Publisher Correction: IGF1R is an entry receptor for respiratory syncytial virus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

IGF1R is an entry receptor for respiratory syncytial virus.

Pneumonia resulting from infection is one of the leading causes of death worldwide. Pulmonary infection by the respiratory syncytial virus (RSV) is a large burden on human health, for which there are few therapeutic options1. RSV targets ciliated epithelial cells in the airways, but how viruses such as RSV interact with receptors on these cells is not understood. Nucleolin is an entry coreceptor for RSV2 and also mediates the cellular entry of influenza, the parainfluenza virus, some enteroviruses and the bacterium that causes tularaemia3,4. Here we show a mechanism of RSV entry into cells in which outside-in signalling, involving binding of the prefusion RSV-F glycoprotein with the insulin-like growth factor-1 receptor, triggers the activation of protein kinase C zeta (PKCζ). This cellular signalling cascade recruits nucleolin from the nuclei of cells to the plasma membrane, where it also binds to RSV-F on virions. We find that inhibiting PKCζ activation prevents the trafficking of nucleolin to RSV particles on airway organoid cultures, and reduces viral replication and pathology in RSV-infected mice. These findings reveal a mechanism of virus entry in which receptor engagement and signal transduction bring the coreceptor to viral particles at the cell surface, and could form the basis of new therapeutics to treat RSV infection.

Role of robot-assisted laparoscopy in deep infiltrating endometriosis with bowel involvement: a systematic review and application of the IDEAL framework.

AIMS: This review aims to evaluate the feasibility of robot-assisted laparoscopic surgery (RALS) as an alternative to standard laparoscopic surgery (SLS) for the treatment of bowel deep-infiltrative endometriosis. Additionally, it aims to provide guidance for future study design, by gaining insight into the current state of research, in accordance with the IDEAL framework. METHOD: A systematic review was conducted to identify relevant studies on RALS for bowel deep infiltrating endometriosis in Medline, Embase, Cochrane Library and PubMed databases up to August 2023 and reported in keeping with PRISMA guidelines. The study was registered with PROSPERO Registration: CRD42022308611 RESULTS: Eleven primary studies were identified, encompassing 364 RALS patients and 83 SLS patients, from which surgical details, operative and postoperative outcomes were extracted. In the RALS group, mean operating time was longer (235 ± 112 min) than in the standard laparoscopy group (171 ± 76 min) (p < 0.01). Patients in the RALS group experienced a shorter hospital stay (5.3 ± 3.5 days vs. 7.3 ± 4.1 days) (p < 0.01), and appeared to have fewer postoperative complications compared to standard laparoscopy. Research evidence for RALS in bowel DE is at an IDEAL Stage 2B of development. CONCLUSION: RALS is a safe and feasible alternative to standard laparoscopy for bowel endometriosis treatment, with a shorter overall length of stay despite longer operating times. Further robust randomized trials recommended to delineate other potential advantages of RALS.

Analgesic effect of local anaesthetic in haemorrhoid banding: systematic review and meta-analysis.

PURPOSE: Rubber band ligation of haemorrhoids can be,painful and there is no consensus regarding the optimal analgesic strategy. This study aims to determine whether there is a difference in post-procedural pain in adults undergoing haemorrhoid banding who have received local anaesthetic, a pudendal nerve block or no regional or local analgesia. METHODS: MEDLINE, Embase, Google Scholar and clinical trial registries were searched for randomised trials of local anaesthetic or pudendal nerve block use in banding. Primary outcomes were patient-reported pain scores. The quality of the evidence was assessed using the GRADE approach. RESULTS: Seven studies were included in the final review. No articles were identified that studied pudendal nerve blocks. The difference in numerical pain scores between treatment groups favoured the local anaesthetic group at all timepoints. The mean difference in scores on a 10-point scale was at 1 h,-1.43 (95% CI-2.30 to-0.56, p < 0.01, n = 342 (175 in treatment group)); 6 h,-0.52 (95% CI-1.04 to 0.01, p = 0.05, n = 250 (130 in treatment group)); and 24 h,-0.31 (95% CI-0.82 to 0.19, p = 0.86, n = 247 (127 in treatment group)). Of reported safety outcomes, vasovagal symptoms proceeded to meta-analysis, with a risk ratio of 1.01 (95% CI 0.64-1.60). The quality of the evidence was rated down to 'low' due to inconsistency and imprecision. CONCLUSION: This review supports the use of LA for reducing early post-procedural pain following haemorrhoid banding. The evidence was limited by small sample sizes and substantial heterogeneity across studies. REGISTRATION: PROSPERO (ID CRD42022322234).

