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069 METABOLICALLY STRESSED MACROPHAGES DISPLAY REDUCED PRO-INFLAMMATORY CYTOKINE PRODUCTION AND INCREASED INTRACELLULAR BACTERIA WHEN CHALLENGED WITH COMMENSAL ESCHERICHIA COLI J Caldwell, DM McKay Epithelial barrier function is a critical component of innate immunity. We have shown that metabolically stressed T84 epithelial cell monolayers display a marked decrease in barrier function upon exposure to non-pathogenic, non-invasive E. coli, resulting in increased transcytosis of the bacteria. In vivo it is unlikely that metabolic stress is restricted to the epithelium alone, but also affects cells within the mucosa such as macrophages. Thus, the affect of metabolic stress on macrophages, as for example a consequence of ischemia or inflammation, could be an altered ability to recognize, handle, and respond to bacteria that breach the epithelial barrier. We tested this supposition using the human THP-1 monocytic cell line that was differentiated into a macrophage-like phenotype by exposure to PMA.
Gastrointestinal Research Group, University of Calgary, Calgary, Alberta
METHODS: THP-1 cells were treated with E. coli (strain HB101, 106 cfu/mL) ± dinitrophenol (DNP; metabolic stressor, 0.1 mM) and 6 h later the following parameters were examined: mitochondrial activity via the MTT assay, caspase-3 cleavage as marker of apoptosis, bacterial internalization by the gentamicin assay (expressed as % extracellular bacterial), TNF-alpha measured by ELISA, and levels of toll-like receptor (TLR)-4 mRNA determined using RT-PCR.
RESULTS: Exposure to DNP evoked significant metabolic stress in the mitochondria, as indicated by a ~50% decrease in the MTT assay (n=3). However, this was not accompanied by evidence of enhanced apoptosis as the levels of total and cleaved caspase-3 were unchanged (n=4). Exposure to metabolic stress, while not affecting the viability of extracellular E. coli, did result in a consistent (n=6) and significant increase in the percentage of viable intracellular bacteria (6.8% in stressed vs. 4.5% in non-stressed (P<0.05)) and a reduction in E. coli-evoked secretion of TNF-alpha (1.7±0.4 in stressed vs. 3.9±0.5 ng/mL in non-stressed THP-1 cells; P<0.05, n=5). Despite the reduced TNF-alpha secretion TLR4 mRNA was unaltered as a consequence of DNP exposure (n=4).
CONCLUSIONS: Metabolic stress that can arise from a variety of insults has profound effects on macrophages resulting in increased intracellular-bacteria, and a considerable, although reduced, production of TNF-alpha which could feedback onto the epithelium and participate in the pathophysiology of IBD. Macrophages are an important component of the intestinal milieu and analysis of their function under normal and stressed conditions will yield significant novel insights on enteric pathophysiological reactions.
Funded by the CCFC and CIHR