Characteristics of Early Antibody Mediated Rejection in Antibody Incompatible Living Donor Kidney Transplantation.

Antibody incompatible transplantation (AIT) may be an only option for highly sensitized patients. Severe form of early antibody mediated rejection (AMR) adversely affects graft survival after AIT. The aim of this study was to identify individuals at risk of AMR. We analyzed 213 living donor AITs performed at our center. Among 120 ABOi, 58 HLAi and 35 DSA + FCXM-negative cases, the rates of early AMR were 6%, 31%, and 9%, respectively (p < 0.001). On multivariate analysis for graft loss, early AMR had a HR of 3.28 (p < 0.001). The HLAi group had worse death-censored graft survival (p = 0.003). In the HLAi group, Patients with aggressive variant AMR (AAMR) had greater percentage of C3d complement fixing DSA, higher baseline class I and total DSA MFI levels and B-cell FCXM RMF. C1q and C3d complement fixing DSA and strong positivity of baseline B- or T-cell FXCM as predictors of AAMR had 100% sensitivity. Early AMR is of significant clinical concern in AIT as it results in poor graft survival and is not well described in literature. An aggressive variant is characterized by massive rise in DSA levels at rejection. Baseline DSA, C1q, and C3d and baseline FCXM values can be used to risk-stratify candidates for AIT.

Complement-Coagulation Cross-talk: Factor H-mediated regulation of the Complement Classical Pathway activation by fibrin clots.

The classical pathway of the complement system is activated by the binding of C1q in the C1 complex to the target activator, including immune complexes. Factor H is regarded as the key downregulatory protein of the complement alternative pathway. However, both C1q and factor H bind to target surfaces via charge distribution patterns. For a few targets, C1q and factor H compete for binding to common or overlapping sites. Factor H, therefore, can effectively regulate the classical pathway activation through such targets, in addition to its previously characterized role in the alternative pathway. Both C1q and factor H are known to recognize foreign or altered-self materials, e.g., bacteria, viruses, and apoptotic/necrotic cells. Clots, formed by the coagulation system, are an example of altered self. Factor H is present abundantly in platelets and is a well-known substrate for FXIIIa. Here, we investigated whether clots activate the complement classical pathway and whether this is regulated by factor H. We show here that both C1q and factor H bind to the fibrin formed in microtiter plates and the fibrin clots formed under in vitro physiological conditions. Both C1q and factor H become covalently bound to fibrin clots, and this is mediated via FXIIIa. We also show that fibrin clots activate the classical pathway of complement, as demonstrated by C4 consumption and membrane attack complex detection assays. Thus, factor H downregulates the activation of the classical pathway induced by fibrin clots. These results elucidate the intricate molecular mechanisms through which the complement and coagulation pathways intersect and have regulatory consequences.

The role of gamma globulin, complement component 1q, factor B, properdin, body temperature, C-reactive protein and serum amyloid alpha to the activity and the function of the human complement system and its pathways.

The complement system plays a crucial role in orchestrating the activation and regulation of inflammation within the human immune system. Three distinct activation pathways-classical, lectin, and alternative-converge to form the common lytic pathway, culminating in the formation of the membrane-attacking complex that disrupts the structure of pathogens. Dysregulated complement system activity can lead to tissue damage, autoimmune diseases, or immune deficiencies. In this study, the antimicrobial activity of human serum was investigated by using a bioluminescent microbe probe, Escherichia coli (pEGFPluxABCDEamp). This probe has previously been used to determine the antimicrobial activity of complement system and the polymorphonuclear neutrophils. In this study, blocking antibodies against key serum activators and components, including IgG, complement component 1q, factor B, and properdin, were utilized. The influence of body temperature and acute phase proteins, such as C reactive protein (CRP) and serum amyloid alpha (SAA), on the complement system was also examined. The study reveals the critical factors influencing complement system activity and pathway function. Alongside crucial factors like C1q and IgG, alternative pathway components factor B and properdin played pivotal roles. Results indicated that the alternative pathway accounted for approximately one third of the overall serum antimicrobial activity, and blocking this pathway disrupted the entire complement system. Contrary to expectations, elevated body temperature during inflammation did not enhance the antimicrobial activity of human serum. CRP demonstrated complement activation properties, but at higher physiological concentrations, it exhibited antagonistic tendencies, dampening the response. On the other hand, SAA enhanced the serum's activity. Overall, this study sheds a light on the critical factors affecting both complement system activity and pathway functionality, emphasizing the importance of a balanced immune response.