Challenges in evaluating pelvic floor physiotherapy based strategies in low anterior resection syndrome: a systematic review and qualitative analysis.

AIM: Physiotherapy is an established treatment strategy for low anterior resection syndrome (LARS). However, data on its efficacy are limited. This is in part due to the inherent challenges in study design in this context. This systematic review aims to analyse the methodology of studies using pelvic floor physiotherapy for treatment of LARS to elucidate the challenges and limitations faced, which may inform the design of future prospective trials. METHODOLOGY: A systematic review of the literature was undertaken through MEDLINE, Embase and Cochrane Library, yielding 345 unique records for screening. Five studies were identified for review. Content thematic analysis of study limitations was carried out using the Braun and Clarke method. Line-by-line coding was used to organize implicit and explicit challenges and limitations under broad organizing categories. RESULTS: Key challenges fell into five overarching categories: patient-related issues, cancer-related issues, adequate symptomatic control, intervention-related issues and measurement of outcomes. Adherence, attrition and randomization contributed to potential bias within these studies, with imbalance in the baseline patient characteristics, particularly gender and baseline pelvic floor function scores. Outcome measurements consisted of patient-reported measures and quality of life measures, where significant improvements in bowel function according to patient-reported outcome measures were not reflected in the quality of life scores. CONCLUSION: Upcoming trial design in the area of pelvic floor physiotherapy for faecal incontinence related to rectal cancer surgery can be cognisant of and design around the challenges identified in this systematic review, including the reduction of bias, exclusion of the placebo effect and the potential cultural differences in attitude towards a sensitive intervention.

Does complete pathological response increase perioperative morbidity risk in rectal cancer?

AIM: The optimal management of patients with clinical complete response after neoadjuvant treatment for rectal cancer is controversial. The aim of this study is to compare the morbidity between patients with locally advanced rectal cancer who have had a pathological complete response (pCR) or not after neoadjuvant chemoradiotherapy (NCRT) and total mesorectal excision (TME). The study hypothesis was that pCR may impact the surgical complication rate. METHOD: A retrospective cohort study was conducted of a prospectively maintained database in Australia and New Zealand, the Binational Colorectal Cancer Audit, that identified patients with locally advanced rectal cancer (<15 cm from anal verge) from 1 January 2007 to 31 December 2019. Patients were included if they had locally advanced rectal cancer and had undergone NCRT and proceeded to surgical resection. RESULTS: There were 4584 patients who satisfied the inclusion criteria, 65% being male. The mean age was 63 years and 11% had a pCR (ypT0N0). TME with anastomosis was performed in 67.8% of patients, and the majority of the cohort received long-course radiotherapy (81.7%). Both major and minor complications were higher in the TME without anastomosis group (17.3% vs. 14.7% and 30.6% vs. 20.8%, respectively), and the 30-day mortality was 1.31%. In the TME with anastomosis group, pCR did not contribute to higher rates of surgical complications, but male gender (p < 0.0012), age (p < 0.0001), preoperative N stage (p = 0.0092) and American Society of Anesthesologists (ASA) score ≥3 (p < 0.0002) did. In addition, pCR had no significant effect (p = 0.44) but male gender (p = 0.0047) and interval to surgery (p = 0.015) contributed to higher rates of anastomotic leak. In the TME without anastomosis cohort, the only variable that contributed to higher rates of complications was ASA score ≥3 (p = 0.033). CONCLUSION: Patients undergoing TME dissection for rectal cancer following NCRT showed no difference in complications whether they had achieved pCR or not.

