Dysregulated C1q and CD47 in the aging monkey brain: association with myelin damage, microglia reactivity, and cognitive decline.

Normal aging, though lacking widespread neurodegeneration, is nevertheless characterized by cognitive impairment in learning, memory, and executive function. The aged brain is spared from neuron loss, but white matter is lost and damage to myelin sheaths accumulates. This myelin damage is strongly associated with cognitive impairment. Although the cause of the myelin damage is not known, microglia dysregulation is a likely contributor. Immunologic proteins interact with microglial receptors to modulate microglia-mediated phagocytosis, which mediates myelin damage clearance and turn-over. Two such proteins, "eat me" signal C1q and "don't eat me" signal CD47, act in opposition with microglia. Both C1q and CD47 have been implicated in Multiple Sclerosis, a demyelinating disease, but whether they play a role in age-related myelin pathology is currently unknown. The present study investigates C1q and CD47 in relation to age-related myelin degeneration using multilabel immunofluorescence, RNAscope, and confocal microscopy in the cingulum bundle of male and female rhesus monkeys across the lifespan. Our findings showed significant age-related elevation in C1q localized to myelin basic protein, and this increase is associated with more severe cognitive impairment. In contrast, CD47 localization to myelin decreased in middle age and oligodendrocyte expression of CD47 RNA decreased with age. Lastly, microglia reactivity increased with age in association with the changes in C1q and CD47. Together, these results suggest disruption in the balance of "eat me" and "don't eat me" signals during normal aging, biasing microglia toward increased reactivity and phagocytosis of myelin, resulting in cognitive deficits.

Immunoglobulin G and Complement as Major Players in the Neurodegeneration of Multiple Sclerosis.

Multiple Sclerosis (MS) is an inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) and is termed as one of the most common causes of neurological disability in young adults. Axonal loss and neuronal cell damage are the primary causes of disease progression and disability. Yet, little is known about the mechanism of neurodegeneration in the disease, a limitation that impairs the development of more effective treatments for progressive MS. MS is characterized by the presence of oligoclonal bands and raised levels of immunoglobulins in the CNS. The role of complement in the demyelinating process has been detected in both experimental animal models of MS and within the CNS of affected MS patients. Furthermore, both IgG antibodies and complement activation can be detected in the demyelinating plaques and cortical gray matter lesions. We propose here that both immunoglobulins and complement play an active role in the neurodegenerative process of MS. We hypothesize that the increased CNS IgG antibodies form IgG aggregates and bind complement C1q with high affinity, activating the classical complement pathway. This results in neuronal cell damage, which leads to neurodegeneration and demyelination in MS.

Circulating Immune Complexes and Complement Activation in Sensitized Kidney Transplant Recipients.

Chronic antibody-mediated rejection in kidney transplantation is a common cause of graft loss in the late post-transplant period. In this process, the role of the classical complement activation pathway is crucial due to the formation of immune complexes between donor-specific antibodies (DSAs) and donor antigens and the attachment of the C1q complement fragment. This study aimed to determine the levels of circulating C1q immunocomplexes (CIC-C1q) and complement activation (CH50), in sensitized kidney transplant recipients (KTRs). In this cross-sectional study we used serum samples from KTRs with de novo or preformed DSAs (n = 14), KTRs without DSAs (n = 28), and 22 subjects with no history of chronic kidney disease (controls). C1q immunocomplexes and CH50 concentration in serum were measured with the enzyme immunoassay (EIA) kit MicroVue CIC-C1q (Quidel, Athens, OH, USA) and EIA kit MicroVue CH50 (Quidel, OH, USA), respectively. Higher concentrations of CIC-C1q was observed in KTRs with DSAs in comparison with controls and with KTRs with no DSAs (6.8 ± 2.7 and 4.8 ± 1.9 vs. 5.0 ± 1.2 µg Eq/mL, respectively, p < 0.01). We found no difference in CIC-C1q between KTRs with no DSAs and controls. CIC-C1q levels were positively correlated with DSA titer. CH50 levels were decreased in KTRs with DSAs in comparison with controls and KTRs with no DSAs (39 ± 15 vs. 68 ± 40 and 71 ± 34 U Eq/mL, respectively, p < 0.01). There was no difference in CH50 between DSA-negative KTRs and controls. Kidney transplant recipients with DSAs had increased serum levels of C1q immunocomplexes and increased classical pathway complement activation.

Single-cell sequencing in diffuse large B-cell lymphoma: C1qC is a potential tumor-promoting factor.

