Single Pathogen Challenge with Agents of the Bovine Respiratory Disease Complex.

Bovine respiratory disease complex (BRDC) is an important cause of mortality and morbidity in cattle; costing the dairy and beef industries millions of dollars annually, despite the use of vaccines and antibiotics. BRDC is caused by one or more of several viruses (bovine respiratory syncytial virus, bovine herpes type 1 also known as infectious bovine rhinotracheitis, and bovine viral diarrhea virus), which predispose animals to infection with one or more bacteria. These include: Pasteurella multocida, Mannheimia haemolytica, Mycoplasma bovis, and Histophilus somni. Some cattle appear to be more resistant to BRDC than others. We hypothesize that appropriate immune responses to these pathogens are subject to genetic control. To determine which genes are involved in the immune response to each of these pathogens it was first necessary to experimentally induce infection separately with each pathogen to document clinical and pathological responses in animals from which tissues were harvested for subsequent RNA sequencing. Herein these infections and animal responses are described.

Antiviral Efficacy of a Respiratory Syncytial Virus (RSV) Fusion Inhibitor in a Bovine Model of RSV Infection.

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants. Effective treatment for RSV infection is a significant unmet medical need. While new RSV therapeutics are now in development, there are very few animal models that mimic the pathogenesis of human RSV, making it difficult to evaluate new disease interventions. Experimental infection of Holstein calves with bovine RSV (bRSV) causes a severe respiratory infection that is similar to human RSV infection, providing a relevant model for testing novel therapeutic agents. In this model, viral load is readily detected in nasal secretions by quantitative real-time PCR (qRT-PCR), and cumulative symptom scoring together with histopathology evaluations of infected tissue allow for the assessment of disease severity. The bovine RSV model was used to evaluate the antiviral activity of an RSV fusion inhibitor, GS1, which blocks virus entry by inhibiting the fusion of the viral envelope with the host cell membrane. The efficacy of GS1, a close structural analog of GS-5806 that is being developed to treat RSV infection in humans was evaluated in two randomized, blind, placebo-controlled studies in bRSV-infected calves. Intravenous administration of GS1 at 4 mg/kg of body weight/day for 7 days starting 24 h or 72 h postinoculation provided clear therapeutic benefit by reducing the viral load, disease symptom score, respiration rate, and lung pathology associated with bRSV infection. These data support the use of the bovine RSV model for evaluation of experimental therapeutics for treatment of RSV.

Equine IgE responses to non-viral vaccine components.

Vaccination of horses is performed annually or semi-annually with multiple viral antigens, either in a combination vaccine or as separate injections. While this practice undoubtedly prevents infection from such diseases as rabies, equine influenza, West Nile virus, and equine herpes virus, the procedure is not without repercussions. Hypersensitivity reactions, including fatal anaphylactic shock, after vaccination, although uncommon, have increased in incidence in recent years. Studies reported herein document the development of IgE antibodies against non-target antigen components of equine viral vaccines. We hypothesize that viral vaccines can induce an IgE response to non-target antigens, which could elicit an adverse response after vaccination with another viral vaccine containing the same component. In one study IgE responses to components of West Nile virus vaccine were evaluated by ELISA before and after vaccination in 30 horses. In a second five-year study 77 horses were similarly tested for IgE antibodies against bovine serum albumin (BSA), a component of most viral vaccines. Mast cell sensitization was evaluated in horses with high, moderate, and negative serum BSA specific IgE using an intradermal skin test with BSA. Over the five-year period high IgE responder horses showed gradually increasing BSA specific serum IgE levels and positive skin test reactivity, yet none had an adverse event. Sera from horses that had developed adverse vaccine reactions were also tested for IgE antibodies. Several of these horses had extremely high levels of BSA-specific IgE. These data suggest that non-essential protein components of vaccines may sensitize horses for future adverse responses to vaccination.

Lipopolysaccharide initiates a positive feedback of epidermal growth factor receptor signaling by prostaglandin E2 in human biliary carcinoma cells.