Evaluating the Educational Value of Cancer Registries - a Systematic Review and Thematic Analysis.

Cancer registries encompass a broad array of functions that underpin cancer control efforts. Despite education being fundamental to improving patient outcomes, little is known regarding the educational value of cancer registries. This review will evaluate the educational value of cancer registries for key stakeholders as reported within published literature and identify opportunities for enhancing their educational value. Four databases (Ovid Medline, Embase, CINAHL and Web of Science) were searched using a predefined search strategy in keeping with the PRISMA statement. Data was extracted and synthesised in narrative format. Themes and frequency of discussion of educational content were explored using thematic content analysis. From 952 titles, ten eligible studies were identified, highlighting six stakeholder groups. Educational outcomes were identified relating to clinicians (6/10), researchers (5/10), patients (4/10), public health organisations (3/10), medical students (1/10) and the public (1/10). Cancer registries were found to educationally benefit key stakeholders despite educational value not being a key focus of any study. Deliberate efforts to harness the educational value of cancer registries should be considered to enable data-driven quality improvement, with the vast amount of data promising ample educational benefit.

Actin cytoskeleton remodeling disrupts physical barriers to infection and presents entry receptors to respiratory syncytial virus.

RSV is the leading cause of infant hospitalizations and a significant cause of paediatric and geriatric morbidity worldwide. Recently, we reported that insulin-like growth factor 1 receptor (IGF1R) was a receptor for respiratory syncytial virus (RSV) in airway epithelial cells and that activation of IGF1R recruited the coreceptor, nucleolin (NCL), to the cell surface. Cilia and mucus that line the airways pose a significant barrier to viral and bacterial infection. The cortical actin cytoskeleton has been shown by others to mediate RSV entry, so we studied the roles of the RSV receptors and actin remodelling during virus entry. We found that IGF1R expression and phosphorylation were associated with the ability of RSV to infect cells. Confocal immunofluorescence imaging showed that actin projections, a hallmark of macropinocytosis, formed around viral particles 30 min after infection. Consistent with prior reports we also found that virus particles were internalized into early endosome antigen-1 positive endosomes within 90 min. Inhibiting actin polymerization significantly reduced viral titre by approximately ten-fold. Inhibiting PI3 kinase and PKCζ in stratified air-liquid interface (ALI) models of the airway epithelium had similar effects on reducing the actin remodelling observed during infection and attenuating viral entry. Actin projections were associated with NCL interacting with RSV particles resting on apical cilia of the ALIs. We conclude that macropinocytosis-like actin projections protrude through normally protective cilia and mucus layers of stratified airway epithelium that helps present the IGF1R receptor and the NCL coreceptor to RSV particles waiting at the surface.

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells.

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.

Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells.

Type III interferons (IFN-lambdas(λ)) are important cytokines that inhibit viruses and modulate immune responses by acting through a unique IFN-λR1/IL-10RB heterodimeric receptor. Until now, the primary antiviral function of IFN-λs has been proposed to be at anatomical barrier sites. Here, we examine the regulation of IFN-λR1 expression and measure the downstream effects of IFN-λ3 stimulation in primary human blood immune cells, compared with lung or liver epithelial cells. IFN-λ3 directly bound and upregulated IFN-stimulated gene (ISG) expression in freshly purified human B cells and CD8+ T cells, but not monocytes, neutrophils, natural killer cells, and CD4+ T cells. Despite similar IFNLR1 transcript levels in B cells and lung epithelial cells, lung epithelial cells bound more IFN-λ3, which resulted in a 50-fold greater ISG induction when compared to B cells. The reduced response of B cells could be explained by higher expression of the soluble variant of IFN-λR1 (sIFN-λR1), which significantly reduced ISG induction when added with IFN-λ3 to peripheral blood mononuclear cells or liver epithelial cells. T-cell receptor stimulation potently, and specifically, upregulated membrane-bound IFNLR1 expression in CD4+ T cells, leading to greater antiviral gene induction, and inhibition of human immunodeficiency virus type 1 infection. Collectively, our data demonstrate IFN-λ3 directly interacts with the human adaptive immune system, unlike what has been previously shown in published mouse models, and that type III IFNs could be potentially utilized to suppress both mucosal and blood-borne viral infections.

