Cell free ACE2 RNA: A potential biomarker of COVID-19 severity.

Despite the downward trend of COVID-19 pandemic and increased immunity of the general population, COVID-19 is still an elusive disease with risks due to emerging variants. Fast and reliable diagnosis of COVID-19 disease would allow better therapeutic interventions for patients at risk to develop more severe outcomes. Cell-free RNAs (cfRNAs) have been proven to be an effective biomarker in cancer and infectious diseases. It has been reported that cfRNAs are amplified in the bloodstream of these patients and at earlier stages of the disease, reflecting tissue damage. Hence, we hypothesize that cfRNAs may serve as a potential indicator of COVID-19 disease severity. To our knowledge, this is the first report to display a significant link between COVID-19 severity and cfRNA of angiotensin converting enzyme-2 (ACE2), the receptor for SARS-CoV-2 virus. qRT-PCR analysis of liquid biopsies from COVID-19 patients (n = 82) displayed a significant increase in ACE2-cfRNA levels in patients with severe manifestations. This finding correlated with blood biomarkers (ANC, WBC, and Creatinine) that were also significantly increased in these patients. We previously showed that bronchial cells from obese subjects express higher ACE2 levels, hence, we further analysed the involvement of obesity as a main contributor to severe outcomes. We confirm a significant increase of ACE2-cfRNA in the plasma of obese/overweight (Ob/Ov) COVID-19 patients compared to lean subjects, with no observed significant change in blood biomarkers. These findings suggest that monitoring ACE2-cfRNAs, as a biomarker, during COVID-19 infection may allow for better disease management, specifically for severe-COVID-19 patients.

Lung Epithelial Cells from Obese Patients Have Impaired Control of SARS-CoV-2 Infection.

Obesity is known to increase the complications of the COVID-19 coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the exact mechanisms of SARS-CoV-2 infection in obese patients have not been clearly elucidated. This study aims to better understand the effect of obesity on the course of SARS-CoV-2 infection and identify candidate molecular pathways involved in the progression of the disease, using an in vitro live infection model and RNA sequencing. Results from this study revealed the enhancement of viral load and replication in bronchial epithelial cells (NHBE) from obese subjects at 24 h of infection (MOI = 0.5) as compared to non-obese subjects. Transcriptomic profiling via RNA-Seq highlighted the enrichment of lipid metabolism-related pathways along with LPIN2, an inflammasome regulator, as a unique differentially expressed gene (DEG) in infected bronchial epithelial cells from obese subjects. Such findings correlated with altered cytokine and angiotensin-converting enzyme-2 (ACE2) expression during infection of bronchial cells. These findings provide a novel insight on the molecular interplay between obesity and SARS-CoV-2 infection. In conclusion, this study demonstrates the increased SARS-CoV-2 infection of bronchial epithelial cells from obese subjects and highlights the impaired immunity which may explain the increased severity among obese COVID-19 patients.

In Silico Bioinformatics Followed by Molecular Validation Using Archival FFPE Tissue Biopsies Identifies a Panel of Transcripts Associated with Severe Asthma and Lung Cancer.

Severe asthma and lung cancer are both heterogeneous pathological diseases affecting the lung tissue. Whilst there are a few studies that suggest an association between asthma and lung cancer, to the best of our knowledge, this is the first study to identify common genes involved in both severe asthma and lung cancer. Publicly available transcriptomic data for 23 epithelial brushings from severe asthmatics and 55 samples of formalin-fixed paraffin-embedded (FFPE) lung cancer tissue at relatively early stages were analyzed by absolute gene set enrichment analysis (GSEA) in comparison to 37 healthy bronchial tissue samples. The key pathways enriched in asthmatic patients included adhesion, extracellular matrix, and epithelial cell proliferation, which contribute to tissue remodeling. In the lung cancer dataset, the main pathways identified were receptor tyrosine kinase signaling, wound healing, and growth factor response, representing the early cancer pathways. Analysis of the enriched genes derived from the pathway analysis identified seven genes expressed in both the asthma and lung cancer sets: BCL3, POSTN, PPARD, STAT1, MYC, CD44, and FOSB. The differential expression of these genes was validated in vitro in the cell lines retrieved from different lung cancer and severe asthma patients using real-time PCR. The effect of the expression of the seven genes identified in the study on the overall survival of lung cancer patients (n = 1925) was assessed using a Kaplan-Meier plot. In vivo validation performed in the archival biopsies obtained from patients diagnosed with both the disease conditions provided interesting insights into the pathogenesis of severe asthma and lung cancer, as indicated by the differential expression pattern of the seven transcripts in the mixed group as compared to the asthmatics and lung cancer samples alone.

