In-Vitro Exposure Of Murine Macrophages To Aspergillus Fumigatus Cell Wall B-Glucan

T. Moua , D. Hebrink , A. H. Limper

Author address: 

Mayo Clinic, Rochester, MN

Abstract: 

Rationale: Aspergillus fumigatus is a notable opportunistic pulmonary pathogen, accounting for relatively high infection-related morbidity in immunocompromised hosts. Previous studies note induction of inflammatory responses during conidial swelling and germ tube formation with exposure to fungal b-glucan. We studied the inflammatory response in mouse macrophages exposed to aspergillus fumigatus cell wall particulate. Methods: Mouse macrophages purchased through ATCC with cell line designation RAW 264.7 were used for experimentation, after growth to confluency in standard T-75 flasks. Passages for experimental cell lines did not exceed 4. Varying concentrations of 50mcg/ml to 200mcg/ml per well of stock aspergillus glucan (2mg/ml) were created for cell challenge. Stock LPS (1mcg/ml) and saccaromyces cell wall glucan (2mg/ml) diluted to varying concentrations were used as controls. Macrophages were cultured onto 96 well sterile culture plates at concentrations of 200,000 cells per well and allowed to sit at 37 degrees for 4hrs before challenge. Varying concentrations of aspergillus glucan, saccaromyces glucan, and LPS controls in triplicates were added to cell cultures and incubated overnight at 37degrees. Plates were then spun and supernatant collected and frozen in 200-300ul aliquots. ELISA was performed on collected supernatant using commercially available eBiosciences kits. Results: TNFa ELISA was elevated, indicating a robust inflammatory Th1 response in mouse macrophages exposed to aspergillus glucan. Th2 and Th17 responses as indicated by repeat Il-4 and Il-23 ELISA at varying concentrations of glucan exposure appeared blunted compared to TNFa. Conclusions: In-vitro exposure to Aspergillus fungal glucan induces a pro-inflammatory Th1 response in mouse macrophages without significant initial Th2 or Th17 responses.
2011

abstract No: 

C24

Full conference title: 

American Thoracic Society
    • ATS 2011