HMGB1 cleavage by complement C1s and its potent anti-inflammatory product.

Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.

Biophysical Characterization of the Oligomeric States of Recombinant Immunoglobulins Type-M and Their C1q-Binding Kinetics by Biolayer Interferometry.

Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs. But, due to their high oligomeric complexity, in vitro production, biochemical characterization, and biophysical characterization are challenging. In this study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (analytical ultracentrifugation, size exclusion chromatography coupled to multi-angle laser light scattering, mass photometry, and transmission electron microscopy). Each IgM construct is defined by a specific expression and purification pattern with different sample quality. Nevertheless, both purified IgMs were able to activate complement in a C1q-dependent manner. More importantly, BioLayer Interferometry (BLI) was used for characterizing the kinetics of C1q binding to recombinant IgMs. We show that recombinant IgMs possess similar C1q-binding properties as IgMs purified from human plasma.

Complement System and Alarmin HMGB1 Crosstalk: For Better or Worse.

Our immune system responds to infectious (PAMPs) and tissue damage (DAMPs) signals. The complement system and alarmin High-Mobility Group Box 1 (HMGB1) are two powerful soluble actors of human host defense and immune surveillance. These systems involve molecular cascades and amplification loops for their signaling or activation. Initially activated as alarm raising systems, their function can be finally switched towards inflammation resolution, where they sustain immune maturation and orchestrate repair mechanisms, opening the way back to homeostasis. However, when getting out of control, these defense systems can become deleterious and trigger serious cellular and tissue damage. Therefore, they can be considered as double-edged swords. The close interaction between the complement and HMGB1 pathways is described here, as well as their traditional and non-canonical roles, their functioning at different locations and their independent and collective impact in different systems both in health and disease. Starting from these systems and interplay at the molecular level (when elucidated), we then provide disease examples to better illustrate the signs and consequences of their roles and interaction, highlighting their importance and possible vicious circles in alarm raising and inflammation, both individually or in combination. Although this integrated view may open new therapeutic strategies, future challenges have to be faced because of the remaining unknowns regarding the molecular mechanisms underlying the fragile molecular balance which can drift towards disease or return to homeostasis, as briefly discussed at the end.

Soluble FAS Ligand Enhances Suboptimal CD40L/IL-21-Mediated Human Memory B Cell Differentiation into Antibody-Secreting Cells.

Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

Insights into the ligand binding specificity of SREC-II (scavenger receptor expressed by endothelial cells).

SREC-II (scavenger receptor expressed by endothelial cells II) is a membrane protein encoded by the SCARF2 gene, with high homology to class F scavenger receptor SR-F1, but no known scavenging function. We produced the extracellular domain of SREC-II in a recombinant form and investigated its capacity to interact with common scavenger receptor ligands, including acetylated low-density lipoprotein (AcLDL) and maleylated or acetylated BSA (MalBSA or AcBSA). Whereas no binding was observed for AcLDL, SREC-II ectodomain interacted strongly with MalBSA and bound with high affinity to AcBSA, a property shared with the SR-F1 ectodomain. SREC-II ectodomain also interacted with two SR-F1-specific ligands, complement C1q and calreticulin, with affinities in the 100 nm range. We proceeded to generate a stable CHO cell line overexpressing full-length SREC-II; binding of MalBSA to these cells was significantly increased compared with nontransfected CHO cells. In contrast, no increase in binding could be detected for C1q and calreticulin. We show for the first time that SREC-II has the capacity to interact with the common scavenger receptor ligand MalBSA. In addition, our data highlight similarities and differences in the ligand binding properties of SREC-II in soluble form and at the cell surface, and show that endogenous protein ligands of the ectodomain of SREC-II, such as C1q and calreticulin, are shared with the corresponding domain of SR-F1.

Molecular Basis of Complement C1q Collagen-Like Region Interaction with the Immunoglobulin-Like Receptor LAIR-1.

