Cockroach (CR) exposure is a predisposing factor for the development of asthma. While many aeroallergens contain protease activity, the role of proteases in modulating the inflammatory response is unknown. We have shown that cockroach extract contains proteolytic activity which is responsible for synergistically increasing tumor necrosis factor (TNF) alpha-mediated interleukin (IL)-8 and IL-6 expression. In addition, we have preliminary data suggesting that cockroach proteases activate the protease activated receptor (PAR)-2 on airway epithelium to elicits signaling cascade to extracellular regulated kinase (ERK) activation. To understand the mechanism by which active cockroach proteases modulate airway inflammation in vitro and in vivo, we propose the following Specific Aims: 1. Establish the role of CR serine protease activity on PARs and its effects on the pro-inflammatory response. We will examine whether TNF alpha stimulation regulates PAR-2 expression by RT-PCR, flow cytometry and immunohistochemistry. We will activate PAR-2 with the activating peptide SLIGKV and investigate its role in cytokine expression. We will overexpress PAR-2 in Cos-1 cells and determine its role in inflammation. We will purify the serine protease from the crude CR extract. 2. Establish the downstream signaling events mediating CR-induced regulation of IL-8. We have evidence that ERK is activated following CR treatment. We will examine the role of transcription factors NF-IL6 and AP-1 on CR-induced synergy. This will be done by promoter deletion studies, DMA binding and transactivation studies. We will determine if NF-IL6 or AP-1 is required or sufficient for the regulation of IL-8. We will determine if crosstalk occurs between ERK and NF-kappa B pathways. Finally we will determine if CR extract transcriptionally regulates other cytokines/chemokines. 3. Establish an in vivo role for cockroach serine protease activity in modulating airway inflammation and reactivity. We will determine the role of proteases in modulating airway inflammation by ex vivo inhibition of the protease activity prior to treatment. We will establish a role for PAR-2 in modulating airway responses using PAR-2 knockout mice. Finally, we will investigate the role of endogenous TNF alpha in the airways using a TNF alpha neutralizing antibody and TNF alpha knockout mice.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL075568-04
Application #
7325685
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Noel, Patricia
Project Start
2004-12-15
Project End
2009-11-30
Budget Start
2007-12-01
Budget End
2008-11-30
Support Year
4
Fiscal Year
2008
Total Cost
$282,558
Indirect Cost
Name
Cincinnati Children's Hospital Medical Center
Department
Type
DUNS #
071284913
City
Cincinnati
State
OH
Country
United States
Zip Code
45229
Day, Scottie B; Ledford, John R; Zhou, Ping et al. (2012) German cockroach proteases and protease-activated receptor-2 regulate chemokine production and dendritic cell recruitment. J Innate Immun 4:100-10
Page, Kristen (2012) Role of cockroach proteases in allergic disease. Curr Allergy Asthma Rep 12:448-55
Lewkowich, Ian P; Day, Scottie B; Ledford, John R et al. (2011) Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation. Respir Res 12:122
Page, Kristen; Zhou, Ping; Ledford, John R et al. (2011) Early immunological response to German cockroach frass exposure induces a Th2/Th17 environment. J Innate Immun 3:167-79
Day, Scottie B; Zhou, Ping; Ledford, John R et al. (2010) German cockroach frass proteases modulate the innate immune response via activation of protease-activated receptor-2. J Innate Immun 2:495-504
Page, Kristen; Ledford, John R; Zhou, Ping et al. (2010) Mucosal sensitization to German cockroach involves protease-activated receptor-2. Respir Res 11:62
Wheeler, Derek S; Chase, Margaret A; Senft, Albert P et al. (2009) Extracellular Hsp72, an endogenous DAMP, is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4. Respir Res 10:31
Page, Kristen; Ledford, John R; Zhou, Ping et al. (2009) A TLR2 agonist in German cockroach frass activates MMP-9 release and is protective against allergic inflammation in mice. J Immunol 183:3400-8
Page, Kristen; Lierl, Kristin M; Hughes, Valerie S et al. (2008) TLR2-mediated activation of neutrophils in response to German cockroach frass. J Immunol 180:6317-24
Chase, Margaret A; Wheeler, Derek S; Lierl, Kristin M et al. (2007) Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism. J Immunol 179:6318-24

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