Cholesterol Crystals Activate the Lectin Complement Pathway via Ficolin-2 and Mannose-Binding Lectin: Implications for the Progression of Atherosclerosis.

Cholesterol crystals (CC) play an essential role in the formation of atherosclerotic plaques. CC activate the classical and the alternative complement pathways, but the role of the lectin pathway is unknown. We hypothesized that the pattern recognition molecules (PRMs) from the lectin pathway bind CC and function as an upstream innate inflammatory signal in the pathophysiology of atherosclerosis. We investigated the binding of the PRMs mannose-binding lectin (MBL), ficolin-1, ficolin-2, and ficolin-3, the associated serine proteases, and complement activation products to CC in vitro using recombinant proteins, specific inhibitors, as well as deficient and normal sera. Additionally, we examined the deposition of ficolin-2 and MBL in human carotid plaques by immunohistochemistry and fluorescence microscopy. The results showed that the lectin pathway was activated on CC by binding of ficolin-2 and MBL in vitro, resulting in activation and deposition of complement activation products. MBL bound to CC in a calcium-dependent manner whereas ficolin-2 binding was calcium-independent. No binding was observed for ficolin-1 or ficolin-3. MBL and ficolin-2 were present in human carotid plaques, and binding of MBL to CC was confirmed in vivo by immunohistochemistry, showing localization of MBL around CC clefts. Moreover, we demonstrated that IgM, but not IgG, bound to CC in vitro and that C1q binding was facilitated by IgM. In conclusion, our study demonstrates that PRMs from the lectin pathway recognize CC and provides evidence for an important role for this pathway in the inflammatory response induced by CC in the pathophysiology of atherosclerosis

C-reactive protein binds to cholesterol crystals and co-localizes with the terminal complement complex in human atherosclerotic plaques

Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are serum proteins associated with increased risk of cardiovascular events and these proteins have been shown to interact with the complement system. Whether the pentraxins binds to CC and mediate downstream complement-dependent inflammatory processes remains unknown. Binding of CRP, PTX3, and SAP to CC was investigated in vitro by flow cytometry and fluorescence microscopy. CRP, PTX3, and SAP bound to CC in a concentration-dependent manner. CRP and PTX3 interacted with the complement pattern recognition molecule C1q on CC by increasing the binding of both purified C1q and C1q in plasma. However, CRP was the strongest mediator of C1q binding and also the pentraxin that most potently elevated C1q-mediated complement activation. In a phagocytic assay using whole blood, we confirmed that phagocytosis of CC is complement dependent and initiated by C1q-mediated activation. The pathophysiological relevance of the in vitro observations was examined in vivo in human atherosclerotic plaques. CRP, PTX3, and SAP were all found in atherosclerotic plaques and were located mainly in the cholesterol-rich necrotic core, but co-localization with the terminal C5b-9 complement complex was only found for CRP. In conclusion, this study identifies CRP as a strong C1q recruiter and complement facilitator on CC, which may be highly relevant for the development of atherosclerosis

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination

Ficolins do not alter host immune responses to lipopolysaccharide-induced inflammation in vivo

Ficolins are a family of pattern recognition molecules that are capable of activating the lectin pathway of complement. A limited number of reports have demonstrated a protective role of ficolins in animal models of infection. In addition, an immune modulatory role of ficolins has been suggested. Yet, the contribution of ficolins to inflammatory disease processes remains elusive. To address this, we investigated ficolin deficient mice during a lipopolysaccharide (LPS)-induced model of systemic inflammation. Although murine serum ficolin was shown to bind LPS in vitro, there was no difference between wildtype and ficolin deficient mice in morbidity and mortality by LPS-induced inflammation. Moreover, there was no difference between wildtype and ficolin deficient mice in the inflammatory cytokine profiles after LPS challenge. These findings were substantiated by microarray analysis revealing an unaltered spleen transcriptome profile in ficolin deficient mice compared to wildtype mice. Collectively, results from this study demonstrate that ficolins are not involved in host response to LPS-induced systemic inflammation

Complementary Roles of the Classical and Lectin Complement Pathways in the Defense against <i>Aspergillus fumigatus</i>

Aspergillus fumigatus infections are associated with a high mortality rate for immunocompromised patients. The complement system is considered to be important in protection against this fungus, yet the course of activation is unclear. The aim of this study was to unravel the role of the classical, lectin and alternative pathways under both immunocompetent and immunocompromised conditions to provide a relevant dual-perspective on the response against A. fumigatus.Conidia (spores) from a clinical isolate of A. fumigatus were combined with various human serum types (including serum deficient of various complement components and serum from umbilical cord blood). We also combined this with inhibitors against C1q, mannose-binding lectin (MBL) and ficolin-2 before complement activation products and phagocytosis were detected by flow cytometry.Our results showed that alternative pathway amplified complement on A. fumigatus, but required classical and/or lectin pathway for initiation. In normal human serum this initiation came primarily from the classical pathway. However, with a dysfunctional classical pathway (C1q-deficient serum), lectin pathway activated complement and mediated opsonophagocytosis through MBL. To model the antibody-decline in a compromised immune system, we used serum from normal umbilical cords and found MBL to be the key complement initiator. In another set of experiments serum from patients with different kinds of immunoglobulin insufficiencies showed that the MBL lectin pathway contribution was highest in the samples with the lowest IgG/IgM binding.In conclusion, lectin pathway appears to be the primary route of complement activation in the absence of anti-A. fumigatus antibodies, whereas in a balanced immune state classical pathway is the main activator. This suggests a crucial role for the lectin pathway in innate immune protection against A. fumigatus in immunocompromised patients