A toolbox for studying respiratory viral infections using air-liquid interface cultures of human airway epithelial cells.

Submerged cultures of primary human airway epithelial cells or human airway epithelial cell lines have been a mainstay of airway epithelial biology research for decades due to their robust in vitro proliferative capacity, relatively low maintenance culture conditions, and clinically translatable results to nasal or bronchial brushings. With the development and improvement of air-liquid interface (ALI) cultures of human airway epithelial cells, such cultures have been considered superior to immortalized cell lines and primary cell monolayers, as such cultures effectively recapitulate in vivo epithelial architecture and cell types. Although ALI culture growth protocols are well-established and widely available, many researchers have avoided their use, as ALI cultures not only take longer to grow but also present technical challenges and limitations that make in vitro intracellular and structural assays taxing. Challenges arise relating to their complex structure, requirements for air exposure, the constraints of transwell growth apparatus, and interference in assays caused by mucus secretion. Although few publications briefly describe technical adaptations for some assays, there is still considerable trial and error required for researchers to establish consistent and reliable assay adaptations, often becoming a deterrent for pursuing mechanistic investigation. We have created a user-friendly toolbox detailing comprehensive protocols for numerous techniques and assay adaptations, particularly focusing on respiratory virus infections. By expanding the repertoire of ALI culture-adapted in vitro assays, we hope to facilitate the widespread adoption of this valuable culture system for mechanistic investigations of respiratory viral infections or other epithelial-pathogen models.

Rhinovirus and Bacteria Synergistically Induce IL-17C Release from Human Airway Epithelial Cells To Promote Neutrophil Recruitment.

Virus-bacteria coinfections are associated with more severe exacerbations and increased risk of hospital readmission in patients with chronic obstructive pulmonary disease (COPD). The airway epithelium responds to such infections by releasing proinflammatory and antimicrobial cytokines, including IL-17C. However, the regulation and role of IL-17C is not well understood. In this study, we examine the mechanisms regulating IL-17C production and its potential role in COPD exacerbations. Human bronchial epithelial cells (HBE) obtained from normal, nontransplanted lungs or from brushings of nonsmokers, healthy smokers, or COPD patients were exposed to bacteria and/or human rhinovirus (HRV). RNA and protein were collected for analysis, and signaling pathways were assessed with pharmacological agonists, inhibitors, or small interfering RNAs. HBE were also stimulated with IL-17C to assess function. HRV-bacterial coinfections synergistically induced IL-17C expression. This induction was dependent on HRV replication and required NF-κB-mediated signaling. Synergy was lost in the presence of an inhibitor of the p38 MAP kinase pathway. HBE exposed to IL-17C show increased gene expression of CXCL1, CXCL2, NFKBIZ, and TFRC, and release CXCL1 protein, a neutrophil chemoattractant. Knockdown of IL-17C significantly reduced induction of CXCL1 in response to HRV-bacterial coinfection as well as neutrophil chemotaxis. HBE from healthy smokers release less IL-17C than cells from nonsmokers, but cells from COPD patients release significantly more IL-17C compared with either nonsmokers or healthy smokers. These data suggest that IL-17C may contribute to microbial-induced COPD exacerbations by promoting neutrophil recruitment.

Rhinovirus replication and innate immunity in highly differentiated human airway epithelial cells.

