was enhanced by 75% in IFNα-treated mice compared to controls. To identify candidate genes involved in regulating γδ IEL motility, we performed RNAseq on γδ IELs isolated from control and IFNα-treated mice. As expected, IFNα induced IFN-stimulated gene expression including CD47 (3.5-fold increase), a transmembrane protein involved in neutrophil transepithelial migration. Stimulation of ex vivo cultured γδ IELs with IFNα resulted in a 3-fold increase in CD47 expression compared to unstimulated controls. Next, to investigate whether IFNα enhances γδ IEL motility in vitro, GFP γδ IELs were co-cultured with enteroids expressing membrane tdTomato and treated with 10ng/ml IFNα for 3h. IFNα increased γδ IEL speed (4.5±0.2 vs 3.4±0.2 μm/min; p&amp;lt;0.05) and displacement (47±3.0 μm vs 34±3.1, p&amp;lt;0.05) relative to untreated controls. Addition of anti-CD47 blocking antibody (MIAP301) abrogated the IFNα-mediated increase in γδ IEL migratory behavior. Taken together, these data demonstrate that IFNα enhances γδ IEL motility via a CD47-mediated mechanism, and suggests that CD47 may represent a conserved mechanism of leukocyte migration within the epithelial barrier.

Insights into the impact of inflammatory acidification on the mucosa

Mucosal acidification is an often-underappreciated aspect of mucosal inflammation. Like other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease (IBD) patients, with reports of colonic pH as low as 4. We recently demonstrated that neutrophil (PMN) transepithelial migration rapidly acidifies the microenvironment resulting in inflammatory acidification of the mucosa. Furthermore, we identified an adaptive tissue response in IEC which buffered extracellular acidification during PMN transepithelial migration, suggesting pH homeostasis is important during acute inflammation. Very little is known in regard to the impact of extracellular acidification on the intestinal epithelial cell (IEC) function. To better understand the impact of extracellular pH on IEC cell function, we investigated the impact of extracellular acidosis on IEC gene expression. Guided by an unbiased RNAseq of T84 IEC exposed to low pH, we identified significant induction of several CREB associated genes. Based on this observation, we examined the phosphorylation of CREB under acidic conditions. Under acidic conditions, CREB is rapidly phosphorylated. We observed CREB phosphorylation within 5 minutes of exposure to acidic conditions and peaks at 30 min. Extending these studies we investigated the mechanism(s) involved in acidosis induced CREB phosphorylation. Interestingly, cAMP signaling, a common signaling pathway for CREB phosphorylation, appears to not be involved in acidosis-induced CREB phosphorylation. Under acidic conditions, there was no significant increase in cAMP levels in IEC and acidosis-induced CREB phosphorylation was still observed in T84 IEC treated with cAMP inhibitors. KEGG analysis of the RNAseq data revealed the induction of several MAPK regulatory genes, including members of the NR4A and DUSP families. Based on this observation we examined several key proteins involved in MAPK signaling, ERK1/2 and MSK1. Under acidic conditions ERK1/2 and MSK1 are rapidly phosphorylation and treatment with a MEK1/2 or MSK1 inhibitor significant attenuated CREB phosphorylation under acidic conditions. Further examination revealed CREB phosphorylation signals through the GaI-linked GPCR, GRP31. Finally, we extended our studies in vivo and examined mucosal acidification in a murine model of ileitis, TNFΔARE mice. Using a novel pH reporting E. coli strain, we identified significant mucosal acidification in the distal ileum of TNFΔARE mice when compared to WT mice. Mucosal acidification was positively correlated with the expression of several acidification associated genes; FosB, NR4A1, and CXCL1. These results identify extracellular acidification as a significant signaling mechanism during tissue inflammation. Thus, regulation of GPR31 or MSK1 may serve as new therapeutic targets to limit acidosis signaling during mucosal inflammation.

Neutrophil dysfunction in cystic fibrosis

BackgroundExcessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that target these mechanisms. MethodsBlood was collected from individuals with CF in the outpatient clinic, CF individuals hospitalized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa-induced neutrophil transepithelial migration using micro-optical coherence technology (µOCT). ResultsParticipants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF individuals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p < 0.05). CF neutrophils, especially Hospitalized CF neutrophils, generated significantly larger aggregates around S. aureus particles over time. Hospitalized CF neutrophils were more likely to have dysfunctional swarming (p < 0.01) and less efficient clearing of C. albicans (p < 0.0001). When comparing trans-epithelial migration towards Pseudomonas aeruginosa epithelial infection, Outpatient CF neutrophils displayed an increase in the magnitude of transmigration and adherence to the epithelium (p < 0.05). ConclusionsAdvanced technologies for characterizing CF neutrophil function reveal significantly altered migratory responses, cell-to-cell clustering, and microbe containment. Future investigations will probe mechanistic basis for abnormal responses in CF to identify potential avenues for novel anti-inflammatory therapeutics.