Simulated Microgravity Influences Immunity-Related Biomarkers in Lung Cancer.

Microgravity is a novel strategy that may serve as a complementary tool to develop future cancer therapies. In lung cancer, the influence of microgravity on cellular processes and the migratory capacity of cells is well addressed. However, its effect on the mechanisms that drive lung cancer progression remains in their infancy. In this study, 13 differentially expressed genes were shown to be associated with the prognosis of lung cancer under simulated microgravity (SMG). Using gene set enrichment analysis, these genes are enriched in humoral immunity pathways. In lieu, alveolar basal-epithelial (A549) cells were exposed to SMG via a 2D clinostat system in vitro. In addition to morphology change and decrease in proliferation rate, SMG reverted the epithelial-to-mesenchymal transition (EMT) phenotype of A549, a key mechanism in cancer progression. This was evidenced by increased epithelial E-cadherin expression and decreased mesenchymal N-cadherin expression, hence exhibiting a less metastatic state. Interestingly, we observed increased expression of FCGBP, BPIFB, F5, CST1, and CFB and their correlation to EMT under SMG, rendering them potential tumor suppressor biomarkers. Together, these findings reveal new opportunities to establish novel therapeutic strategies for lung cancer treatment.

Derangement of cell cycle markers in peripheral blood mononuclear cells of asthmatic patients as a reliable biomarker for asthma control.

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity.

Asthma severity as a contributing factor to cancer incidence: A cohort study.

BACKGROUND: A putative link between asthma and asthma severity with the occurrence of cancer has been suggested but has not been fully investigated. The objective of this study is to assess the incidence of all types of cancer in a cohort of asthmatic patients. METHODS AND FINDINGS: A single center cohort retrospective study was conducted to investigate the role of asthma as a potential risk factor for various cancers. Participants were followed for a period of 9 years from 01/01/2010 to 30/12/2018 and cancer incidence and its determinants were collected in asthmatic patients and controls from the same population source but without any respiratory disease. Overall, 2,027 asthma patients and 1,637 controls were followed up for an average of 9 years. The statistical analysis showed that 2% of asthma patients were diagnosed with various cancers, resulting in an incidence rate of cancer of 383.02 per 100,000 persons per year which is significantly higher than the 139.01 per 100,000 persons per year observed in matched controls (p-value < 0.001). The top four cancers reported among asthmatics were breast, colon, lung and prostate cancer. Lung cancer in asthmatics had the longest diagnosis period with a mean of 36.6 years compared to the shortest with prostate cancer with 16.5 years. CONCLUSIONS: This study shows that asthma patients are at increased risk of different types of cancers with asthma severity and goiter as the main factors that may increase the risk of developing cancers among asthmatic patients.

Regulation of Angiotensin- Converting Enzyme 2 in Obesity: Implications for COVID-19.