BACKGROUND: Human rhinovirus (HRV) infections are the primary cause of the common cold and are a major trigger for exacerbations of lower airway diseases, such as asthma and chronic obstructive pulmonary diseases. Although human bronchial epithelial cells (HBE) are the natural host for HRV infections, much of our understanding of how HRV replicates and induces host antiviral responses is based on studies using non-airway cell lines (e.g. HeLa cells). The current study examines the replication cycle of HRV, and host cell responses, in highly differentiated cultures of HBE. METHODS: Highly differentiated cultures of HBE were exposed to initial infectious doses ranging from 104 to 101 50% tissue culture-infective dose (TCID50) of purified HRV-16, and responses were monitored up to 144 h after infection. Viral genomic RNA and negative strand RNA template levels were monitored, along with levels of type I and II interferons and selected antivirals. RESULTS: Regardless of initial infectious dose, relatively constant levels of both genomic and negative strand RNA are generated during replication, with negative strand copy numbers being10,000-fold lower than those of genomic strands. Infections were limited to a small percentage of ciliated cells and did not result in any overt signs of epithelial death. Importantly, regardless of infectious dose, HRV-16 infections were cleared by HBE in the absence of immune cells. Levels of type I and type III interferons (IFNs) varied with initial infectious dose, implying that factors other than levels of double-stranded RNA regulate IFN induction, but the time-course of HRV-16 clearance HBE was the same regardless of levels of IFNs produced. Patterns of antiviral viperin and ISG15 expression suggest they may be generated in an IFN-independent manner during HRV-16 infections. CONCLUSIONS: These data challenge a number of aspects of dogma generated from studies in HeLa cells and emphasize the importance of appropriate cell context when studying HRV infections.

Toll-like receptor 3 blockade in rhinovirus-induced experimental asthma exacerbations: A randomized controlled study.

BACKGROUND: Human rhinoviruses (HRVs) commonly precipitate asthma exacerbations. Toll-like receptor 3, an innate pattern recognition receptor, is triggered by HRV, driving inflammation that can worsen asthma. OBJECTIVE: We sought to evaluate an inhibitory mAb to Toll-like receptor 3, CNTO3157, on experimental HRV-16 inoculation in healthy subjects and asthmatic patients. METHODS: In this double-blind, multicenter, randomized, parallel-group study in North America and Europe, healthy subjects and patients with mild-to-moderate stable asthma received single or multiple doses of CNTO3157 or placebo, respectively, and were then inoculated with HRV-16 within 72 hours. All subjects were monitored for respiratory symptoms, lung function, and nasal viral load. The primary end point was maximal decrease in FEV1 during 10 days after inoculation. RESULTS: In asthmatic patients (n = 63) CNTO3157 provided no protection against FEV1 decrease (least squares mean: CNTO3157 [n = 30] = -7.08% [SE, 8.15%]; placebo [n = 25] = -5.98% [SE, 8.56%]) or symptoms after inoculation. In healthy subjects (n = 12) CNTO3157 versus placebo significantly attenuated upper (P = .03) and lower (P = .02) airway symptom scores, with area-under-the-curve increases of 9.1 (15.1) versus 34.9 (17.6) and 13.0 (18.4) versus 50.4 (25.9) for the CNTO3157 (n = 8) and placebo (n = 4) groups, respectively, after inoculation. All of the severe and 4 of the nonserious asthma exacerbations occurred while receiving CNTO3157. CONCLUSION: In summary, CNTO3157 was ineffective in attenuating the effect of HRV-16 challenge on lung function, asthma control, and symptoms in asthmatic patients but suppressed cold symptoms in healthy subjects. Other approaches, including blockade of multiple pathways or antiviral agents, need to be sought for this high unmet medical need.

TLR2 Activation Limits Rhinovirus-Stimulated CXCL-10 by Attenuating IRAK-1-Dependent IL-33 Receptor Signaling in Human Bronchial Epithelial Cells.

