Purification and Characterization of Trypsin-Like Protease(s) from Organic Dust and Studies of Their Effects on Induction of Inflammatory Mediator Expression in Lung Epithelial Cells

Details

• Personal Author:
  Boggaram V ; Keshava S ; Kusampudi S ; Meganathan V
• Description:
  Rationale. Exposure to aerosolized organic dust in concentrated animal feeding operations (CAFOs) is a risk factor for the development of respiratory diseases. Recently, proteases in organic dusts were found to induce lung inflammatory mediator expression. To gain a better understanding of the role of organic dust-derived proteases in lung inflammation, trypsin-like protease(s) were purified from poultry organic dust and characterized and their effects on inflammatory mediator expression in lung epithelial cells studied. Methods. Trypsin-like proteases were purified from aqueous extracts of poultry organic dust by batch-wise affinity chromatography using Benzamidine Sepharose 6B. Trypsin and elastase activities were determined by colorimetric assay using Na-Benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA) and N-succinyl-Ala-Ala pro-Phe p-nitroanilide (SAPNA), respectively. Purified protease(s) was analyzed by SDS-PAGE and silver staining and by gelatin zymography. Mass spectrometry was used for identification of purified protease(s) and its amino terminus. The effects of purified protease(s) on inflammatory gene expression in Beas2B and normal human bronchial epithelial (NHBE) cells were analyzed by real-time qRT-PCR, ELISA, and western blotting. Cellular and mitochondrial reactive oxygen species (ROS) were probed using 2',7'-dichlorofluorescein diacetate (DCFDA) and MitoSOX Red labeling, respectively. Protease activated receptor activation (PAR) was analyzed by intracellular calcium release using Fluo-4AM dye by confocal microscopy. Results. Affinity purification of proteases resulted in a single major protein band of approx. 25 kDa and a minor band of approx. 22 kDa. Purified protease(s) displayed activity in gelatin zymography. Purified protease(s) displayed trypsin but not elastase activity that was inhibited by serine-protease inhibitors. Analysis by mass spectrometry identified the purified protein(s) as chicken trypsin II-P29 with trace amounts of chicken trypsin I-P38. Treatment of Beas2B and NHBE cells with the purified protease(s) increased pro-interleukin(IL)-1β, ICAM-1, IL-6 and IL-8 protein and mRNA levels that were suppressed by serine protease inhibitors. Purified protease(s) treated Beas2B cells exhibited increase in cellular and mitochondrial ROS production that were attenuated by serine protease inhibitors, antioxidants and MitoTEMPO. Antioxidants were also found to suppress induction of inflammatory mediators by purified protease(s). Calcium imaging studies with protease activated receptor (PAR)-peptide agonists, thrombin and human trypsin suggested activation of PAR 1-4 by purified protease(s). Conclusions. Our studies have shown that chicken trypsin II-P29 and trypsin I-P38 are constituents of poultry organic dust that activate ROS production to induce inflammatory mediator expression in lung epithelial cells. [Description provided by NIOSH]
• Subjects:
  Agriculture Dust Food Industry Immune System Occupational Health Respiratory System Respiratory Tract Diseases Safety
• Keywords:
  Immune Reaction Organic Dusts Poultry Respiratory Diseases
• ISSN:
  1073-449X
• Document Type:
  Text
• Funding:
  Cooperative Agreement
• Genre:
  Abstract or Summary
• Place as Subject:
  OSHA Region 6 ; Texas
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Volume:
  205
• NIOSHTIC Number:
  nn:20070057
• Citation:
  Am J Respir Crit Care Med 2022 May; 205(Abstract Issue):A3582
• Email:
  shilpa.kusampudi@uthct.edu
• Federal Fiscal Year:
  2022
• NORA Priority Area:
  Agriculture, Forestry and Fishing
• Performing Organization:
  University of Texas Health Center at Tyler
• Peer Reviewed:
  False
• Start Date:
  20010930
• Source Full Name:
  American Journal of Respiratory and Critical Care Medicine
• Supplement:
  Abstract Issue
• End Date:
  20270929
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:281ec70eeff2f4f9dc021fc0dcba6c4a897618c161aa885109bdf564071ee772f73d3487316eecf4811e5c54afe33288dec03d38ec366e784e1b2fa76dad46a8
• Download URL:
  https://stacks.cdc.gov/view/cdc/208355/cdc_208355_DS1.pdf
• File Type:
  [PDF - 29.57 KB ]