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George Maiti, Jihane Frikeche, Ye Eun Jeong, Shukti Chakravarti; Corneal proteoglycan lumican modulates lipid rafts and innate immunity. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2064. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Lumican, a member of the SLRP family is widely present in the cornea, conjunctiva and other parts of the eye, and has a significant role in modulating the local inflammatory milieu. However, the underlying toll-like receptor 4 (TLR4) mediated molecular mechanisms are not fully understood. Our prior work shows that lumican enhances TLR4 mediated host response to bacterial lipopolysaccharides (LPS) through its interactions with CD14, an LPS-adaptor present in Caveolin-1 (Cav-1) enriched lipid rafts on neutrophil and macrophage cell surfaces. We hypothesize that lumican localizes to these Cav-1 enriched lipid microdomains and regulates TLR4 recruitment to lipid rafts.
Wild type (WT) and lumican-null (Lum-/-) mouse macrophages (MØ), unstimuated or treated with LPS, were stained with Alexa-Fluor 488® cholera toxin B and examined by confocal microscopy. Fluorescently stained rafts were quantified from 5 independent fields. To assess lipid raft composition, and whether, in WT MØ, lumican localizes in these plasma membrane extracts, WT and Lum-/- MØ were fractionated by sucrose gradient density centrifugation and analyzed by SDS-PAGE and immunoblotting for Cav-1, CD14, TLR4 and lumican. Also, RAW 264.7, a macrophage-like cell line was treated with and without LPS and recombinant lumican (rLum) and raft composition analyzed by sucrose gradients as described. Surface TLR4 was quantified on peritoneal macrophages from Wt and Lum-/- mice by FACS analysis.
Confocal microscopy showed Lum-/- MØ to have lower fluorescently labeled cell surface rafts compared to WT MØ. Immunoblotting of sucrose gradient fractions showed increased enrichment of Cav-1 raft fractions in WT MØ and in RAW cells treated with rLum. We also tested lumican regulation of MyD88 dependent (p65 nuclear translocation) versus TRIF-dependent (phospho-IRF3) down stream TLR4 signal transduction. Exposure to rLum increased both axes of LPS response in mouse MØ and RAW cells. FACS analysis showed higher surface TLR4 on WT MØ compared to Lum-/- MØ.
Lumican may be interacting directly with Cav-1 to modulate lipid raft structure and functions and promote both MyD88 and TRIF driven signaling arms downstream of LPS recognition. Further studies are underway to determine whether lumican promotes surface retention of TLR4 in lpid rafts and its interatcions with LPS.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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