Airway epithelial cells are the major target for rhinovirus (RV) infection and express proinflammatory chemokines and antiviral cytokines that play a role in innate immunity. Previously, we demonstrated that RV interaction with TLR2 causes ILR-associated kinase-1 (IRAK-1) depletion in both airway epithelial cells and macrophages. Further, IRAK-1 degradation caused by TLR2 activation was shown to inhibit ssRNA-induced IFN expression in dendritic cells. Therefore, in this study, we examined the role of TLR2 and IRAK-1 in RV-induced IFN-ß, IFN-λ1, and CXCL-10, which require signaling by viral RNA. In airway epithelial cells, blocking TLR2 enhanced RV-induced expression of IFNs and CXCL-10. By contrast, IRAK-1 inhibition abrogated RV-induced expression of CXCL-10, but not IFNs in these cells. Neutralization of IL-33 or its receptor, ST2, which requires IRAK-1 for signaling, inhibited RV-stimulated CXCL-10 expression. In addition, RV induced expression of both ST2 and IL-33 in airway epithelial cells. In macrophages, however, RV-stimulated CXCL-10 expression was primarily dependent on TLR2/IL-1R. Interestingly, in a mouse model of RV infection, blocking ST2 not only attenuated RV-induced CXCL-10, but also lung inflammation. Finally, influenza- and respiratory syncytial virus-induced CXCL-10 was also found to be partially dependent on IL-33/ST2/IRAK-1 signaling in airway epithelial cells. Together, our results indicate that RV stimulates CXCL-10 expression via the IL-33/ST2 signaling axis, and that TLR2 signaling limits RV-induced CXCL-10 via IRAK-1 depletion at least in airway epithelial cells. To our knowledge, this is the first report to demonstrate the role of respiratory virus-induced IL-33 in the induction of CXCL-10 in airway epithelial cells.

Human Rhinovirus Infection of Epithelial Cells Modulates Airway Smooth Muscle Migration.

Airway remodeling, a characteristic feature of asthma, begins in early life. Recurrent human rhinovirus (HRV) infections are a potential inciting stimulus for remodeling. One component of airway remodeling is an increase in airway smooth muscle cell (ASMC) mass with a greater proximity of the ASMCs to the airway epithelium. We asked whether human bronchial epithelial cells infected with HRV produced mediators that are chemotactic for ASMCs. ASMC migration was investigated using the modified Boyden Chamber and the xCELLigence Real-Time Cell Analyzer (ACEA Biosciences Inc., San Diego, CA). Multiplex bead analysis was used to measure HRV-induced epithelial chemokine release. The chemotactic effects of CCL5, CXCL8, and CXCL10 were also examined. Supernatants from HRV-infected epithelial cells caused ASMC chemotaxis. Pretreatment of ASMCs with pertussis toxin abrogated chemotaxis, as did treatment with formoterol, forskolin, or 8-bromo-cAMP. CCL5, CXCL8, and CXCL10 were the most up-regulated chemokines produced by HRV-infected airway epithelial cells. When recombinant CCL5, CXCL8, and CXCL10 were used at levels found in epithelial supernatants, they induced ASMC chemotaxis similar to that seen with epithelial cell supernatants. When examined individually, CCL5 was the most effective chemokine in causing ASMC migration, and treatment of supernatant from HRV-infected epithelial cells with anti-CCL5 antibodies significantly attenuated ASMC migration. These findings suggest that HRV-induced CCL5 can induce ASMC chemotaxis and thus may contribute to the pathogenesis of airway remodeling in patients with asthma.

Rhinovirus-bacteria coexposure synergistically induces CCL20 production from human bronchial epithelial cells.

Exacerbations of chronic obstructive pulmonary disease are triggered by viral or bacterial pathogens, with human rhinovirus (HRV) and nontypeable Hemophilus influenzae (NTHI) among the most commonly detected pathogens. Patients who suffer from concomitant viral and bacterial infection have more severe exacerbations. The airway epithelial cell is the initial site of viral and bacterial interactions, and CCL20 is an epithelial chemokine that attracts immature dendritic cells to the airways and can act as an antimicrobial. As such, it contributes to innate and adaptive immune responses to infection. We used primary cultures of human bronchial epithelial cells and the BEAS-2B cell line to examine the effects of bacterial-viral coexposure, as well as each stimulus alone, on epithelial expression of CXCL8 and, in particular, CCL20. HRV-bacterial coexposure induced synergistic production of CXCL8 and CCL20 compared with the sum of each stimulus alone. Synergistic induction of CCL20 did not require viral replication and occurred with two different HRV serotypes that use different viral receptors. Synergy was also seen with either NTHI or Pseudomonas aeruginosa Synergistic induction of CCL20 was transcriptionally regulated. Although NF-κB was required for transcription, it did not regulate synergy, but NF-IL-6 did appear to contribute. Among MAPK inhibitors studied, neither SB203580 nor PD98059 had any effect on synergy, whereas U0126 prevented synergistic induction of CCL20 by HRV and bacteria, apparently via "off-target" effects. Thus bacterial-viral coexposure synergistically increases innate immune responses compared with individual infections. We speculate that this increased inflammatory response leads to worse clinical outcomes.