Elevated Complement Activation Fragments and C1q-Binding Circulating Immune Complexes in Varied Phases of Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is a causative agent of a disease continuum, ranging from an acute transient chikungunya fever to chronic incapacitating viral arthralgia. The interaction between anti-CHIKV antibodies and the complement system has recently received attention. However, the contribution of complement activation in CHIKV-induced pathologies has not been fully elucidated. The present study was undertaken to delineate the possible contribution of complement activation in CHIKV-induced disease progression. In this study, using plasma specimens of chikungunya patients in the acute, chronic, and recovered phases of infection, we explicated the involvement of complement activation in CHIKV disease progression by ELISAs and Bio-Plex assays. Correlation analysis was carried out to demonstrate interrelation among C1q-binding IgG-containing circulating immune complexes (CIC-C1q), complement activation fragments (C3a, C5a, sC5b-9), and complement-modulated pro-inflammatory cytokines (IL-1ß, IL-18, IL-6, and TNF-α). We detected elevated complement activation fragments, CIC-C1q, and complement-modulated cytokines in the varied patient groups compared with the healthy controls, indicating persistent activation of the complement system. Furthermore, we observed statistically significant correlations among CIC-C1q with complement activation fragments and C3a with complement modulatory cytokines IL-1ß, IL-6, and IL-18 during the CHIKV disease progression. Taken together, the current data provide insight into the plausible association between CICs, complement activation, subsequent complement modulatory cytokine expression, and CHIKV etiopathology.

Anti-C1q antibodies: a biomarker for diagnosis and management of lupus nephritis. A narrative review.

Nephritis is a frequent and severe complication of Systemic Lupus Erythematous (SLE). The clinical course of lupus nephritis (LN) is usually characterized by alternating phases of remission and exacerbation. Flares of LN can lead to deterioration of kidney function, necessitating timely diagnosis and therapy. The presence of autoantibodies against C1q (anti-C1qAb) in the sera of SLE patients has been reported in various studies. Some research suggests that the presence and changes in the titer of anti-C1qAb may be associated with the development of LN, as well as with LN activity and renal flares. However, the exact role of anti-C1qAb in LN remains a subject of debate. Despite variability in the results of published studies, anti-C1qAb hold promise as noninvasive markers for assessing LN activity in SLE patients. Measuring anti-C1qAb levels could aid in diagnosing and managing LN during periods of both inactive disease and renal flares. Nevertheless, larger controlled trials with standardized laboratory assays are necessary to further establish the utility of anti-C1qAb in predicting the reactivation and remission of LN and guiding treatment strategies.

Endothelial dysfunction and complement activation are independently associated with disease duration in patients with systemic vasculitis.

OBJECTIVES: Systemic vasculitis is a heterogenous group of autoimmune diseases characterized by enhanced cardiovascular mortality. Endothelial dysfunction is associated with accelerated vascular damage, representing a core pathophysiologic mechanism contributing to excess CV risk. Recent studies have also shown that complement activation holds significant role in the pathogenesis of Anti-Neutrophilic Cytoplasmic Autoantibody (ANCA) -associated vasculitis (AAV). Given the potential crosstalk between the endothelium and complement, we aimed to assess, for the first time simultaneously, easily accessible biomarkers of endothelial dysfunction and complement activation in SV. METHODS: We measured circulating endothelial microvesicles (EMVs) and soluble complement components representative of alternative, classical and terminal activation (C5b-9, C1q, Bb fragments, respectively) in a meticulously selected group of patients with systemic vasculitis, but without cardiovascular disease. Individuals free from systemic diseases, who were matched with patients for cardiovascular risk factors(hypertension, diabetes, smoking, dyslipidemia), comprised the control group. RESULTS: We studied 60 individuals (30 in each group). Patients with systemic vasculitis had elevated EMVs, higher levels of C5b-9 [536.4(463.4) vs 1200.94457.3), p = 0.003] and C1q [136.2(146.5 vs 204.2(232.9), p = 0.0129], compared to controls [232.0 (243.5) vs 139.3(52.1), p < 0.001]. In multivariate analysis both EMVs and C5b-9 were independently associated with disease duration (p = 0.005 and p = 0.004 respectively), yet not with disease activity. CONCLUSION: Patients with systemic vasculitis exhibit impaired endothelial function and complement activation, both assessed by easily accessible biomarkers, even in the absence of cardiovascular disease manifestations. EMVs and soluble complement components such as C5b-9 and C1q could be used as early biomarkers of endothelial dysfunction and complement activation, respectively, in clinical practice during the course of SV, yet their predictive value in terms of future cardiovascular disease warrants further verification in appropriately designed studies.