Bacterial products (e.g., LPS) are viewed as critical stimuli in inflammation-associated cancer. Cyclooxygenase 2 (COX-2), a major effector of LPS, and EGFR, are key to carcinogenesis, notably in the hepatobiliary tract. In this study, we tested the hypothesis that LPS can initiate an interaction between the epidermal growth factor receptor (EGFR) and COX-2 pathways. We examined the effect of LPS in biliary carcinoma cells that displayed constitutive COX-2 expression and PGE(2) production and in normal human biliary epithelial cells in which COX-2/PGE(2) expression was virtually absent. LPS induced early phosphorylation of EGFR and ERK1/2 in both types of cells, which reached maximum levels within 30 min (first phase). However, only the carcinoma cells showed a second significant rise in both EGFR and ERK phosphorylation 6 h after exposure to LPS (second phase). Inhibition of COX-2/PGE(2) production prevented the second, but not the first, phase of EGFR and ERK1/2 phosphorylation, implicating COX-2/PGE(2) in the second phase of phosphorylation. LPS induced COX-2-derived PGE2 production at 4 h, which was before the rise in the second phosphorylation that occurred at 6 h. Exogenous PGE(2) also caused EGFR activation via a signaling pathway involving TACE-dependent TGF-alpha release. Inhibition of the second phase of EGFR phosphorylation with EGFR or COX-2 inhibitor prevented LPS-induced cell invasion in vitro, demonstrating the biological importance of this COX-2 feedback signaling in cancer cells. We conclude that LPS triggers a positive feedback loop involving COX-2/PGE(2) in biliary carcinoma cells and that this second phase of EGFR phosphorylation is implicated in cell invasion by LPS.

Multiple TLRs activate EGFR via a signaling cascade to produce innate immune responses in airway epithelium.

Toll-like receptors (TLRs) are critical for the recognition of inhaled pathogens that deposit on the airway epithelial surface. The epithelial response to pathogens includes signaling cascades that activate the EGF receptor (EGFR). We hypothesized that TLRs communicate with EGFR via epithelial signaling to produce certain innate immune responses. Airway epithelium expresses the highest levels of TLR2, TLR3, TLR5, and TLR6, and here we found that ligands for these TLRs increased IL-8 and VEGF production in normal human bronchial epithelial cells. These effects were prevented by treatment with a selective inhibitor of EGFR phosphorylation (AG-1478), a metalloprotease (MP) inhibitor, a reactive oxygen species (ROS) scavenger, and an NADPH oxidase inhibitor. In an airway epithelial cell line (NCI-H292), TNF-alpha-converting enzyme (TACE) small interfering RNA (siRNA) was used to confirm that TACE is the MP involved in TLR ligand-induced IL-8 and VEGF production. We show that transforming growth factor (TGF)-alpha is the EGFR ligand in this signaling cascade by using TGF-alpha neutralizing antibody and by showing that epithelial production of TGF-alpha occurs in response to TLR ligands. Dual oxidase 1 (Duox1) siRNA was used to confirm that Duox1 is the NADPH oxidase involved in TLR ligand-induced IL-8 and VEGF production. We conclude that multiple TLR ligands induce airway epithelial cell production of IL-8 and VEGF via a Duox1--> ROS--> TACE--> TGF-alpha--> EGFR phosphorylation pathway. These results show for the first time that multiple TLRs in airway epithelial cells produce innate immune responses by activating EGFR via an epithelial cell signaling cascade.

Regulation of interleukin-8 via an airway epithelial signaling cascade.