Role of human rhinovirus in triggering human airway epithelial-mesenchymal transition.

BACKGROUND: Structural changes in the airways, collectively referred to as airway remodeling, are a characteristic feature of asthma, and are now known to begin in early life. Human rhinovirus (HRV)-induced wheezing illnesses during early life are a potential inciting stimulus for remodeling. Increased deposition of matrix proteins causes thickening of the lamina reticularis, which is a well-recognized component of airway remodeling. Increased matrix protein deposition is believed to be due to the presence of increased numbers of activated mesenchymal cells (fibroblasts/myofibroblasts) in the subepithelial region of asthmatic airways. The origin of these increased mesenchymal cells is not clear, but one potential contributor is the process of epithelial-mesenchymal transition (EMT). We hypothesized that HRV infection may help to induce EMT. METHODS: We used the BEAS-2B human bronchial epithelial cells line, which uniformly expresses the major group HRV receptor, to examine the effects of stimulation with HRV alone, transforming growth factor-ß1 (TGF-ß1), alone, and the combination, on induction of changes consistent with EMT. Western blotting was used to examine expression of epithelial and mesenchymal phenotypic marker proteins and selected signaling molecules. Cell morphology was also examined. RESULTS: In this study, we show that two different strains of HRV, which use two different cellular receptors, are each capable of triggering phenotypic changes consistent with EMT. Moreover, both HRV serotypes synergistically induced changes consistent with EMT when used in the presence of TGF-ß1. Morphological changes were also most pronounced with the combination of HRV and TGF-ß1. Viral replication was not essential for phenotypic changes. The synergistic interactions between HRV and TGF-ß1 were mediated, at least in part, via activation of mitogen activated protein kinase pathways, and via induction of the transcription factor SLUG. CONCLUSIONS: These data support a role for HRV in the induction of EMT, which may contribute to matrix protein deposition and thickening of the lamina reticularis in airways of patients with asthma.

Chemokine release from human rhinovirus-infected airway epithelial cells promotes fibroblast migration.

BACKGROUND: Thickening of the lamina reticularis, a feature of remodeling in the asthmatic airways, is now known to be present in young children who wheeze. Human rhinovirus (HRV) infection is a common trigger for childhood wheezing, which is a risk factor for subsequent asthma development. We hypothesized that HRV-infected epithelial cells release chemoattractants to recruit fibroblasts that could potentially contribute to thickening of the lamina reticularis. OBJECTIVE: We sought to investigate whether conditioned medium from HRV-infected epithelial cells can trigger directed migration of fibroblasts. METHODS: Human bronchial epithelial cells were exposed to medium alone or infected with HRV-16. Conditioned medium from both conditions were tested as chemoattractants for human bronchial fibroblasts in the xCELLigence cell migration apparatus. RESULTS: HRV-conditioned medium was chemotactic for fibroblasts. Treatment of fibroblasts with pertussis toxin, an inhibitor of Gαi-coupled receptors, prevented their migration. Production of epithelial chemoattractants required HRV replication. Multiplex analysis of epithelial supernatants identified CXCL10, CXCL8, and CCL5 as Gαi-coupled receptor agonists of potential interest. Subsequent analysis confirmed that fibroblasts express CXCR3 and CXCR1 receptors and that CXCL10 and, to a lesser extent, CXCL8, but not CCL5, are major contributors to fibroblast migration caused by HRV-conditioned medium. CONCLUSION: CXCL10 and CXCL8 produced from HRV-infected epithelial cells are chemotactic for fibroblasts. This raises the possibility that repeated HRV infections in childhood could contribute to the initiation and progression of airway remodeling in asthmatic patients by recruiting fibroblasts that produce matrix proteins and thicken the lamina reticularis.