Crystal structure of Trichinella spiralis calreticulin and the structural basis of its complement evasion mechanism involving C1q.

Helminths produce calreticulin (CRT) to immunomodulate the host immune system as a survival strategy. However, the structure of helminth-derived CRT and the structural basis of the immune evasion process remains unclarified. Previous study found that the tissue-dwelling helminth Trichinella spiralis produces calreticulin (TsCRT), which binds C1q to inhibit activation of the complement classical pathway. Here, we used x-ray crystallography to resolve the structure of truncated TsCRT (TsCRTΔ), the first structure of helminth-derived CRT. TsCRTΔ was observed to share the same binding region on C1q with IgG based on the structure and molecular docking, which explains the inhibitory effect of TsCRT on C1q-IgG-initiated classical complement activation. Based on the key residues in TsCRTΔ involved in the binding activity to C1q, a 24 amino acid peptide called PTsCRT was constructed that displayed strong C1q-binding activity and inhibited C1q-IgG-initiated classical complement activation. This study is the first to elucidate the structural basis of the role of TsCRT in immune evasion, providing an approach to develop helminth-derived bifunctional peptides as vaccine target to prevent parasite infections or as a therapeutic agent to treat complement-related autoimmune diseases.

The C1q and gC1qR axis as a novel checkpoint inhibitor in cancer.

Understanding at the molecular level of the cell biology of tumors has led to significant treatment advances in the past. Despite such advances however, development of therapy resistance and tumor recurrence are still unresolved major challenges. This therefore underscores the need to identify novel tumor targets and develop corresponding therapies to supplement existing biologic and cytotoxic approaches so that a deeper and more sustained treatment responses could be achieved. The complement system is emerging as a potential novel target for cancer therapy. Data accumulated to date show that complement proteins, and in particular C1q and its receptors cC1qR/CR and gC1qR/p33/HABP1, are overexpressed in most cancer cells and together are involved not only in shaping the inflammatory tumor microenvironment, but also in the regulation of angiogenesis, metastasis, and cell proliferation. In addition to the soluble form of C1q that is found in plasma, the C1q molecule is also found anchored on the cell membrane of monocytes, macrophages, dendritic cells, and cancer cells, via a 22aa long leader peptide found only in the A-chain. This orientation leaves its 6 globular heads exposed outwardly and thus available for high affinity binding to a wide range of molecular ligands that enhance tumor cell survival, migration, and proliferation. Similarly, the gC1qR molecule is not only overexpressed in most cancer types but is also released into the microenvironment where it has been shown to be associated with cancer cell proliferation and metastasis by activation of the complement and kinin systems. Co-culture of either T cells or cancer cells with purified C1q or anti-gC1qR has been shown to induce an anti-proliferative response. It is therefore postulated that in the tumor microenvironment, the interaction between C1q expressing cancer cells and gC1qR bearing cytotoxic T cells results in T cell suppression in a manner akin to the PD-L1 and PD-1 interaction.

An approach based on a combination of toxicological experiments and in silico predictions to investigate the adverse outcome pathway (AOP) of paraquat neuro-immunotoxicity.

Paraquat (PQ) exposure is strongly associated with neurotoxicity. However, research on the neurotoxicity mechanisms of PQ varies in terms of endpoints of toxic assessment, resulting in a great challenge to understand the early neurotoxic effects of PQ. In this study, we developed an adverse outcome pathway (AOP) to investigate PQ-induced neuro-immunotoxicity from an immunological perspective, combining of traditional toxicology methods and computer simulations. In vivo, PQ can microstructurally lead to an early synaptic loss in the brain mice, which is a large degree regarded as a main reason for cognitive impairment to mice behavior. Both in vitro and in vivo demonstrated synapse loss is caused by excessive activation of the complement C1q/C3-CD11b pathway, which mediates microglial phagocytosis dysfunction. Additionally, the interaction between PQ and C1q was validated by molecular simulation docking. Our findings extend the AOP framework related to PQ neurotoxicity from a neuro-immunotoxic perspective, highlighting C1q activation as the initiating event for PQ-induced neuro-immunotoxicity. In addition, downstream complement cascades induce abnormal microglial phagocytosis, resulting in reduced synaptic density and subsequent non-motor dysfunction. These findings deepen our understanding of neurotoxicity and provide a theoretical basis for ecological risk assessment of PQ.