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q's CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.

Anti-Ficolin-2 and Anti-Ficolin-3 Autoantibody Detection by ELISA.

Ficolins are recognition proteins of the lectin pathway of the complement system and also play an important role in innate immunity and in the maintenance of tissue homeostasis. They deserve special attention in the context of autoimmunity since they are involved in the uptake of dying cells. Because the monitoring of systemic lupus erythematosus (SLE) patients is particularly difficult, it is crucial to find new relevant serum biomarkers. The ability to detect autoantibodies in the patients' sera provides a diagnostic and prognostic advantage. We describe in this chapter quantitative enzyme linked immunosorbent assays (ELISA) to detect the presence of autoantibodies targeting ficolin-2 and ficolin-3 in human sera. Recombinant ficolins produced in a mammalian expression system are used as coating antigens. The described in-house ELISAs provide a valuable tool to efficiently quantify anti-ficolin autoantibodies in the sera of SLE patients.

Analysis of the Ligand Recognition Specificities of Human Ficolins Using Surface Plasmon Resonance.

Ficolins are innate immune recognition proteins involved in activation of the lectin complement pathway. These oligomeric lectin-like proteins are assembled from subunits consisting of a collagen-like triple helix and a trimeric fibrinogen-like recognition domain. In humans, three ficolins coexist: they differ in their ligand binding specificities, but share the capacity to associate with proteases through their collagen-like stalks and trigger complement activation. We describe methods to decipher the recognition specificities of ficolins, based on surface plasmon resonance, an optical technique allowing real-time and label-free monitoring of biomolecular interactions. This technique was mainly used to characterize and compare binding of the three recombinant full-length ficolins and of their isolated recognition domains to various immobilized BSA-glycoconjugates, acetylated BSA or biotinylated heparin. The avidity phenomenon that enhances the apparent affinity of interactions between oligomeric lectin-like proteins and the multivalent ligands is also discussed.

Functional recombinant human complement C1q with different affinity tags.

Complement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q.

Headless C1q: a new molecular tool to decipher its collagen-like functions.

Complement component C1q, a soluble defense collagen, is the recognition protein of the classical complement pathway. C1q is able to recognize and interact with multiple targets and, via the subsequent activation of its cognate serine proteases C1r and C1s, initiates the complement cascade. C1q is made up of six ABC heterotrimers each containing two different functional regions, an N-terminal collagen-like region (CLR) and a C-terminal globular region (GR). These heterotrimers assemble via their N-terminal regions, resulting in the characteristic 'bouquet-like' shape of C1q with an N-terminal bundle of collagen fibers with six diverging stems each exhibiting a C-terminal globular head. The GRs are responsible for the versatile recognition of multiple C1q targets, whereas the CLRs trigger immune response through interacting with several cellular or soluble partners. We report here the generation of the first recombinant form of human C1q without its recognition globular heads. The noncollagenous domain 2 (nc2) of type IX collagen has been substituted for the C1q GR in order to control the correct registering of the collagen triple helices of C1q chains A, B, and C. The resulting CLR_nc2 recombinant protein produced in stably transfected EXPI293 mammalian cells was correctly assembled and folded, as demonstrated by mass spectrometry, mass photometry, and electron microscopy experiments. Its interaction properties were investigated using surface plasmon resonance analysis with known CLR ligands: the tetramer of C1r and C1s dimers and MBL-associated protein MAp44. Comparison with the interaction properties of native serum-derived C1q and CLR revealed that recombinant CLR_nc2 retains C1q CLR functional properties.

Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases.

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.

Molecular and Cellular Interactions of Scavenger Receptor SR-F1 With Complement C1q Provide Insights Into Its Role in the Clearance of Apoptotic Cells.