BACKGROUND: Complement component 1q (C1q) is central to the classical complement pathway. High C1q expression has been linked to poor prognosis in patients with cancer. However, the precise mechanism via which C1q contributes to diffuse large B-cell lymphoma (DLBCL) is still unknown. We aimed to explore the potential mechanism by which C1qC promoting DLBCL. METHODS: Using multiplex immunohistochemistry (mIHC) to identify immunocyte subgroups associated with prognosis in DLBCL tissues. Constructing a risk prediction model based on immunocytes using least absolute shrinkage and selection operator (LASSO) regression. Single-cell sequencing detects the expression level of C1qC in immunocytes in the DLBCL microenvironment. Using Wb and qPCR to detect markers of M2 macrophages after knocking down C1qC, and exploring the interactions between lymphoma cells and macrophages through co-culture. Analyzing clinical data from DLBCL patients to investigate the clinical significance of C1qC+ M2 macrophages. Lastly, using bioinformatics in conjunction with mIHC to elucidate the potential pro-tumor mechanism of C1qC. RESULTS: First, we found T cell subtypes, neutrophils, and M2 macrophages are associated with prognosis. Subsequently, the risk model identified C1qC as a differential gene relevant to DLBCL prognosis. Furthermore, single-cell sequencing suggested high C1qC expression in M2 macrophages. The expression level of CD163 is significantly lower following siC1qC. Co-culture experiments have shown that M2 macrophages can promote the proliferation of tumor cells and reduce their drug sensitivity. Furthermore, as an independent predictive indicator, high expression of C1qC+ M2 macrophages is associated with poor prognosis in patients. Finally, a positive correlation between increased C1qC expression and immune checkpoints, as well as an increase in the infiltration of regulatory T cells (Tregs) and M2 macrophages. CONCLUSIONS: C1qC offering new insights into pathogenesis and presenting a potential therapeutic target in DLBCL.

C1q Binding Ability for Prior Risk Assessment of Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation.

In ABO blood group incompatible kidney transplantation (ABO-I), potential issues on acute antibody-mediated rejection (ABMR) remain to be solved. This study aimed to assess the risk factors of acute ABMR using recipient- or donor-derived specimens. Quantitative analysis of A/B antigen expression was conducted in 104 donor kidney tissues (Kt), platelets (Plt), and red blood cells (RBC) by immunohistochemical staining or flow cytometry (FCM). ABO-I pre-transplant recipient serum samples (ABMR = 12, non-ABMR = 27) were extracted by propensity score matching. Anti-A antibody titers of IgM, IgG and IgG subclasses, and C1q binding ability (%) on antibody were measured using RBC-FCM. No association was observed between ABMR and A/B antigen expression levels in donor's Plt, RBC, or Kt. In recipient's sample, C1q-IgG binding ability was significantly higher in the ABMR group than in the non-ABMR group (C1q-IgG: 9.04% vs. 5.93% p = 0.049). Neither the A/B antigen expression level in donors (grafts) nor anti-blood group IgG/IgM antibodies in recipient sera before desensitization seemed to influence ABMR incidence in ABO-I. In contrast, C1q-IgG binding ability could be a potential predictor for ABMR in ABO-I.

The prognostic role of activation of the complement pathways in the progression of advanced IgA nephropathy to end-stage renal disease.

INTRODUCTION: The role of complement system in late stage of IgA nephropathy (IgAN) remains unknown. We therefore investigated the effects of complement system on worsening kidney function in advanced (stage 4 CKD) IgAN. METHODS: Renal specimens of 69 IgAN patients who underwent renal biopsy during stage 4 CKD between 2010 and 2021, were stained using immunofluorescence (IF) and immunohistochemistry (IHC) for glomerular complement components. The primary outcome was progression to end-stage renal disease (ESRD). Associations of complement components with baseline clinicopathological characteristics and outcomes were assessed using multivariable Cox regression and Spearman analyses. RESULTS: During a median follow-up of 18.0 months, 26 (37.7%) patients progressed to ESRD and none died. C1q and C3 deposition were detected in 12 and 66 patients, respectively. Higher eGFR [hazards ratio (HR), 0.852, 95% confidence interval (CI), 0.756-0.959; P = 0.008], higher C3 intensity (HR, 2.955, 95%CI, 1.063-8.220; P = 0.038) and T1-2 score (HR, 2.576, 95%CI, 1.205-5.576, P = 0.015) were predictive of time to ESRD in CKD 4 stage IgAN. Significant expressions of C1q (P = 0.005), C4d (P < 0.001), factor B (P < 0.001), C3 (P = 0.042) and C5b-9 (P = 0.004) were identified in ESRD group than in non-ESRD group by IHC, while MBL expression was low. Although they were not associated with baseline 24 h-UP, higher factor B and C1q expressions were both correlated with a lower baseline eGFR (P < 0.001 and = 0.04, respectively) and the deterioration of kidney function during follow-up (P = 0.046 and 0.015, respectively). CONCLUSION: Complement deposition in IgAN patients with stage 4 CKD portends a faster deterioration of kidney function. Activation of classical and alternative complement pathways plays a major role in this stage.

