An Extracellular Matrix Protein Lumican Regulates Innate Immune Response to Bacterial Lps Through the TLR4 Pathway

Abstract

Purpose:: Lumican (Lum) is a major leucine-rich repeat (LRR) ECM proteoglycan of the cornea. Mice deficient in lumican show delayed healing of LPS-keratitis. Here we demonstrate that lumican regulates innate immune response and the sensing of gram-negative bacterial endotoxin lipopolysaccharide (LPS) by interacting with the CD14-TLR4 pathway.

Methods:: Lum expression was measured in primary macrophages induced with LPS by qRT-PCR and ELISA. Response of Lum-null mice to LPS-mediated septic shock was determined after intraperitoneal injection of E, coli LPS (16.7 microgram/gram body weight). Proinflammatory cytokines were measured in the serum 36 hrs after the LPS challenge by Beadlyte multiplex cytokine analysis. To test innate immune response in macrophages, primary cultures were treated with LPS or a spectrum pf other PAMPs, and TNF alpha and IL6 determined by ELISA. NF-kappa B activation was measured in bone marrow-derived macrophages by EMSA. His-tagged recombinant lumican (rLum) was prepared in pSecTag2 Hek 293 cells (Invitrogen). Binding of rLum to fluorescent FITC-LPS was measured in solid phased binding assays. Binding of rLum to CD14 was tested in solution by pull down assays and CD14 bound to rLum was detected by western blotting.

Results:: Primary macrophages express lumican after LPS-stimulation. Immunohistology shows the presence of lumican on macrophages. In a LPS-septic shock model, Lum+/+ mice die within 48 to 72 hrs, but Lum-/- mice show increased survival, with less than normal pro-inflammatory cytokines in the serum. In culture Lum-/- macrophages produce significantly lower levels of TNF alpha and IL6 in response to LPS, but not the other PAMPS. In EMSA Lum-/- bone marrow-derived macrophages showed delayed induction of NF-kappa B. In solid phased binding assays rLum shows dose-dependent binding of FITC-LPS. In the pull down assays rLum specifically retained and bound CD14.

Conclusions:: The results suggest that lumican is present in the pericellular ECM of macrophages where it binds LPS and CD14 of the CD14-TLR4 receptor complex and specifically regulates innate immune response to this bacterial pathogen, thus uncovering a novel role for an ECM proteoglycan in regulating host innate immune response.

Keywords: cornea: basic science • extracellular matrix • inflammation