Abstract
Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.
Highlights
Macrophages and other sentinel cells detect infections with the use of pattern recognition receptors, which recognize compounds produced by entire groups of related pathogens, by not by host cells, the so-called pathogen-associated molecular patterns (PAMPs)
BS-LPS binding to metabolically poisoned peritoneal exudate macrophages (PEMs) at 37°C was strongly inhibited by anti-CD14 mAb (Fig 1C), but unaffected by CD36 or scavenger receptor (SR)-A deficiency (S1A Fig)
LPS-binding protein (LBP) seems to be the serum component responsible for the acceleration of biotinylated S-LPS (bS-LPS) binding to rCD14, because the addition of 200 ng/ml rLBP to Bovine serum albumin (BSA)-phosphate-buffered saline (PBS) increased the 1-h binding to levels even exceeding those observed in the presence of serum (S2C Fig)
Summary
Macrophages and other sentinel cells detect infections with the use of pattern recognition receptors, which recognize compounds produced by entire groups of related pathogens, by not by host cells, the so-called pathogen-associated molecular patterns (PAMPs). Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, is the major PAMP signifying infections caused by these pathogens It is recognized through the heterodimer of Toll-like receptor 4 (TLR4) with the secreted protein MD-2 [1]. TLR4/MD-2/LPS complexes undergo dynamin-dependent endocytosis through clathrin-coated pits, and within endosomes they induce the second wave of signaling, mediated by the adaptor pair TRAM/TRIF [4,5,6] This TRIF-dependent pathway mediates activation of interferon-regulatory factor 3 and delayed activation of NF-κB, and is responsible for the induction of the majority of LPS-inducible genes, including type I interferons, interferon-inducible genes and some chemokines, such as RANTES [4, 7]
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