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers. We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Getting Your Neutrophil: Neutrophil Transepithelial Migration in the Lung.

Neutrophil transepithelial migration is a fundamental process that facilitates the rapid trafficking of neutrophils to inflammatory foci and occurs across a diverse range of tissues. For decades there has been widespread interest in understanding the mechanisms that drive this migratory process in response to different pathogens and organ systems. This has led to the successful integration of key findings on neutrophil transepithelial migration from the intestines, lungs, liver, genitourinary tract, and other tissues into a single, cohesive model. However, recent studies have identified organ specific differences in neutrophil transepithelial migration. These findings support a model where the tissue in concert with the pro-inflammatory stimuli dictate a unique collection of signals that drive neutrophil trafficking. This review focuses on the mechanisms that drive neutrophil transepithelial migration in response to microbial infection of a single organ, the lung. Herein we provide a detailed analysis of the adhesion molecules and chemoattractants that contribute to the recruitment of neutrophil into the airways. We also highlight important advances in experimental models for studying neutrophil transepithelial migration in the lung over the last decade.

A Microengineered Airway Lung Chip Models Key Features of Viral-induced Exacerbation of Asthma.

Viral-induced exacerbation of asthma remains a major cause of hospitalization and mortality. New human-relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks and to advance treatment development. Here, we describe a new human-relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium and neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. Specifically, a microengineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a T-helper cell type 2 asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. We observed that the infection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a T-helper cell type 2 microenvironment through exogenous IL-13 stimulation induced features of asthmatic airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High-resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered IL-6, IFN-λ1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, whereas treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. In conclusion, our microengineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts' ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response.

Neutrophil expressed CD47 regulates CD11b/CD18-dependent neutrophil transepithelial migration in the intestine in vivo.

Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.

880 Targeted IL-17RA antagonism ameliorates histological and transcriptomic features of hidradenitis suppurativa: A proof of concept study

Hidradenitis Suppurativa is an autoinflammatory disorder of keratinization with a major inflammatory contribution from the Th17 axis. We have previously shown that in addition to previously described IL-17A & IL-17F; IL-17C is found at higher levels by both IHC and rt-PCR in lesional HS skin. We asked whether the IL-17 axis mediates disease activity by using an IL-17RA antagonist Brodalumab to examine histological and transcriptomic features of disease in 10 individuals with HS. Biopsies as per previously published consensus criteria were taken at Baseline and at Weeks 4 and 12 after treatment with an IL-17RA antagonist. Elevated levels keratinocyte derived genes (S100A9: logFCH =10.19; CXCL1: logFCH=5.46; CXCL8: logFCH=5.2), B-cell associated genes (IGHG3: logFCH=14.49) and cell trafficking chemokines (CXCl13: logFCH=7.62) were significantly elevated compared to normal site matched controls. IL-17RA antagonism resulted in statistically significant reductions in CXCL1, CXCL8, IL-36a to the level of unaffected HS patient tissue as confirmed by rt-PCR by Week 4. Histology demonstrates a significant reduction in CD11+ and CD3+ dermal cells (measured by quantitative IHC) by week 4 (p<0.01) continuing on to Week 12. Significant reductions in transepithelial neutrophil trafficking (measured by quantitative IHC) into dermal epithelialized tunnels was also noted. Morphological changes in epidermal psoriasiform hyperplasia were seen by Week 12 of therapy. Epidermal psoriasiform hyperplasia and trans-epithelial neutrophil migration in HS epidermis and epithelialized tunnels have parallels with Psoriasis Vulgaris and Generalized Pustular Psoriasis. IL17RA antagonism demonstrates rapid (by week 4) reduction in expression of B and T cell trafficking chemokines as measured by RT-PCR and significant alterations in epidermal morphology by Week 12. This highlights the role of IL-17 mediated feed-forward inflammation as in psoriasis and transepithelial neutrophil trafficking in dermal tunnels in HS.

