TLR4 INDUCED AUTOPHAGY INCREASES BACTERIAL INTERNALIZATION BY ENTEROCYTES
Matthew D. Neal, MD, Chhinder P. Sodhi, PhD, Ward M. Richardson, MD, Amin Afrazi, BS, Richard Siggers, PhD, David J. Hackam, MD, PhD, Division of Pediatric Surgery, Department of Surgery, University of Pittsburgh Medical Center
Introduction: We have recently identified a critical role for the LPS receptor, Toll-like receptor 4 (TLR4) in the pathogenesis of intestinal inflammation, and we have shown that enterocytes are capable of TLR4 mediated internalization of Gram negative bacteria (SIS 2004). The cellular processes that mediate internalization of bacteria are unknown. TLR4 has recently been shown to be an environmental sensor for autophagy in macrophages, and autophagy has been shown to be critical in the processing of internalized bacteria by epithelial cells. We now hypothesize that enterocytes undergo TLR4 induced autophagy and that autophagy plays a role in processing of internalized bacteria.
Methods: Enterocytes lacking the ability to undergo autophagy were engineered using siRNA to the critical autophagy gene, ATG16 (ATG16-/-). TLR4 knock-out cells were generated with shRNA (TLR4-/-). Wild-type intestinal epithelial cells (IEC-6), TLR4-/-, and ATG16-/- were pre-treated with the autophagy inducer, rapamycin (50nmol/L, 18hrs), the TLR4 agonist, LPS (25ug/ml, 18hrs) and the autophagy inhibitor, chloroquine (10uM, 3hrs). To assess for internalization, cells were incubated with E.coli that were pre-labeled with green fluorescence and biotin. Following internalization (2hrs, 37C), cells were incubated with rhodamine-streptavidin to distinguish bound bacteria (red and green) from internalized (green alone). Quantification of internalization was performed by flow cytometry and confocal microscopy. Immunofluorescence was performed for the autophagosome marker, LC3 using anti-LC3 antibodies. IEC-6 cells transfected with mCherry-LC3 were incubated with LPS-coated latex beads and imaged using live-cell fluorescent microscopy.
Results: Activation of TLR4 with LPS increased LC3 expression in IEC-6 cells demonstrating that TLR4 activation leads to autophagy in enterocytes. IEC-6 were capable of internalizing E. coli, confirming our earlier findings (SIS 2004). Strikingly, activation of autophagy with both rapamycin and LPS increased internalization (control 11.5, rapa 18, LPS 16.7, p<0.05, recorded as % internalization). Inhibition of autophagy with chloroquine did not significantly change internalization (11.7 vs. 11.5, p=0.71). Enterocytes deficient in autophagy (ATG16-/-) were able to internalize but had no change with rapamycin, LPS, or choloquine (control 11.8, rapa 11.1, LPS 11.6, p=NS). Internalization was significantly inhibited in TLR4 knock-out cells with no increase after stimulation of autophagy (control 10.6, TLR4-/- 0.9, rapa-TLR4-/- 1.1, LPS-TLR4-/- 0.7, p<0.01), indicating the critical role for TLR4. LC3 was found to co-localize with E. coli and LPS-coated beads, indicating that autophagosomes form around internalized bacteria.
Conclusion: These data prove a novel and unexpected link between TLR4 activation and autophagy in enterocytes leading to the internalization of gram negative bacteria. This new paradigm linking autophagy with bacterial internalization may have broad implications in our understanding of the factors leading to the regulation of intestinal inflammation and systemic sepsis.
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