Identification of potential C1-binding sites in the immunoglobulin CL domains.

Immunoglobulin G (IgG) molecules that bind antigens on the membrane of target cells spontaneously form hexameric rings, thus recruiting C1 to initiate the complement pathway. However, our previous report indicated that a mouse IgG mutant lacking the Cγ1 domain activates the pathway independently of antigen presence through its monomeric interaction with C1q via the CL domain, as well as Fc. In this study, we investigated the potential interaction between C1q and human CL isoforms. Quantitative single-molecule observations using high-speed atomic force microscopy revealed that human Cκ exhibited comparable C1q binding capabilities with its mouse counterpart, surpassing the Cλ types, which have a higher isoelectric point than the Cκ domains. Nuclear magnetic resonance and mutation experiments indicated that the human and mouse Cκ domains share a common primary binding site for C1q, centred on Glu194, a residue conserved in the Cκ domains but absent in the Cλ domains. Additionally, the Cγ1 domain, with its high isoelectric point, can cause electrostatic repulsion to the C1q head and impede the C1q-interaction adjustability of the Cκ domain in Fab. The removal of the Cγ1 domain is considered to eliminate these factors and thus promote Cκ interaction with C1q with the potential risk of uncontrolled activation of the complement pathway in vivo in the absence of antigen. However, this research underscores the presence of potential subsites in Fab for C1q binding, offering promising targets for antibody engineering to refine therapeutic antibody design.

Canonical and noncanonical functions of complement in systemic lupus erythematosus.

For many years complement activation in systemic lupus erythematosus (SLE) was viewed as a major cause of tissue injury. However, human and murine studies showed that complement plays a protective as well as a proinflammatory role in tissue damage. A hierarchy is apparent with early classical pathway components, particularly C1q, exerting the greatest influence. Understanding the mechanisms underlying the protective function(s) of complement remains an important challenge for the future and has implications for the use of complement therapy in SLE. We review recent advances in the field and give a new perspective on the complement conundrum in SLE.

Major low-energy trauma results in non-specific immunoglobulin generation without evidence for specific autoantibody production: A prospective cohort study.

Cellular debris resulting from large trauma might overwhelm the scavenger mechanisms and lead to autoimmune reactions. We analysed whether a major well-defined trauma in humans induces laboratory signs of transient autoimmunity in the months after the insult. We included 50 patients with pertrochanteric femur fracture undergoing intramedullary nail osteosynthesis in a prospective cohort study and followed them at 3-4 days, 6 weeks, 12 weeks and 12 months postoperatively. By standard techniques, we assessed levels of total immunoglobulins, anti-nuclear antibodies (ANA), anti-cardiolipin antibodies, anti-dsDNA antibodies and anti-C1q antibodies, as well as antibodies against cytomegalovirus (CMV) as a control. Blood leukocyte differential and lymphocyte subpopulations were determined at baseline and in the first two postoperative samples. The mean age of the patients reached 80.1 years, and 23 (46%) completed all visits. Serum concentrations of total IgG, IgM and IgA increased at all follow-up time points. The ANA fluorescence light intensity units increased at 12 weeks and 12 months postoperatively (p < 0.0001), but the proportion of ANA-positive patients did not change (35%). The values of anti-C1q mildly increased at all follow-up visits, but not the ratio to total IgG. Anti-dsDNA remained negative in all patients, and anti-cardiolipin IgG/IgM antibodies did not change. Anti-CMV IgG antibodies increased significantly at all follow-up visits, without change in the ratio to total IgG. Flow cytometry showed an increased proportion of B-cells 3-4 days postoperatively. In conclusion, major musculoskeletal trauma in elderly patients induces a generalized non-specific increase in immunoglobulin production without laboratory signs for enhanced systemic autoimmunity.