The scavenger receptor SR-F1 binds to and mediates the internalization of a wide range of ligands, and is involved in several immunological processes. We produced recombinant SR-F1 ectodomain and fragments deleted from the last 2 or 5 C-terminal epidermal growth factor-like modules and investigated their role in the binding of acetylated low density lipoprotein (AcLDL), complement C1q, and calreticulin (CRT). C1q measured affinity was in the 100 nM range and C1q interaction occurs via its collagen-like region. We identified two different binding regions on SR-F1: the N-terminal moiety interacts with C1q and CRT whereas the C-terminal moiety binds AcLDL. The role of SR-F1 N-linked glycans was also tested by mutating each of the three glycosylated asparagines. The three mutants retained binding activities for both AcLDL and C1q. A stable THP-1 cell line overexpressing SR-F1 was generated and C1q was shown to bind more strongly to the surface of SR-F1 overexpressing macrophages, with C1q/SR-F1 colocalization observed in some membrane areas. We also observed a higher level of CRT internalization for THP-1 SR-F1 cells. Increasing SR-F1 negatively modulated the uptake of apoptotic cells. Indeed, THP-1 cells overexpressing SR-F1 displayed a lower phagocytic capacity as compared with mock-transfected cells, which could be partially restored by addition of C1q in the extracellular milieu. Our data shed some light on the role of SR-F1 in efferocytosis, through its capacity to bind C1q and CRT, two proteins involved in this process.

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease.

The complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions.

Recombinant C1q variants modulate macrophage responses but do not activate the classical complement pathway.

Complement protein C1q plays a dual role in a number of inflammatory diseases such as atherosclerosis. While in later stages classical complement pathway activation by C1q exacerbates disease progression, C1q also plays a beneficial role in early disease. Independent of its role in complement activation, we and others have identified a number of potentially beneficial interactions of C1q with phagocytes in vitro, including triggering phagocytosis of cellular and molecular debris and polarizing macrophages toward an anti-inflammatory phenotype. These interactions may also be important in preventing autoimmunity. Here, we characterize variants of recombinant human C1q (rC1q) which no longer initiate complement activation, through mutation of the C1r2C1s2 interaction site. For insight into the structural location of the site of C1q that is important for interaction with phagocytes, we investigated the effect of these mutations on phagocytosis and macrophage inflammatory polarization, as compared to wild-type C1q. Phagocytosis of antibody coated sheep erythrocytes and oxidized LDL was measured in human monocytes and monocyte-derived macrophages (HMDM) respectively that had interacted with rC1q wild-type or variants. Secreted levels of cytokines were also measured in C1q stimulated HMDM. All variants of C1q increased phagocytosis in HMDM compared to controls, similar to native or wild-type rC1q. In addition, levels of certain pro-inflammatory cytokines and chemokines secreted by HMDM were modulated in cells that interacted with C1q variants, similar to wild-type rC1q and native C1q. This includes downregulation of IL-1α, IL-1ß, TNFα, MIP-1α, and IL-12p40 by native and rC1q in both resting and M1-polarized HMDM. This suggests that the site responsible for C1q interaction with phagocytes is independent of the C1r2C1s2 interaction site. Further studies with these classical pathway-null variants of C1q should provide greater understanding of the complement-independent role of C1q, and allow for potential therapeutic exploitation.

Complement C1r serine protease contributes to kidney fibrosis.