Update on hereditary C1q deficiency: pathophysiology, clinical presentation, genotype and management.

PURPOSE OF REVIEW: C1q deficiency is a rare inborn error of immunity characterized by susceptibility to severe infections and profound immune dysregulation, with a systemic lupus erythematosus-like phenotype. The management of patients with C1q deficiency is challenged by the rarity of this condition and the wide clinical variability. This review aims to emphasize the importance of a thorough immunological and clinical characterization to help guide a personalized and comprehensive approach to patients. RECENT FINDINGS: We focus on the concept of C1q deficiency as a bridge between the monogenic form of systemic lupus erythematosus and the Mendelian type I interferonopathies. Moreover, we explore the role of new treatment strategies such as Janus-associated kinase (JAK) inhibitors and allogeneic stem cell transplantation. SUMMARY: In this narrative review, we provide a systematic overview of C1q deficiency, starting with the description of the pathophysiological background and the variable clinical phenotype, and then exploring the different prognoses, the consequent treatment strategies and future directions.

Hematopoietic Stem Cell Transplantation for C1q Deficiency: A Study on Behalf of the EBMT Inborn Errors Working Party.

C1q deficiency is a rare inborn error of immunity characterized by increased susceptibility to infections and autoimmune manifestations mimicking SLE, with an associated morbidity and mortality. Because C1q is synthesized by monocytes, to date, four patients treated with allogeneic HSCT have been reported, with a positive outcome in three. We conducted an international retrospective study to assess the outcome of HSCT in C1q deficiency. Eighteen patients, fourteen previously unreported, from eleven referral centres, were included. Two patients had two HSCTs, thus 20 HSCTs were performed in total, at a median age of 10 years (range 0.9-19). Indications for HSCT were autoimmune manifestations not controlled by ongoing treatment in seventeen, and early development of MALT lymphoma in one patient. Overall survival (OS) was 71% and event-free survival was 59% at two years (considering an event as acute GvHD ≥ grade III, disease recurrence and death). In eleven patients HSCT led to resolution of autoimmune features and discontinuation of immunosuppressive treatments (follow-up time range 3-84 months). Five patients died due to transplant-related complications. Patients with a severe autoimmune phenotype, defined as neurological and/or renal involvement, had the worst OS (40% vs 84%; p = 0.034). Reviewing data of 69 genetically confirmed C1q deficient patients, we found that anti-Ro antibodies are associated with neurologic involvement, and anti-RNP and anti-DNA antibodies with renal involvement. In conclusion, HSCT may be a valid curative option for C1q deficiency, but careful selection of patients, with an accurate assessment of risk and benefit, is mandatory.

Intracellular C1q - an unexpected player in neuronal proteostasis.

Extrahepatic, cell-autonomous, and/or intracellularly active complement components are increasingly recognized as key orchestrators of cell physiological processes. A recent study by Scott-Hewitt et al. demonstrates that microglia-derived C1q unexpectedly associates with the ribosomes of neurons in the aging murine brain, where it impacts protein translation and impairs the extinction of conditioned fear responses.

Randomized trial of influence of vitamin D on the prevention and improvement of symptomatic COVID-19.