Ginsenoside Rg3 ameliorates acute exacerbation of COPD by suppressing neutrophil migration

Acute Exacerbation of Chronic Obstructive Pulmonary Disease (AECOPD) is an irreversible inflammatory airways disease responsible for global health burden, involved with a complex condition of immunological change. Exacerbation-mediated neutrophilia is an important factor in the pathogenesis of cigarette smoke-induced AECOPD. Ginsenoside Rg3, a red–ginseng–derived compound, has multiple pharmacological properties such as anti-inflammatory and antitumor activities. Here, we investigated a protective role of Rg3 against AECOPD, focusing on neutrophilia. 14-week-cigarette smoke (CS) exposure and non–typeable Haemophilus inflenzae (NTHi) infection were used to establish the AECOPD murine model. Rg3 (10, 20, 40 mg/kg) was administered intragastrically from the 12th week of CS exposure before infection, and this led to improved lung function and lung morphology, and reduced neutrophilic inflammation, indicating a suppressive effect on neutrophil infiltration by Rg3. Further investigations on the mechanism of Rg3 on neutrophils were carried out using bronchial epithelial cell (BEAS-2B) and neutrophil co-culture and transepithelial migration model. Pre–treatment of neutrophils with Rg3 reduced neutrophil migration, which seemed to be the result of inhibition of phosphatidylinositol (PtdIns) 3–kinases (PI3K) activation within neutrophils. Thus, Rg3 could inhibit exacerbation-induced neutrophilia in COPD by negatively regulating PI3K activities in neutrophils. This study provides a potential natural drug against AECOPD neutrophil inflammation.

Insights into the mechanisms of inflammatory acidification within the mucosa

Tissue acidification is commonly associated with a number of inflammatory states. The primary mechanism of extracellular acidification is thought to be increased accumulation of lactic acid resulting from enhanced glycolysis. Like other chronic inflammatory diseases, tissue acidification has been observed in inflammatory bowel disease patients, with reports of colonic pH as low as 4. We have recently demonstrated that neutrophil (PMN) transepithelial migration rapidly acidifies the microenvironment resulting in inflammatory acidification in the mucosa. Very little is known with regard to the mechanism(s) involved in this acidification or the impact of such acidification on the intestinal epithelial cell (IEC) function. Initial observations showed that physical interaction between IEC and PMN was required for inflammatory acidification; however, when transmigration was blocked using anti‐CD11b antibodies, we observed significant extracellular acidification in the absence of PMN transepithelial migration. This observation suggests that binding of the CD11b receptor plays a crucial role promoting extracellular acidification. Expanding upon these observations, we analyzed extracellular lactate secretion. We observed that lactic acid was released by IEC 4 to 5 hours after the addition of PMN and in quantities not high enough to account for the extracellular acidification observed. Additionally, the concentration of lactic acid released in the anti‐CD11b treated cells was similar to control IEC in the absence of PMN. When combined, these data indicate lactate is not a significant mediator of extracellular acidification following PMN transmigration. Given the large number of PMNs present and chronic tissue acidification observed during active inflammation, we investigated the impact of extracellular acidification on the regulation of epithelial derived pro‐inflammatory markers. When T84 IEC were cultured in pH 6.0 media for 3 or 6 hours we observed increases in pro‐inflammatory markers, including IL‐8. To better understand how extracellular acidification alters IEC gene regulation, we utilized a transcription factor array to examine changes in transcription factor expression under acidic conditions. This screen identified CREB as one of the major transcription factors controlling responses to acidification. Further examination revealed that CREB phosphorylation occurs rapidly, is cAMP independent, and requires MSK1. Inhibition of MSK1 prevents the induction of IL‐8 and other acidification associated gene transcription. Theses result identified extracellular acidification as a significant signaling mechanism during tissue inflammation. Targeted regulation of MSK1 may serve as a new therapeutic approach to limit pro‐inflammatory signaling during mucosal inflammation.Support or Funding InformationR01 DK095491, NIH

β2-integrin LFA1 mediates airway damage following neutrophil transepithelial migration during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) bronchiolitis is the most common cause of infant hospital admissions, but there is limited understanding of the mechanisms of disease, and no specific antiviral treatment. Using a novel in vitro primary transepithelial neutrophil migration model and innovative imaging methods, we show that RSV infection of nasal airway epithelium increased neutrophil transepithelial migration and adhesion to infected epithelial cells, which is associated with epithelial cell damage and reduced ciliary beat frequency, but also with a reduction in infectious viral load.Following migration, RSV infection results in greater neutrophil activation, degranulation and release of neutrophil elastase into the airway surface media compared to neutrophils that migrated across mock-infected nasal epithelial cells. Blocking of the interaction between the ligand on neutrophils (the β2-integrin LFA-1) for intracellular adhesion molecule (ICAM)-1 on epithelial cells reduced neutrophil adherence to RSV-infected cells and epithelial cell damage to pre-infection levels, but did not reduce the numbers of neutrophils that migrated or prevent the reduction in infectious viral load.These findings have provided important insights into the contribution of neutrophils to airway damage and viral clearance, which are relevant to the pathophysiology of RSV bronchiolitis. This model is a convenient, quantitative preclinical model that will further elucidate mechanisms that drive disease severity and has utility in antiviral drug discovery.