Single-cell analyses highlight the proinflammatory contribution of C1q-high monocytes to Behçet's disease.

Behçet's disease (BD) is a chronic vasculitis characterized by systemic immune aberrations. However, a comprehensive understanding of immune disturbances in BD and how they contribute to BD pathogenesis is lacking. Here, we performed single-cell and bulk RNA sequencing to profile peripheral blood mononuclear cells (PBMCs) and isolated monocytes from BD patients and healthy donors. We observed prominent expansion and transcriptional changes in monocytes in PBMCs from BD patients. Deciphering the monocyte heterogeneity revealed the accumulation of C1q-high (C1qhi) monocytes in BD. Pseudotime inference indicated that BD monocytes markedly shifted their differentiation toward inflammation-accompanied and C1qhi monocyte-ended trajectory. Further experiments showed that C1qhi monocytes enhanced phagocytosis and proinflammatory cytokine secretion, and multiplatform analyses revealed the significant clinical relevance of this subtype. Mechanistically, C1qhi monocytes were induced by activated interferon-γ (IFN-γ) signaling in BD patients and were decreased by tofacitinib treatment. Our study illustrates the BD immune landscape and the unrecognized contribution of C1qhi monocytes to BD hyperinflammation, showing their potential as therapeutic targets and clinical assessment indexes.

Lipoproteome screening of the Lyme disease agent identifies inhibitors of antibody-mediated complement killing.

Spirochetal pathogens, such as the causative agent of Lyme disease, Borrelia burgdorferi sensu lato, encode an abundance of lipoproteins; however, due in part to their evolutionary distance from more well-studied bacteria, such as Proteobacteria and Firmicutes, few spirochetal lipoproteins have assigned functions. Indeed, B. burgdorferi devotes almost 8% of its genome to lipoprotein genes and interacts with its environment primarily through the production of at least 80 surface-exposed lipoproteins throughout its tick vector­vertebrate host lifecycle. Several B. burgdorferi lipoproteins have been shown to serve roles in cellular adherence or immune evasion, but the functions for most B. burgdorferi surface lipoproteins remain unknown. In this study, we developed a B. burgdorferi lipoproteome screening platform utilizing intact spirochetes that enables the identification of previously unrecognized host interactions. As spirochetal survival in the bloodstream is essential for dissemination, we targeted our screen to C1, the first component of the classical (antibody-initiated) complement pathway. We identified two high-affinity C1 interactions by the paralogous lipoproteins, ElpB and ElpQ (also termed ErpB and ErpQ, respectively). Using biochemical, microbiological, and biophysical approaches, we demonstrate that ElpB and ElpQ bind the activated forms of the C1 proteases, C1r and C1s, and represent a distinct mechanistic class of C1 inhibitors that protect the spirochete from antibody-mediated complement killing. In addition to identifying a mode of complement inhibition, our study establishes a lipoproteome screening methodology as a discovery platform for identifying direct host­pathogen interactions that are central to the pathogenesis of spirochetes, such as the Lyme disease agent.

Fc-engineered antibodies with immune effector functions completely abolished.

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been described in the literature but none of them completely eliminates binding to all of the Fcγ receptors. Here we describe a set of novel variants having specific amino acid substitutions in the Fc region at L234 and L235 combined with the substitution G236R. They show no detectable binding to Fcγ receptors or to C1q, are inactive in functional cell-based assays and do not elicit inflammatory cytokine responses. Meanwhile, binding to FcRn, manufacturability, stability and potential for immunogenicity are unaffected. These variants have the potential to improve the safety and efficacy of therapeutic antibodies and Fc fusion proteins.

A 56-Year-Old Man With Emphysema, Rash, and Arthralgia.