The ongoing COVID-19 pandemic is caused by the novel coronavirus SARS-CoV-2. Age, smoking, obesity, and chronic diseases such as cardiovascular disease and diabetes have been described as risk factors for severe complications and mortality in COVID-19. Obesity and diabetes are usually associated with dysregulated lipid synthesis and clearance, which can initiate or aggravate pulmonary inflammation and injury. It has been shown that for viral entry into the host cell, SARS-CoV-2 utilizes the angiotensin-converting enzyme 2 (ACE2) receptors present on the cells. We aimed to characterize how SARS-CoV-2 dysregulates lipid metabolism pathways in the host and the effect of dysregulated lipogenesis on the regulation of ACE2, specifically in obesity. In our study, through the re-analysis of publicly available transcriptomic data, we first found that lung epithelial cells infected with SARS-CoV-2 showed upregulation of genes associated with lipid metabolism, including the SOC3 gene, which is involved in the regulation of inflammation and inhibition of leptin signaling. This is of interest as viruses may hijack host lipid metabolism to allow the completion of their viral replication cycles. Furthermore, a dataset using a mouse model of diet-induced obesity showed a significant increase in Ace2 expression in the lungs, which negatively correlated with the expression of genes that code for sterol response element-binding proteins 1 and 2 (SREBP). Suppression of Srebp1 showed a significant increase in Ace2 expression in the lung. Moreover, ACE2 expression in human subcutaneous adipose tissue can be regulated through changes in diet. Validation of the in silico data revealed a higher expression of ACE2, TMPRSS2 and SREBP1 in vitro in lung epithelial cells from obese subjects compared to non-obese subjects. To our knowledge this is the first study to show upregulation of ACE2 and TMPRSS2 in obesity. In silico and in vitro results suggest that the dysregulated lipogenesis and the subsequently high ACE2 expression in obese patients might be the mechanism underlying the increased risk for severe complications in those patients when infected by SARS-CoV-2.

Action of 1,25(OH)2D3 on Human Asthmatic Bronchial Fibroblasts: Implications for Airway Remodeling in Asthma.

BACKGROUND: Airway fibroblasts are major contributors to the histopathological feature of airway remodeling in asthma by their implication in the cell invasiveness and profibrogenic secretory phenotype observed in subepithelial fibrosis. 1,25 Dihydroxy vitamin D3 (1,25(OH)2D3) is an important therapeutic agent that blocks many features of airway remodeling induced by profibrogenic mediators, such as transforming growth factor beta 1 (TGF-ß1) or T helper type 1 inflammatory cytokines. OBJECTIVE: We hypothesized that 1,25(OH)2D3 opposes the TGF-ß1 or tumor necrosis factor alpha (TNF-α)-Interleukin 1 beta (IL-1ß) stimulation on airway fibroblast profibrogenic secretory phenotype observed in severe asthmatic patients. Our aim was to investigate the anti-fibrogenic effect of 1,25(OH)2D3 in TGF-ß1 or TNF-α-IL-1ß-stimulated human bronchial fibroblast cells (HBFCs) from severe asthmatic compared with non-asthmatic subjects. PATIENTS AND METHODS: All experiments were performed on primary HBFCs from asthmatic (DHBFCs, n=4) and non-asthmatic subjects (NHBFCs, n=4). mRNA expression and protein quantification of key fibrogenic markers were analyzed by RT-qPCR and ELISA, comparing HBFCs from asthmatic and non-asthmatic subjects. Vitamin D receptor (VDR) mRNA expression and its functionality in HBFCs were assessed by RT-qPCR. HBFCs proliferation was assessed by flow cytometry using BrdU-FITC/7AAD bivariate staining, while HBFCs apoptosis by Annexin V-FITC/7AAD. RESULTS: VDR is constitutively expressed in HBFCs and the addition of 1,25(OH)2D3 significantly increased mRNA expression of CYP24A1 (a direct VDRs' target gene) in both HBFCs groups. DHBFCs cultured in the presence of TGF-ß1 or TNF-α-IL-1ß showed increased mRNA expression and protein secretion of fibrogenic markers when compared to NHBFCs. Additionally, we observed decreased mRNA expression of FN 1, LUM, BGN, MMP2, COL5A1, TIMP1 and CC-chemokines (CCL2, CCL5, CCL11) in response to 1,25(OH)2D3 addition to the TGF-ß1 or TNF-α-IL-1ß-stimulated HBFCs. Cell culture media obtained from TGF-ß1 or TNF-α-IL-1ß-stimulated DHBFCs showed decreased protein secretion (fibronectin 1, lumican, MCP1, RANTES and eotaxin-1) in response to 1,25(OH)2D3 when compared to NHBFCs. 1,25(OH)2D3 inhibited proliferation in TGF-ß1-stimulated HBFCs through G0/G1 cell cycle arrest and these effects were not correlated with the induction of apoptosis. CONCLUSION: DHBFCs under TGF-ß1 or TNF-α-IL-1ß stimulation showed higher fibrogenic capacity when compared to NHBFCs. 1,25(OH)2D3 significantly blocked these effects and highlight 1,25(OH)2D3 as a possible therapeutic target for severe asthma.

