Abstract
Endogenous and exogenous crystalline structures are involved in various pathologies and diseases in humans by inducing sterile inflammation, mechanical stress, or obstruction of excretory organs. The best studied of these diseases is gout, in which crystallization of uric acid in the form of monosodium urate (MSU) mainly in synovial fluid of the joints leads to sterile inflammation. Though some of these diseases have been described for centuries, little is known about if and how the immune system recognizes the associated crystals. Thus, in this study we aimed at identifying possible recognition molecules of MSU using liquid chromatography-mass spectrometry (LC-MS) analysis of MSU-binding serum proteins. Among the strongest binding proteins, we unexpectedly found two transmembrane receptors, namely macrophage receptor with collagenous structure (MARCO) and low-density lipoprotein (LDL) receptor (LDLR). We show that recombinant versions of both human and mouse MARCO directly bind to unopsonized MSU and several other disease-associated crystals. Recombinant LDLR binds many types of crystals mainly when opsonized with serum proteins. We show that this interaction is predominantly mediated by LDL, which we found to bind to all crystalline structures tested except for cholesterol crystals. However, murine macrophages lacking LDLR expression do neither show altered phagocytosis nor interleukin-1β (IL-1β) production in response to opsonized crystals. Binding of LDL to MSU has previously been shown to inhibit the production of reactive oxygen species (ROS) by human neutrophils. We extend these findings and show that LDL inhibits neutrophil ROS production in response to most crystals tested, even cholesterol crystals. The inhibition of neutrophil ROS production only partly correlated with the inhibition of IL-1β production by peripheral blood mononuclear cells (PBMCs): LDL inhibited IL-1β production in response to large MSU crystals, but not small MSU or silica crystals. This may suggest distinct upstream signals for IL-1β production depending on the size or the shape of the crystals. Together, we identify MARCO and LDLR as potential crystal recognition receptors, and show that LDL binding to diverse disease-associated crystalline structures has variable effects on crystal-induced innate immune cell activation.
Highlights
Crystallopathies are a diverse set of medical conditions where the formation of crystals is the basis of or at least a contributing factor to the disease [1, 2]
In an extension of previous studies we compared the serum proteins bound to monosodium urate (MSU) crystals to those bound to the microbial pattern zymosan
Within the bound serum proteins we identified the transmembrane receptors macrophage receptor with collagenous structure (MARCO), LDLR, and CD14 as proteins binding to MSU or zymosan
Summary
Crystallopathies are a diverse set of medical conditions where the formation of crystals is the basis of or at least a contributing factor to the disease [1, 2]. Deposition of monosodium urate (MSU) crystals leads to gout [3] and formation of calcium pyrophosphate dihydrate (CPPD) induces pseudogout [4], while the accumulation of cholesterol crystals in blood vessel walls contributes to atherosclerosis development. Crystalline structures induce inflammation in various ways: MSU crystals cause activation of neutrophils [6] leading to formation of neutrophil extracellular traps (NETs) [7, 8] and activation of the complement system [9, 10]. Monocytes respond to MSU crystals with strong pro-inflammatory cytokine production such as interleukin-1b (IL-1b), IL-6, or tumor necrosis factor-a (TNF-a) [11,12,13], while macrophages require a priming signal e.g., C5a binding [14]. Recent studies suggested necroptosis (a programmed form of necrosis/inflammatory cell death) as another molecular mechanism behind the crystal-induced inflammation [16]
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