We aimed to investigate the preventive effect of vitamin D2 on COVID-19 and the improvement of symptoms after COVID-19 infection. The study recruited 228 health care workers who tested negative PCR or antigen for COVID-19. Subjects were randomly allocated to vitamin D2 or non-intervention at a ratio 1:1. Subjects recorded PCR or antigen tests and the symptoms of COVID-19 twice a week during the follow-up visit. The concentration of serum 25-hydroxyvitamin D (25(OH)D), C-reaction protein (CRP), complement component C1q and inflammatory cytokines were measured. The rates of COVID-19 infection were 50.5% in the vitamin D2 group and 52.4% in the non-intervention group (P = 0.785). There was no difference in the COVID-19 symptoms between the two groups. The mean 25(OH)D level significantly increased from 14.1 to 31.1 ng/mL after administration (P < 0.001). The difference between the two groups was not significant for the concentrations of CRP, C1q and inflammatory cytokines on the thirtieth day of the trial. According to the second level of vitamin D, there was a 14.3% difference in positive infection rates between the vitamin D adequate (> 30 ng/mL) and deficient groups (< 20 ng/mL). Adequate vitamin D had a tendency to prevent COVID-19.Trial registration: ClinicalTrials.gov NCT05673980, dated: 12/2022.

Macrophages Promote Atherosclerosis Development by Inhibiting CD8T Cell Apoptosis.

Background: Atherosclerosis is an inflammatory cardiovascular disease. However, whether the association of immune cells in plaques promotes the progression of this disease has not yet been completely elucidated. Materials and Methods: Thus, this study aimed to investigate the relationship between C1q+ macrophages and CD8T cells through scRNA-seq data reanalysis, quantitative real-time PCR, and flow cytometry. Chromatin immunoprecipitation-quantitative polymerase chain reaction, western blot, and antibody-blocking experiments were performed to investigate the role of macrophage-CD8T interaction in atherosclerosis. An atherosclerotic mouse model was developed to confirm our findings. Results: Mechanistically, Spi1 expression induced by granulocyte-macrophage colony-stimulating factor promoted C1q expression in the macrophages. Moreover, C1q+ macrophages suppressed CD8T cell apoptosis by upregulating Slc7a7 expression to enhance the L-arginine uptake of CD8T cells. CD8T-derived interferon-γ promoted macrophage activation to induce atherosclerosis. Blockade of the C1q-C1qbp axis attenuated atherosclerosis. Conclusion: In conclusion, macrophages interacting with CD8T promote atherosclerosis development via the C1q-C1qbp axis.

Identifying High Recurrence Risk in Breast Carcinoma Patients Through Spatial Transcriptomic Analysis.

BACKGROUND/AIM: Comparing gene expression profiles according to recurrence risk using spatially resolved transcriptomic analysis has not been reported. This study aimed to identify distinct genetic features of breast carcinoma associated with a high Oncotype DX Recurrence Score (ORS). PATIENTS AND METHODS: Patients were categorized into two groups, ORS-high (ORS-H; two patients) and ORS-non-high (ORS-NH; five patients). We performed digital spatial profiling and bioinformatic analyses to investigate the spatial transcriptomic profiles. RESULTS: Lysozyme (LYZ), complement C1q C chain (C1QC), and complement C1q B chain (C1QB) exhibited the highest fold changes in the stromal compartment of the ORS-H group. Gene ontology enrichment analysis of the ORS-H group revealed significant up-regulation of genes associated with immune response in the stromal compartment, including lymphocyte-mediated immunity, adaptive immune response related to the immunoglobulin superfamily, and leukocyte-mediated immunity. Gene set enrichment analysis showed significant positive enrichment of gene sets associated with interferon (IFN) response and complement pathways in the stromal compartment. CONCLUSION: This study highlights significant differences in gene expression profiles and spatially resolved transcriptional activities between ORS-H and ORS-NH breast carcinomas. The significant up-regulation of genes and pathways associated with cell-mediated immunity, IFN response, and complement C1q in the stromal compartment of the ORS-H group warrants further evaluation with larger population cohorts.

Hereditary C1q Deficiency is Associated with Type 1 Interferon-Pathway Activation and a High Risk of Central Nervous System Inflammation.

Hereditary C1q deficiency (C1QDef) is a rare monogenic disorder leading to defective complement pathway activation and systemic lupus erythematosus (SLE)-like manifestations. The link between impairment of the complement cascade and autoimmunity remains incompletely understood. Here, we assessed type 1 interferon pathway activation in patients with C1QDef. Twelve patients with genetically confirmed C1QDef were recruited through an international collaboration. Clinical, biological and radiological data were collected retrospectively. The expression of a standardized panel of interferon stimulated genes (ISGs) in peripheral blood was measured, and the level of interferon alpha (IFNα) protein in cerebrospinal fluid (CSF) determined using SIMOA technology. Central nervous system (encompassing basal ganglia calcification, encephalitis, vasculitis, chronic pachymeningitis), mucocutaneous and renal involvement were present, respectively, in 10, 11 and 2 of 12 patients, and severe infections recorded in 2/12 patients. Elevated ISG expression was observed in all patients tested (n = 10/10), and serum and CSF IFNα elevated in 2/2 patients. Three patients were treated with Janus-kinase inhibitors (JAKi), with variable outcome; one displaying an apparently favourable response in respect of cutaneous and neurological features, and two others experiencing persistent disease despite JAKi therapy. To our knowledge, we report the largest original series of genetically confirmed C1QDef yet described. Additionally, we present a review of all previously described genetically confirmed cases of C1QDef. Overall, individuals with C1QDef demonstrate many characteristics of recognized monogenic interferonopathies: particularly, cutaneous involvement (malar rash, acral vasculitic/papular rash, chilblains), SLE-like disease, basal ganglia calcification, increased expression of ISGs in peripheral blood, and elevated levels of CSF IFNα.