Airways Expression of SARS-CoV-2 Receptor, ACE2, and TMPRSS2 Is Lower in Children Than Adults and Increases with Smoking and COPD.

It has been reported that angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) are the main cell entry proteins for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and play a critical role in causing coronavirus disease 2019 (COVID-19). To investigate the expression level of these SARS-CoV-2 host cell entry genes in the lung airway, we used public gene expression datasets. We have found a differential expression of ACE2 and TMPRSS2 in nasal and bronchial airways relative to age and diseases status. Children were found to have significantly lower expression of COVID-19 receptors in the upper and lower airways (nasal and bronchial). Moreover, the lung airway expression of both ACE2 and TMPRSS2 was found to be significantly upregulated in smokers compared with non-smokers, and in patients with chronic obstructive pulmonary disease (COPD) compared with healthy subjects. No difference was observed in the blood expression levels of ACE2 and TMPRSS2 between children and adults, or in COPD or diabetic patients. However, a significant increase in blood expression levels of these genes was observed in patients with essential hypertension, whereas only ACE2 was upregulated in the blood of asthmatics. These results suggest that the observed difference in COVID-19 severity between children and adults could, in part, be attributed to the difference in ACE2 and TMPRSS2 airways tissue expression levels.

Confounding Patient Factors Affecting the Proper Interpretation of the Periostin Level as a Biomarker in Asthma Development.

INTRODUCTION: The proper use of serum periostin (POSTN) as a biomarker for asthma is hindered by inconsistent performance in different clinical settings. OBJECTIVE: To explore patient's factors that may affect POSTN expression locally and systematically and its utility as a biomarker for asthma development. MATERIALS AND METHODS: Here we used bioinformatics analysis of publicly available transcriptomics data to confirm that POSTN is an asthma specific gene involved in core signaling pathways enriched in the bronchial epithelium during asthma. We then explored a large number of datasets to identify possible confounders that may affect the POSTN gene expression and consequently, its interpretation as a reliable biomarker for asthma. Plasma and saliva levels of POSTN were determined in locally recruited asthmatic patients (mild, moderate and severe) compared to healthy controls to confirm the bioinformatics findings. RESULTS: Our bioinformatics results confirmed that POSTN was consistently upregulated in the bronchial epithelium in asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) bronchial epithelium. In asthma, its mRNA expression was affected by gender, sample anatomical site and type, steroid therapy, and smoking. In our cohort, plasma POSTN was upregulated in severe and non-severe asthmatic patients. Saliva POSTN was significantly higher in non-severe asthmatic patients compared to healthy and severe asthmatic patients (specifically those who are not on Xolair (omalizumab)). Patients' BMI, inhaled steroid use and Xolair treatment affected POSTN plasma levels. CONCLUSION: Up to our knowledge, this is the first study examining the level of POSTN in the saliva of asthmatic patients. Both plasma and saliva POSTN levels can aid in early diagnosis of asthma. Saliva POSTN level was more sensitive than plasma POSTN in differentiating between severe and non-severe asthmatics. Patients' characteristics like BMI, the use of inhaled steroids, or Xolair treatment should be carefully reviewed before any meaningful interpretation of POSTN level in clinical practice.

Effect of bronchial thermoplasty on structural changes and inflammatory mediators in the airways of subjects with severe asthma.