We have previously reported that complement activation precedes the development of kidney fibrosis; however, little is known about the cellular mechanisms involved in this transition. We hypothesized that increased expression of C1 complex protease C1r, the initiator of complement activation, contributes to tubulointerstitial fibrosis and tested this idea in mice with global deletion of C1r. Although expression of C1r in untreated wild-type (WT) mice was higher in the liver compared with kidney tissue, administration of folic acid (FA) led to upregulation of C1r mRNA and protein levels only in kidney tissue. Immunohistochemistry and in situ hybridization experiments localized increased expression of C1r and C1s proteases to renal tubular epithelial cells. C1r-null mice had reduced acute tubular injury and inflammation measured 2 days after FA administration compared with WT mice. C1r deletion reduced expression of C1s, C3 fragment formation, and organ fibrosis measured 14 days after FA administration. Differential gene expression performed in kidney tissue demonstrated that C1r-null mice had reduced expression of genes associated with the acute phase response, complement, proliferation of connective tissue cells (e.g., platelet-derived growth factor receptor-ß), and reduced expression of genes associated with inflammation compared with FA-treated WT mice. In vitro experiments in renal epithelial cells demonstrated that C1s expression is dependent on increased C1r expression and that interferon-γ induces the expression of these two proteases. We conclude that increased expression of C1 complex proteases is associated with increased tissue inflammation and complement C3 formation and represents an important pathogenic mechanism leading to FA-mediated tubulointerstitial fibrosis.

Interaction of C1q With Pentraxin 3 and IgM Revisited: Mutational Studies With Recombinant C1q Variants.

Pentraxins and complement defense collagens are soluble recognition proteins that sense pathogens and altered-self elements, and trigger immune responses including complement activation. PTX3 has been shown to interact with the globular recognition domains (gC1q) of the C1q protein of the classical complement pathway, thereby modulating complement activity. The C1q-PTX3 interaction has been characterized previously by site-specific mutagenesis using individual gC1q domains of each of the three C1q chains. The present study is aimed at revisiting this knowledge taking advantage of full-length recombinant C1q. Four mutations targeting exposed amino acid residues in the gC1q domain of each of the C1q chains (LysA200Asp-LysA201Asp, ArgB108Asp-ArgB109Glu, TyrB175Leu, and LysC170Glu) were introduced in recombinant C1q and the interaction properties of the mutants were analyzed using surface plasmon resonance. All C1q mutants retained binding to C1r and C1s proteases and mannose-binding lectin-associated serine proteases, indicating that the mutations did not affect the function of the collagen-like regions of C1q. The effect of these mutations on the interaction of C1q with PTX3 and IgM, and both the PTX3- and IgM-mediated activation of the classical complement pathway were investigated. The LysA200Asp-LysA201Asp and LysC170Glu mutants retained partial interaction with PTX3 and IgM, however they triggered efficient complement activation. In contrast, the ArgB108Asp-ArgB109Glu mutation abolished C1q binding to PTX3 and IgM, and significantly decreased complement activation. The TyrB175Leu mutant exhibited decreased PTX3- and IgM-dependent complement activation. Therefore, we provided evidence that, in the context of the full length C1q protein, a key contribution to the interaction with both PTX3 and IgM is given by the B chain Arg residues that line the side of the gC1q heterotrimer, with a minor participation of a Lys residue located at the apex of gC1q. Furthermore, we generated recombinant forms of the human PTX3 protein bearing either D or A at position 48, a polymorphic site of clinical relevance in a number of infections, and observed that both allelic variants equally recognized C1q.

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes.

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

Recognition protein C1q of innate immunity agglutinates nanodiamonds without activating complement.

Nanodiamonds are promising nanomedicines for diagnostic and therapeutic applications. As nanodiamonds are mainly administered intravenously, it is critical to understand the humoral immune response upon exposure to nanodiamonds. Here, we report the interactions of pristine, oxidized, and PEG-functionalized nanodiamonds with human complement, an important part of our humoral innate immunity. In particular, we report the nanodiamond binding properties of the recognition protein of the classical complement pathway: C1q, which also takes part in many other physiological and pathological processes. Our results show similar trends in the effects of C1q on the three types of nanodiamonds. Complement activation assays using human serum show that the nanodiamonds trigger slight activities via the alternative pathway and no response via the classical pathway. Nevertheless, surface plasmon resonance shows that C1q binds the nanodiamonds and transmission electron microscopy reveals their agglutination. Studies with macrophages further show that C1q attachment affects their phagocytosis and cytokine response.