Tumor necrosis factor α decreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription.

Inflammatory diseases of the gut are associated with altered electrolyte and water transport, leading to the development of diarrhea. Epithelially expressed aquaporins (AQPs) are downregulated in inflammation, although the mechanisms involved are not known. We hypothesized that AQP3 expression in intestinal epithelial cells is altered in intestinal inflammation and that these changes are driven by tumor necrosis factor (TNF) α Human colonic adenocarcinoma (HT-29) cells were treated with TNFα to investigate signaling mechanisms in vitro. AQP3 expression was assessed by real-time PCR and radiolabeled glycerol uptake, with select inhibitors and a luciferase reporter construct used to further elucidate intracellular signaling. AQP3 expression was downregulated in HT-29 cells treated with TNFα Luciferase reporter construct experiments revealed that TNFα downregulated constitutive transcriptional activity of the AQP3 promoter, and inhibition of MEK/ERK and nuclear factor κB (NF-κB) signaling prevented the decrease in AQP3 mRNA expression. Constitutive AQP3 expression was suppressed by specificity protein (Sp) 3, and knockdown of this transcription factor bound to the AQP3 promoter was able to partially prevent the TNFα-induced downregulation of AQP3. TNFα signals through MEK/ERK and NF-κB to enhance the negative transcriptional control of AQP3 expression exerted by Sp3. Similar mechanisms regulate numerous ion channels, suggesting a common mechanism by which both ion and water transport are altered in inflammation.

Interferon-γ suppresses intestinal epithelial aquaporin-1 expression via Janus kinase and STAT3 activation.

Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway.

Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes.

Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

Human rhinovirus-induced epithelial production of CXCL10 is dependent upon IFN regulatory factor-1.

Human rhinovirus (HRV) infections are associated with exacerbations of lower-airway diseases. HRV-induced production of proinflammatory chemokines, such as CXCL10, from infected airway epithelial cells may play a role in the pathogenesis of exacerbations. We have previously shown that the MAP/ERK kinase (MEK) pathway selectively down-regulates HRV-16-induced epithelial production of CXCL10 by modulating nuclear translocation and/or binding of IFN regulatory factor (IRF)-1 with the CXCL10 promoter. Using primary human bronchial epithelial cells (HBEs) and the BEAS-2B bronchial epithelial cell line, we have further evaluated the role of IRF-1 in HRV-16-induced epithelial CXCL10 production. We demonstrate that HRV-16 induced the expression of both IRF-1 mRNA and protein in a time-dependent manner. Interestingly, MEK1 pathway inhibition with PD98059 or U0126 significantly enhanced HRV-16-induced IRF-1 mRNA levels in BEAS-2B cells and HBEs, although IRF-1 protein expression was only enhanced in HBEs. Using short interfering RNA (siRNA), we both inhibited HRV-16-induced IRF-1 expression and reduced nuclear translocation and/or binding of IRF-1 to the CXCL10 promoter. Knockdown of IRF-1 also led to a significant reduction in HRV-16-induced CXCL10 production, confirming that IRF-1 is directly involved in HRV-16-induced CXCL10 expression in epithelial cells. Moreover, pronounced IRF-1 knockdown abrogated the enhancement of CXCL10 normally induced by inhibitors of the MEK1 pathway. Phosphatase experiments indicate that IRF-1 binding to the CXCL10 promoter is not dependent upon its phosphorylation state. We conclude that HRV-16-induced CXCL10 production is dependent upon IRF-1, and that the MEK1 pathway-dependent suppression of CXCL10 expression is also mediated via effects on IRF-1.

Nitric oxide inhibits IFN regulatory factor 1 and nuclear factor-kappaB pathways in rhinovirus-infected epithelial cells.