BACKGROUND: Bronchial thermoplasty (BT) is a novel technique used in the treatment of subjects with severe refractory asthma. Radiofrequency is provided to airway walls during bronchoscopy in order to reduce airway remodeling. Several clinical studies have reported an improvement in subjects' symptoms following BT. However, how BT affects the airway architectures and inflammatory mediators in the airways has not been yet fully elucidated. METHODS: Fourteen subjects with severe asthma were recruited in this study according to the criteria of ATS severe asthma definition. The study subjects undertook bronchial biopsy during the bronchoscopy procedure at baseline and 6 weeks after the initial BT treatment. The obtained samples were stained with antibodies for α-smooth muscle actin (α-SMA); protein gene product (PGP) 9.5, a specific nerve marker; von Willebrand factor (vWF), a marker for blood vessels; interleukin-17A (IL-17A) and transforming growth factor-ß1 (TGF-ß1). RESULTS: The expression of α-SMA and PGP9.5 were significantly reduced post-BT. There was no significant difference in the number of blood vessels between baseline and post-BT. In addition, BT did not affect the production of IL-17A and TGF-ß1 in the airways. The changes in the expression of α-SMA and PGP9.5 had no significant correlation with the improvement of pulmonary function. CONCLUSION: and Clinical Relevance: This study suggests that BT reduces airway smooth muscle mass and the airway innervation without affecting vasculature and the production of inflammatory mediators in the airways of subjects with severe asthma.

The TLR4-TRIF pathway can protect against the development of experimental allergic asthma.

The Toll-like receptor (TLR) adaptor proteins myeloid differentiating factor 88 (MyD88) and Toll, interleukin-1 receptor and resistance protein (TIR) domain-containing adaptor inducing interferon-ß (TRIF) comprise the two principal limbs of the TLR signalling network. We studied the role of these adaptors in the TLR4-dependent inhibition of allergic airway disease and induction of CD4+ ICOS+ T cells by nasal application of Protollin™, a mucosal adjuvant composed of TLR2 and TLR4 agonists. Wild-type (WT), Trif-/- or Myd88-/- mice were sensitized to birch pollen extract (BPEx), then received intranasal Protollin followed by consecutive BPEx challenges. Protollin's protection against allergic airway disease was TRIF-dependent and MyD88-independent. TRIF deficiency diminished the CD4+ ICOS+ T-cell subsets in the lymph nodes draining the nasal mucosa, as well as their recruitment to the lungs. Overall, TRIF deficiency reduced the proportion of cervical lymph node and lung CD4+ ICOS+ Foxp3- cells, in particular. Adoptive transfer of cervical lymph node cells supported a role for Protollin-induced CD4+ ICOS+ cells in the TRIF-dependent inhibition of airway hyper-responsiveness. Hence, our data demonstrate that stimulation of the TLR4-TRIF pathway can protect against the development of allergic airway disease and that a TRIF-dependent adjuvant effect on CD4+ ICOS+ T-cell responses may be a contributing mechanism.

Nanotubes connect CD4+ T cells to airway smooth muscle cells: novel mechanism of T cell survival.

Contact between airway smooth muscle (ASM) cells and activated CD4(+) T cells, a key interaction in diseases such as asthma, triggers ASM cell proliferation and enhances T cell survival. We hypothesized that direct contact between ASM and CD4(+) T cells facilitated the transfer of anti-apoptotic proteins via nanotubes, resulting in increased survival of activated CD4(+) T cells. CD4(+) T cells, isolated from PBMCs of healthy subjects, when activated and cocultured with ASM cells for 24 h, formed nanotubes that were visualized by immunofluorescence and atomic force microscopy. Cell-to-cell transfer of the fluorescent dye calcein-AM confirmed cytoplasmic communication via nanotubes. Immunoreactive B cell lymphoma 2 (Bcl-2) and induced myeloid leukemia cell differentiation protein (Mcl-1), two major anti-apoptotic proteins, were present within the nanotubes. Downregulation of Mcl-1 by small interfering RNA in ASM cells significantly increased T cell apoptosis, whereas downregulation of Bcl-2 had no effect. Transfer of GFP-tagged Mcl-1 from ASM cells to CD4(+) T cells via the nanotubes confirmed directionality of transfer. In conclusion, activated T cells communicate with ASM cells via nanotube formation. Direct transfer of Mcl-1 from ASM to CD(+) T cells via nanotubes is involved in T cell survival. This study provides a novel mechanism of survival of CD4(+) T cells that is dependent on interaction with a structural cell.