Airways function as an innate immune organ against airborne bacteria that are inhaled and deposited in airways. One of the mechanisms of host defense is to recruit neutrophils into airways to clear the invaders. Airway epithelial cells produce neutrophil chemoattractant interleukin (IL)-8 in response to invading bacteria. In this study we show a signaling pathway on the plasma surface of human airway epithelial NCI-H292 cells that regulate IL-8 production in response to a model inflammatory stimulus, phorbol 12-myristate 13-acetate, and a pathophysiological stimulus, gram-negative bacterial lipopolysaccharide. First, we show that EGF receptor (EGFR) and MAP kinase ERK1/2 are involved in IL-8 expression by these stimuli. Second, we show that EGFR ligand transforming growth factor (TGF)-alpha mediates IL-8 production. Third, we show that tumor necrosis factor-alpha-converting enzyme (TACE) is required for IL-8 production by cleaving EGFR proligand proTGF-alpha into soluble TGF-alpha, activating EGFR. Last, we show that dual oxidase 1 (Duox1), a homolog of NADPH oxidase in airways, mediates TACE activation and IL-8 expression via generation of reactive oxygen species. In summary, we describe a signaling pathway, Duox1-TACE-TGF-alpha-EGFR, on the surface of airway epithelial (NCI-H292) cells that mediates airway epithelial defense against bacterial infection by producing IL-8. This pathway, which also regulates mucin production in human airways, provides mechanisms for killing foreign organisms and for their clearance.

Pseudomonas lipopolysaccharide accelerates wound repair via activation of a novel epithelial cell signaling cascade.

The surface of the airway epithelium represents a battleground in which the host intercepts signals from pathogens and activates epithelial defenses to combat infection. Wound repair is an essential function of the airway epithelium in response to injury in chronic airway diseases, and inhaled pathogens such as Pseudomonas bacteria are implicated in the pathobiology of several of these diseases. Because epidermal growth factor receptor (EGFR) activation stimulates wound repair and because LPS activates EGFR, we hypothesized that LPS accelerates wound repair via a surface signaling cascade that causes EGFR phosphorylation. In scrape wounds of NCI-H292 human airway epithelial cells, high concentrations of LPS were toxic and decreased wound repair. However, lower concentrations of LPS accelerated wound repair. This effect was inhibited by treatment with a selective inhibitor of EGFR phosphorylation (AG 1478) and by an EGFR neutralizing Ab. Metalloprotease inhibitors and TNF-alpha-converting enzyme (TACE) small interfering RNA inhibited wound repair, implicating TACE. Additional studies implicated TGF-alpha as the active EGFR ligand cleaved by TACE during wound repair. Reactive oxygen species scavengers, NADPH oxidase inhibitors, and importantly small interfering RNA of dual oxidase 1 inhibited LPS-induced wound repair. Inhibitors of protein kinase C isoforms alphabeta and a TLR-4 neutralizing Ab also inhibited LPS-induced wound repair. Normal human bronchial epithelial cells responded similarly. Thus, LPS accelerates wound repair in airway epithelial cells via a novel TLR-4-->protein kinase C alphabeta-->dual oxidase 1-->reactive oxygen species-->TACE-->TGF-alpha-->EGFR phosphorylation pathway.

Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme.

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.

Dual oxidase 1-dependent MUC5AC mucin expression in cultured human airway epithelial cells.

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases. MUC5AC mucin is a major component of airway mucus, and its expression is modulated by a TNF-alpha-converting enzyme (TACE)-EGF receptor pathway that can be activated by reactive oxygen species (ROS). Dual oxidase 1 (Duox1), a homologue of glycoprotein p91(phox), is expressed in airway epithelium and generates ROS. We hypothesize that Duox1 activates TACE, cleaving pro-TGF-alpha into soluble TGF-alpha, resulting in mucin expression. To examine this hypothesis, we stimulated both normal human bronchial epithelial cells and NCI-H292 airway epithelial cells with phorbol 12-myristate 13-acetate and with human neutrophil elastase. These stimuli induced TACE activation, TGF-alpha release, and mucin expression, effects that were inhibited by ROS scavengers, implicating ROS in TACE activation. Inhibition of epithelial NADPH oxidase or knockdown of Duox1 expression with small interfering RNA prevented ROS generation, TGF-alpha release, and mucin expression by these stimuli, implicating Duox1 in TACE activation and mucin expression. Furthermore, the PKCdelta/PKC inhibitor rottlerin prevented the effects induced by phorbol 12-myristate 13-acetate and human neutrophil elastase, suggesting that PKCdelta and PKC are involved in Duox1 activation. From these results, we conclude that Duox1 plays a critical role in mucin expression by airway epithelial cells through PKCdelta/PKC-Duox1-ROS-TACE-pro-ligand-EGF receptor cascade.

Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells.

Chronic obstructive pulmonary disease (COPD) is one of the leading causes of death in the U.S. Because cigarette smoking is so importantly implicated in the pathogenesis of COPD and because mucus hypersecretion plays such an important role in COPD, understanding of the mechanisms of smoking-induced mucus hypersecretion could lead to new therapies for COPD. Cigarette smoke causes mucin overproduction via EGF receptor (EGFR) in airway epithelial cells, but the cellular mechanism remains unknown. Airway epithelial cells contain EGFR proligands on their surfaces, which can be cleaved by metalloprotease and subsequently bind to EGFR resulting in mucin production. We hypothesize that TNF-alpha-converting enzyme (TACE) is activated by cigarette smoke, resulting in increased shedding of EGFR proligand, leading to EGFR phosphorylation and mucin induction in human airway epithelial (NCI-H292) cells. Here we show that cigarette smoke increases MUC5AC production in NCI-H292 cells, an effect that is prevented by an EGFR-neutralizing antibody and by specific knockdown of transforming growth factor-alpha (TGF-alpha) using small interfering RNA (siRNA) for TGF-alpha, implicating TGF-alpha-dependent EGFR activation in the responses. Cigarette smoke increases TGF-alpha shedding, EGFR phosphorylation, and mucin production, which are prevented by metalloprotease inhibitors (GM-6001 and TNF-alpha protease inhibitor-1) and by specific knockdown of TACE with TACE siRNA, implicating TACE in smoking-induced responses. Furthermore, pretreatment with antioxidants prevents smoking-induced TGF-alpha shedding and mucin production, suggesting that reactive oxygen species is involved in TACE activation. These results implicate TACE in smoking-induced mucin overproduction via the TACE-proligand-EGFR signal pathway in NCI-H292 cells.

Tumor necrosis factor alpha-converting enzyme mediates MUC5AC mucin expression in cultured human airway epithelial cells.

Ectodomain shedding of epidermal growth factor receptor (EGFR) ligands [e.g., transforming growth factor type alpha (TGF-alpha)] and EGFR phosphorylation are implicated in mucin production in airway epithelial cells. Tumor necrosis factor alpha-converting enzyme (TACE) is reported to cleave precursor of TGF-alpha, with release of soluble mature TGF-alpha in various epithelial tissues. We hypothesized that TACE increases the shedding of TGF-alpha, resulting in EGFR phosphorylation and inducing mucin production in human airway epithelial (NCI-H292) cells. To examine this hypothesis, we stimulated NCI-H292 cells with phorbol 12-myristate 13-acetate (PMA, an activator of TACE) and pathophysiologic stimuli [lipopolysaccharide (LPS) and supernatant from the Gram-negative bacterium Pseudomonas aeruginosa (PA sup)]. PMA, PA sup, and LPS increased MUC5AC gene expression and mucin protein production, effects that were prevented by pretreatment with AG1478, a selective inhibitor of EGFR phosphorylation and by preincubation with an EGFR-neutralizing Ab or with a TGF-alpha-neutralizing Ab, implicating ligand (TGF-alpha)-dependent EGFR phosphorylation in mucin production. These stimuli induced release of soluble TGF-alpha, EGFR phosphorylation, and MUC5AC expression, which were blocked by the metalloprotease inhibitors tumor necrosis factor-alpha protease inhibitor-1 and tissue inhibitor of metalloprotease-3. We specifically knocked down the expression of metalloprotease TACE by using small interfering RNA for TACE. Knockdown of TACE inhibited PMA-, PA sup-, and LPS-induced TGF-alpha shedding, EGFR phosphorylation, and mucin production. From these results, we conclude that TACE plays a critical role in mucin production by airway epithelial cells by means of a TACE ligand-EGFR cascade in response to various stimuli.