CASE PRESENTATION: A 56-year-old man presented to the pulmonary clinic with dyspnea and hypoxemia on exertion. He was an avid biker and skier who had noticed a significant decrease in high-level physical activity over the past 3 years. He reported dyspnea, desaturations at altitudes higher than 9,000 feet, dry cough, tachycardia, and palpitations with exercise. Review of systems was also notable for gluten-intolerance, Raynaud's phenomenon, recurrent skin lesions and joint swelling, pain, and stiffness in the areas overlying the jaw, wrists, knees, and ankles (after capsaicin exposure). He denied fever, chills, anorexia, weight loss, hair loss, ocular symptoms, jaw claudication, chest pain, or lower extremity swelling. He had a five pack-year smoking history, no history of prematurity, childhood asthma, recurrent infections, or environmental and occupational exposure. Based on pulmonary function tests from an outside provider, he had received a diagnosis of exercise-induced asthma and had been prescribed an albuterol inhaler to use on an as-needed basis, which failed to improve his symptoms. He was later prescribed a mometasone-formoterol inhaler, still with no symptomatic improvement.

Contribution of classical complement activation and IgM to the control of Rickettsia infection.

Pathogenic Rickettsia are obligate intracellular bacteria and the etiologic agents of many life-threatening infectious diseases. Due to the serious nature of these infections, it is imperative to both identify the responsive immune sensory pathways and understand the associated immune mechanisms that restrict Rickettsia proliferation. Previous studies have demonstrated that the mammalian complement system is both activated during Rickettsia infection and contributes to the immune response to infection. To further define this component of the mammalian anti-Rickettsia immune response, we sought to identify the mechanism(s) of complement activation during Rickettsia infection. We have employed a series of in vitro and in vivo models of infection to investigate the role of the classical complement activation pathway during Rickettsia infection. Depletion or elimination of complement activity demonstrates that both C1q and pre-existing IgM contribute to complement activation; thus implicating the classical complement system in Rickettsia-mediated complement activation. Elimination of the classical complement pathway from mice increases susceptibility to R. australis infection with both increased bacterial loads in multiple tissues and decreased immune activation markers. This study highlights the role of the classical complement pathway in immunity against Rickettsia and implicates resident Rickettsia-responsive IgM in the response to infection.

Development and Characterization of a Multiplex Assay to Quantify Complement-Fixing Antibodies against Dengue Virus.

Antibodies capable of activating the complement system (CS) when bound with antigen are referred to as "complement-fixing antibodies" and are involved in protection against Flaviviruses. A complement-fixing antibody test has been used in the past to measure the ability of dengue virus (DENV)-specific serum antibodies to activate the CS. As originally developed, the test is time-consuming, cumbersome, and has limited sensitivity for DENV diagnosis. Here, we developed and characterized a novel multiplex anti-DENV complement-fixing assay based on the Luminex platform to quantitate serum antibodies against all four serotypes (DENV1-4) that activate the CS based on their ability to fix the complement component 1q (C1q). The assay demonstrated good reproducibility and showed equivalent performance to a DENV microneutralization assay that has been used to determine DENV serostatus. In non-human primates, antibodies produced in response to primary DENV1-4 infection induced C1q fixation on homologous and heterologous serotypes. Inter-serotype cross-reactivity was associated with homology of the envelope protein. Interestingly, the antibodies produced following vaccination against Zika virus fixed C1q on DENV. The anti-DENV complement fixing antibody assay represents an alternative approach to determine the quality of functional antibodies produced following DENV natural infection or vaccination and a biomarker for dengue serostatus, while providing insights about immunological cross-reactivity among different Flaviviruses.

Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve.

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion of C1q, C3, or CR3 attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3+ microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENT Despite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.

Anti-Idiotype scFv Localizes an Autoepitope in the Globular Domain of C1q.

We addressed the issue of C1q autoantigenicity by studying the structural features of the autoepitopes recognized by the polyclonal anti-C1q antibodies present in Lupus Nephritis (LN) sera. We used six fractions of anti-C1q as antigens and selected anti-idiotypic scFv antibodies from the phage library "Griffin.1". The monoclonal scFv A1 was the most potent inhibitor of the recognition of C1q and its fragments ghA, ghB and ghC, comprising the globular domain gC1q, by the lupus autoantibodies. It was sequenced and in silico folded by molecular dynamics into a 3D structure. The generated 3D model of A1 elucidated CDR similarity to the apical region of gC1q, thus mapping indirectly for the first time a globular autoepitope of C1q. The VH CDR2 of A1 mimicked the ghA sequence GSEAD suggested as a cross-epitope between anti-DNA and anti-C1q antibodies. Other potential inhibitors of the recognition of C1q by the LN autoantibodies among the selected recombinant antibodies were the monoclonal scFv F6, F9 and A12.