Interplay between host humoral pattern recognition molecules controls undue immune responses against Aspergillus fumigatus.

Pentraxin 3 (PTX3), a long pentraxin and a humoral pattern recognition molecule (PRM), has been demonstrated to be protective against Aspergillus fumigatus, an airborne human fungal pathogen. We explored its mode of interaction with A. fumigatus, and the resulting implications in the host immune response. Here, we demonstrate that PTX3 interacts with A. fumigatus in a morphotype-dependent manner: (a) it recognizes germinating conidia through galactosaminogalactan, a surface exposed cell wall polysaccharide of A. fumigatus, (b) in dormant conidia, surface proteins serve as weak PTX3 ligands, and (c) surfactant protein D (SP-D) and the complement proteins C1q and C3b, the other humoral PRMs, enhance the interaction of PTX3 with dormant conidia. SP-D, C3b or C1q opsonized conidia stimulated human primary immune cells to release pro-inflammatory cytokines and chemokines. However, subsequent binding of PTX3 to SP-D, C1q or C3b opsonized conidia significantly decreased the production of pro-inflammatory cytokines/chemokines. PTX3 opsonized germinating conidia also significantly lowered the production of pro-inflammatory cytokines/chemokines while increasing IL-10 (an anti-inflammatory cytokine) released by immune cells when compared to the unopsonized counterpart. Overall, our study demonstrates that PTX3 recognizes A. fumigatus either directly or by interplaying with other humoral PRMs, thereby restraining detrimental inflammation. Moreover, PTX3 levels were significantly higher in the serum of patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA), supporting previous observations in IPA patients, and suggesting that it could be a potential panel-biomarker for these pathological conditions caused by A. fumigatus.

Bioinformatics screening and verification of ischemic stroke-related key genes and drug prediction.

Stroke is one of the major causes of disability and death worldwide. The lack of effective medical treatment for stroke heightens the need for new therapeutic targets. In this study, we obtained two microarray data sets from the Gene Expression Omnibus (GEO) database and identified differential genes (DEGs) between MCAO and control groups. Then, enrichment analysis of the DEGs was performed using DAVID and Metascape. The results show 27 DEGs shared between the two datasets. The functional enrichment analysis showed that these genes are mainly enriched in immune response, complement and coagulation cascades, apoptotic processes. The four hub genes (C1qc, Fcgr2b, C1qb, and Cd14) were screened out using the Cytoscape. Next, real-time PCR and Western blot analysis showed that expression of C1q and CD14 increased at 14 days after tMCAO. Furthermore, we took eight small molecule compounds with the lowest score using Cmap and studied their background characteristics. These results are built on a meta-analysis of data, which are generally accessible from the online space. Finally, we evaluated the protective effect of the rolipram through behavior tests after tMCAO, and results showed that the rolipram significantly attenuated neurobehavioral dysfunction at 14 days after brain ischemia. The present results provide novel insights into the biological process and potential therapeutic drugs involved in stroke.

D-Glutamate production by stressed Escherichia coli gives a clue for the hypothetical induction mechanism of the ALS disease.

In the search for the origin of Amyotrophic Lateral Sclerosis disease (ALS), we hypothesized earlier (Monselise, 2019) that D-amino acids produced by stressed microbiome may serve as inducers of the disease development. Many examples of D-amino acid accumulation under various stress conditions were demonstrated in prokaryotic and eukaryotic cells. In this work, wild-type Escherichia coli, members of the digestive system, were subjected to carbon and nitrogen starvation stress. Using NMR and LC-MS techniques, we found for the first time that D-glutamate accumulated in the stressed bacteria but not in control cells. These results together with the existing knowledge, allow us to suggest a new insight into the pathway of ALS development: D-glutamate, produced by the stressed microbiome, induces neurobiochemical miscommunication setting on C1q of the complement system. Proving this insight may have great importance in preventive medicine of such MND modern-age diseases as ALS, Alzheimer, and Parkinson.