T cell-induced airway smooth muscle cell proliferation via the epidermal growth factor receptor.

Allergic asthma is a heterogeneous disease with no curative therapies. T cells infiltrate the airway smooth muscle (ASM) layer and may be implicated in airway remodeling and the increase of ASM mass, a cardinal feature of asthma. The mechanism by which CD4(+) T cells drive airway remodeling remains unknown. This study sought to determine the T cell-mediated mechanism of ASM cell proliferation. We hypothesized that CD4(+) T cells adhere to ASM cells via CD44, and induce ASM cell proliferation through the activation of the epidermal growth factor receptor (EGFR). A coculture model showed that the contact of antigen-stimulated CD4(+) T cells with ASM cells induced high levels of EGFR ligand expression in CD4(+) T cells and the activation of matrix metalloproteinase (MMP)-9, required for the shedding of EGFR ligands. The inhibition of EGFR and MMP-9 prevented the increase of ASM cell proliferation after coculture. The hyaluronan receptor CD44 is the dominant mediator of the tight adherence of T cells to ASM and is colocalized with MMP-9 on the cell surface. Moreover, the neutralization of CD44 prevents ASM cell hyperplasia. These data provide a novel mechanism by which antigen-stimulated CD4(+) T cells induce the remodeling indicative of a direct trophic role for CD4(+) T cells.

Insights into asthmatic airway remodelling through murine models.

Asthma is a chronic disorder of the airways associated in many instances with structural changes of the airways, termed airway remodelling. Irritant and allergen-induced murine models have been used to further understand the mechanisms of airway remodelling. The infiltration of the airways by inflammatory cells, such as T lymphocytes, mast cells, eosinophils, neutrophils and macrophages after repeated allergen challenges may be important effectors in the initiation and perpetuation of airway remodelling through the release of inflammatory mediators and growth factors. Interleukins-4 and -13 have been widely studied in experimental models, and have been shown to play a significant role in airway remodelling. Recently, a role for Th17 cells has been established. Other mediators involved in this process are ligands of the epidermal growth factor receptor, matrix metalloproteases and cysteinyl leukotrienes. A better understanding of the mechanisms leading to airway remodelling in allergic diseases may lead to the identification of novel therapeutic strategies but validation in human subjects is required for potential targets.

EGF receptor activation during allergic sensitization affects IL-6-induced T-cell influx to airways in a rat model of asthma.

EGF receptor (EGFR) is involved in cell differentiation and proliferation in airways and may trigger cytokine production by T cells. We hypothesized that EGFR inhibition at the time of allergic sensitization may affect subsequent immune reactions. Brown Norway rats were sensitized with OVA, received the EGFR tyrosine kinase inhibitor, AG1478 from days 0 to 7 and OVA challenge on day 14. OVA-specific IgE in serum and cytokines and chemokines in BAL were measured 24 h after challenge. To evaluate effects on airway hyperresponsiveness (AHR), rats were sensitized, treated with AG1478, intranasally challenged, and then AHR was assessed. Furthermore chemotactic activity of BALF for CD4(+) T cells was examined. The eosinophils, neutrophils and lymphocytes in BAL were increased by OVA and only the lymphocytes were reduced by AG1478. OVA significantly enhanced IL-6 concentration in BAL, which was inhibited by AG1478. However AHR, OVA-specific IgE and IL-4 mRNA expression in CD4(+) T cells were not affected by AG1478. BALF from OVA-sensitized/challenged rats induced CD4(+) T-cell migration, which was inhibited by both AG1478 treatment in vivo and neutralization of IL-6 in vitro. EGFR activation during sensitization may affect the subsequent influx of CD4(+) T cells to airways, mainly mediated through IL-6.