BACKGROUND: Nitric oxide (NO) has previously been shown to inhibit human rhinovirus (HRV) replication in airway epithelial cells and to inhibit rhinovirus-induced epithelial cytokine and chemokine production independently of its effects on viral replication by modulating nuclear translocation and binding of transcription factors. OBJECTIVE: To define the molecular mechanisms by which NO inhibits HRV-16-induced epithelial production of CXCL10 by affecting nuclear translocation and binding of nuclear factor-kappaB (NF-kappaB) and IFN regulatory factor 1 (IRF-1). METHODS: Cultured human airway epithelial cells were infected with HRV-16 in the absence or presence of a NO donor, or were preincubated with 2 highly selective inhibitors of inhibitor of kappaB kinase (IKK)beta and then infected with HRV-16. Effects on the NF-kappaB and IRF-1 pathways were examined by using electrophoretic mobility shift assays, Western blotting, and real-time RT-PCR. RESULTS: Nitric oxide directly inhibited the binding of both recombinant NF-kappaB p50 protein and recombinant IRF-1 to their recognition sequences from the CXCL10 promoter. NO also inhibited phosphorylation of the NF-kappaB inhibitor, IkappaBalpha, in HRV-16-infected cells. In addition, both NO and inhibitors of IKKbeta inhibited viral induction of IRF-1 mRNA and protein. CONCLUSIONS: Nitric oxide blocks rhinovirus-mediated activation and nuclear translocation of both NF-kappaB and IRF-1. NO also directly inhibits the binding of each of these transcription factors to their respective recognition sites in the CXCL10 promoter. In addition, the ability of HRV-16 to induce epithelial expression of IRF-1 is dependent, at least in part, on viral activation of NF-kappaB.

Selective transcriptional down-regulation of human rhinovirus-induced production of CXCL10 from airway epithelial cells via the MEK1 pathway.

Human rhinovirus (HRV) infections can trigger exacerbations of lower airway diseases. Infection of airway epithelial cells induces production of a number of proinflammatory chemokines that may exacerbate airway inflammation, including CXCL10, a chemoattractant for type 1 lymphocytes and NK cells. Primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line were used to examine the role of MAPK pathways in HRV-16-induced production of CXCL10. Surprisingly, PD98059 and U0126, two inhibitors of the MEK1/2-ERK MAPK pathway, significantly enhanced HRV-16-induced CXCL10 mRNA and protein. This enhancement was not seen with IFN-beta-induced production of CXCL10. Studies using small interfering RNA revealed that knockdown of MEK1, but not MEK2, was associated with enhanced HRV-induced CXCL10 production. Promoter construct studies revealed that PD98059 and U0126 enhanced HRV-16-induced transcriptional activation of CXCL10. HRV-16-induced promoter activation was regulated by two NF-kappaB binding sites, kappaB1 and kappaB2, and by an IFN-stimulated response element. Inhibitors of the MEK1/2-ERK pathway did not alter HRV-16-induced activation of tandem repeat kappaB1 or kappaB2 constructs, nor did they alter HRV-16-induced nuclear translocation/binding of NF-kappaB to either kappaB1 or kappaB2 recognition sequences. Furthermore, PD98059 and U0126 did not alter phosphorylation or degradation of IkappaBalpha. In contrast, inhibitors of the MEK1/2-ERK pathway, and small interfering RNA knockdown of MEK1, enhanced nuclear translocation/binding of IFN regulatory factor (IRF)-1 to the IFN-stimulated response element recognition sequence in HRV-16 infected cells. We conclude that activation of MEK1 selectively down-regulates HRV-16-induced expression of CXCL10 via modulation of IRF-1 interactions with the gene promoter in human airway epithelial cells.

Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells.

BACKGROUND: Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease. Nitric oxide (NO) inhibits HRV replication in human airway epithelial cells and suppresses HRV-induced epithelial production of several cytokines and chemokines. OBJECTIVE: We sought to delineate the mechanisms by which NO inhibits HRV-induced epithelial production of CXCL10, a chemoattractant for type 1 T cells and natural killer cells. METHODS: Primary human bronchial epithelial cells or cells of the BEAS-2B human bronchial epithelial cell line were exposed to HRV-16 in the presence or absence of the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate). A cGMP analogue and an inhibitor of soluble guanylyl cyclase were used to examine the role of the cyclic guanosine monophosphate (cGMP) pathway in the actions of NO. BEAS-2B cells were transfected with CXCL10 promoter-luciferase constructs and the effects of PAPA NONOate were examined to study mechanisms of transcriptional regulation. Electrophoretic mobility shift assays were also used. RESULTS: PAPA NONOate inhibited HRV-16-induced increases in CXCL10 mRNA and protein. Inhibition of CXCL10 production occurred through a cGMP-independent pathway. PAPA NONOate inhibited HRV-16-induced CXCL10 transcription by blocking nuclear translocation, binding, or both of both nuclear factor kappaB and IFN response factors (IRFs) to their respective recognition elements in the CXCL10 promoter. CONCLUSIONS: NO inhibits HRV-16-induced production of CXCL10 by inhibiting viral activation of nuclear factor kappaB and of IRFs, including IRF-1, through a cGMP-independent pathway. The broad-ranging inhibition of HRV-induced epithelial cytokine and chemokine production by NO suggests a potential therapeutic utility of NO donors in viral exacerbations of asthma and chronic obstructive pulmonary disease.