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OBJECTIVES: To study the correlation of anti-C1q antibodies with active systemic lupus erythematosus (SLE) and lupus nephritis (LN) in children, as well as their diagnostic value for active SLE and LN. METHODS: A retrospective selection of 90 hospitalized children with SLE at the Children's Medical Center of Second Xiangya Hospital, Central South University from January 2016 to March 2019 as the SLE group, all of whom were tested for anti-C1q antibodies. A control group was formed by collecting 70 hospitalized children with other autoimmune diseases (OAD) during the same period. The differences in anti-C1q antibody levels were compared between two groups.The correlation of anti-C1q antibodies with various indicators of SLE and LN was analyzed, and the diagnostic value of anti-C1q in SLE and LN was evaluated. RESULTS: The serum levels of anti-C1q antibodies in the SLE group were higher than those in the OAD group (P<0.05). The SLE disease activity index score was positively correlated with anti-C1q antibodies (rs=0.371, P<0.001) and positively correlated with anti-double-stranded DNA antibodies (rs=0.370, P<0.001). The sensitivity and specificity of anti-C1q antibodies for diagnosing active SLE were 89.90% and 53.90%, respectively, with an area under the curve of 0.720 (P<0.05) and a critical value of 5.45 U/mL. The sensitivity and specificity of anti-C1q antibody levels for diagnosing active LN were 58.50% and 85.00%, respectively, with an area under the curve of 0.675 (P<0.05) and a critical value of 22.05 U/mL. CONCLUSIONS: Anti-C1q antibodies can serve as non-invasive biomarkers for evaluating the activity of SLE or predicting the activity of LN in children.

Evaluating the complement C1q levels in serum and cerebrospinal fluid in multiple sclerosis patients: Could it serve as a valuable marker in clinical practice?

Immunohistochemical studies have identified complement component C1q in MS lesions. We aimed to compare serum (sC1q) and CSF (csfC1q) levels in a large cohort of MS patients (pwMS) (n = 222) with those of healthy controls (HC, n = 52), individuals with other immune (IND, n = 14), and non-immune neurological disorders (nIND, n = 15), and to analyze their correlation with other biomarkers. pwMS were divided into three series based on their origin. CSF samples were unavailable for HC. All three pwMS cohorts had lower sC1q levels compared to HC and IND. csfC1q was higher in one pwMS cohort, with a trend in another, and correlated with IgG, Free Kappa Light Chains, GFAP, and Chitinase-3 Like Protein-1 in CSF. Our findings suggest a significant role for C1q in MS pathophysiology, potentially serving as a biomarker for disease identification.

C1q/MASP Complexes-Hybrid Complexes of Classical and Lectin Pathway Proteins Are Found in the Circulation.

Complement pathways, traditionally regarded as separate entities in vitro, are increasingly noted for cross-communication and bypass mechanisms. Among these, the MBL/ficolin/CL-associated serine protease (MASP)-3, a component of lectin pathway pattern recognition molecules, has shown the ability to process critical substrates such as pro-factor D and insulin growth factor binding protein-5. Given shared features between lectin pathway pattern recognition molecules and C1q from the classical pathway, we hypothesized that C1q might be a viable in vivo binding partner for the MASPs. We used microscale thermophoresis, ELISA, and immunoprecipitation assays to detect C1q/MASP complexes and functionally assessed the complexes through enzymatic cleavage assays. C1q/MASP-3 complexes were detected in human serum and correlated well with MASP-3 serum levels in healthy individuals. The binding affinity between MASP-3 and C1q in vitro was in the nanomolar range, and the interaction was calcium-dependent, as demonstrated by their dissociation in the presence of EDTA. Furthermore, most of the circulating C1q-bound MASP-3 was activated. Based on immunoprecipitation, also C1q/MASP-2 complexes appeared to be present in serum. Finally, C1q/MASP-2 and C1q/MASP-3 in vitro complexes were able to cleave C4 and pro-factor D, respectively. Our study reveals the existence of C1q/MASP complexes in the circulation of healthy individuals, and both C1q/MASP-2 and C1q/MASP-3 complexes display proteolytic activity. Hence, this study uncovers a crosstalk route between complement pathways not previously described.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.