Human rhinovirus infection enhances airway epithelial cell production of growth factors involved in airway remodeling.

BACKGROUND: Childhood human rhinovirus (HRV) infections are associated with an increased risk of asthma. We reasoned that HRV infections might be important in the pathogenesis of airway remodeling, thereby providing a mechanism by which these children are at risk of asthma. OBJECTIVE: We sought to determine whether HRV infection of airway epithelial cells regulates production of growth factors associated with airway remodeling and to determine whether vascular endothelial growth factor (VEGF) was upregulated in airways during HRV-induced natural colds. METHODS: Cultured human airway epithelial cells were infected with HRV. Amphiregulin, activin A, and VEGF protein levels were assayed by means of ELISA, and VEGF mRNA was quantified by using real-time RT-PCR. Pharmacologic inhibitors were used to assess the role of mitogen-activated protein kinase and nuclear factor kappaB pathways. Nasal lavage samples from subjects with confirmed natural HRV infections were assayed for VEGF protein and compared with baseline levels and with control levels. RESULTS: HRV infection upregulated amphiregulin, activin A, and VEGF protein levels compared with control media (P < .05). VEGF gene expression was maximally induced 3 hours after infection. HRV-induced generation of VEGF was regulated by p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways but did not depend on nuclear factor kappaB activation. In subjects with HRV infections, VEGF levels during peak cold symptoms were significantly higher than at baseline (P = .005) or in control subjects (P < .01). CONCLUSION: HRV-16 infection upregulates amphiregulin, activin A, and VEGF in airway epithelial cells, and HRV infections in vivo upregulate airway VEGF production.

Human airway epithelial cells produce IP-10 (CXCL10) in vitro and in vivo upon rhinovirus infection.

Human rhinovirus (HRV) infections trigger exacerbations of asthma and chronic obstructive pulmonary disease (COPD) and are associated with lymphocytic infiltration of the airways. We demonstrate that infection of primary cultures of human airway epithelial cells, or of the BEAS-2B human bronchial epithelial cell line, with human rhinovirus type 16 (HRV-16) induces expression of CXCL10 [IFN-gamma-inducible protein 10 (IP-10)], a ligand for the CXCR3 receptor found on activated type 1 T lymphocytes and natural killer cells. IP-10 mRNA reached maximal levels 24 h after HRV-16 infection then declined, whereas protein levels peaked 48 h after infection with no subsequent new synthesis. Cytosolic levels of AU-rich factor 1, a protein associated with mRNA destabilization, increased beginning 24 h after HRV-16 infection. Generation of IP-10 required virus capable of replication but was not dependent on prior induction of type 1 interferons. Transfection of synthetic double-stranded RNA into epithelial cells induced robust production of IP-10, whereas transfection of single-stranded RNA had no effect. Induction of IP-10 gene expression by HRV-16 depended upon activation of NF-kappaB, as well as other transcription factor recognition sequences further upstream in the IP-10 promoter. In vivo infection of human volunteers with HRV-16 strikingly increased IP-10 protein in nasal lavages during symptomatic colds. Levels of IP-10 correlated with symptom severity, viral titer, and numbers of lymphocytes in airway secretions. Thus IP-10 may play a role in the pathogenesis of HRV-induced colds and in HRV-induced exacerbations of COPD and asthma.