Tumor Cell-Derived Complement Component C1r Acts as a Prognostic Biomarker and Promotes Esophageal Squamous Cell Carcinoma Progression.

BACKGROUND: Mounting evidence indicates that complement components play a crucial role in cancer progression. Recent findings indicate that certain complement components display a significant rise in expression within esophageal squamous cell carcinoma (ESCC). However, the specific tumorigenic functions of these components remain unclear. This study focuses on investigating the expression pattern of C1r, elucidating a role for C1r in ESCC, as well as exploring underlying mechanisms controlled by C1r. METHODS: The expression of C1r in ESCC tissues, malignant epithelial cells, and its relationship with survival were analyzed using the Gene Expression Omnibus (GEO) database and tissue microarrays. Single-cell RNA sequencing (scRNA-seq) was used to study the expression of C1r in malignant epithelial cells. C1r knockdown or C1r overexpression in cultured ESCC cells were used to assess the effects of C1r on proliferation, migration, invasion, cell-matrix adhesion, apoptosis, and growth of xenografted tumors in immunocompromised (nude) mice. Western blotting was used to detect the expression of MMP-1 and MMP-10 in C1r knockdown or C1r overexpressing ESCC cells. RESULTS: C1r was highly expressed in ESCC tissues, malignant epithelial cells, and cultured ESCC cell lines. High C1r expression indicated a poor prognosis. Knockdown of C1r significantly suppressed the proliferation, migration, invasion, cell-matrix adhesion, and promoted apoptosis in cultured ESCC cells. Additionally, knockdown of C1r markedly inhibited tumor growth in nude mice. Overexpression of C1r had the opposite effects. C1r induced the expression of MMP-1 and MMP-10. CONCLUSIONS: C1r is highly expressed in ESCC and promotes the progression of this tumor type. Our findings suggest that C1r may serve as a novel prognostic biomarker and therapeutic target in ESCC.

Autoantibodies against Complement Classical Pathway Components C1q, C1r, C1s and C1-Inh in Patients with Lupus Nephritis.

Autoantibodies against the complement component C1q (anti-C1q) are among the main biomarkers in lupus nephritis (LN) known to contribute to renal injury. C1q, the recognition subcomponent of the complement classical pathway, forms a heterotetrameric complex with C1r and C1s, and can also associate a central complement regulator and C1 Inhibitor (C1-Inh). However, the frequency and the pathogenic relevance of anti-C1r, anti-C1s and anti-C1-Inh autoantibodies remain poorly studied in LN. In this paper, we screened for anti-C1q, anti-C1r, anti-C1s and anti-C1-Inh autoantibodies and evaluated their association with disease activity and severity in 74 LN patients followed up for 5 years with a total of 266 plasma samples collected. The presence of anti-C1q, anti-C1r, anti-C1s and anti-C1-Inh was assessed by ELISA. IgG was purified by Protein G from antigen-positive plasma and their binding to purified C1q, C1r and C1s was examined by surface plasmon resonance (SPR). The abilities of anti-C1q, anti-C1r and anti-C1s binding IgG on C1 complex formation were analyzed by ELISA. The screening of LN patients' plasma revealed 14.9% anti-C1q positivity; only 4.2%, 6.9% and 0% were found to be positive for anti-C1r, anti-C1s and anti-C1-Inh, respectively. Significant correlations were found between anti-C1q and anti-dsDNA, and anti-nuclear antibodies, C3 and C4, respectively. High levels of anti-C1q antibodies were significantly associated with renal histologic lesions and correlated with histological activity index. Patients with the most severe disease (A class according to BILAG Renal score) had higher levels of anti-C1q antibodies. Anti-C1r and anti-C1s antibodies did not correlate with the clinical characteristics of the LN patients, did not interfere with the C1 complex formation, and were not measurable via SPR. In conclusion, the presence of anti-C1q, but not anti-C1s or anti-C1r, autoantibodies contribute to the autoimmune pathology and the severity of LN.

C1R, CCL2, and TNFRSF1A Genes in Coronavirus Disease-COVID-19 Pathway Serve as Novel Molecular Biomarkers of GBM Prognosis and Immune Infiltration.

Glioblastoma multiforme (GBM) is a prevalent intracranial brain tumor associated with a high rate of recurrence and treatment difficulty. The prediction of novel molecular biomarkers through bioinformatics analysis may provide new clues into early detection and eventual treatment of GBM. Here, we used data from the GTEx and TCGA databases to identify 1923 differentially expressed genes (DEGs). GO and KEGG analyses indicated that DEGs were significantly enriched in immune response and coronavirus disease-COVID-19 pathways. Survival analyses revealed a significant correlation between high expression of C1R, CCL2, and TNFRSF1A in the coronavirus disease-COVID-19 pathway and the poor survival in GBM patients. Cell experiments indicated that the mRNA expression levels of C1R, CCL2, and TNFRSF1A in GBM cells were very high. Immune infiltration analysis revealed a significant difference in the proportion of immune cells in tumor and normal tissue, and the expression levels of C1R, CCL2, and TNFRSF1A were associated with immune cell infiltration of GBM. Additionally, the protein-protein interaction networks of C1R, CCL2, and TNFRSF1A involved a total of 65 nodes and 615 edges. These results suggest that C1R, CCL2, and TNFRSF1A may be used as molecular biomarkers of prognosis and immune infiltration in GBM patients in the future.

C1r Upregulates Production of Matrix Metalloproteinase-13 and Promotes Invasion of Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. Previous studies have shown the role of the complement system in cSCC progression. In this study, we have investigated the mechanistic role of serine proteinase C1r, a component of the classical pathway of the complement system, in cSCC. Knockout of C1r in cSCC cells using CRISPR/Cas9 resulted in a significant decrease in their proliferation, migration, and invasion through collagen type I compared with that of wild-type cSCC cells. Knockout of C1r suppressed the growth and vascularization of cSCC xenograft tumors and promoted apoptosis of tumor cells in vivo. mRNA-sequencing analysis after C1r knockdown revealed significantly regulated Gene Ontology terms cell-matrix adhesion, extracellular matrix component, basement membrane, and metalloendopeptidase activity and Kyoto Encyclopedia of Genes and Genomes pathway extracellular matrix‒receptor interaction. Among the significantly regulated genes were invasion-associated matrix metalloproteinases (MMPs) MMP1, MMP13, MMP10, and MMP12. Knockout of C1r resulted in decreased production of MMP-1, MMP-13, MMP-10, and MMP-12 by cSCC cells in culture. Knockout of C1r inhibited the expression of MMP-13 by tumor cells, suppressed invasion, and reduced the amount of degraded collagen in vivo in xenografts. These results provide evidence for the role of C1r in promoting the invasion of cSCC cells by increasing MMP production.

Multi-omics analysis reveals the interaction between the complement system and the coagulation cascade in the development of endometriosis.

Endometriosis (EMS) is a disease that shows immune dysfunction and chronic inflammation characteristics, suggesting a role of complement system in its pathophysiology. To find out the hub genes and pathways involved in the pathogenesis of EMs, three raw microarray datasets were recruited from the Gene Expression Omnibus database (GEO). Then, a series of bioinformatics technologies including gene ontology (GO), Hallmark pathway enrichment, protein-protein interaction (PPI) network and gene co-expression correlation analysis were performed to identify hub genes. The hub genes were further verified by the Real-time quantitative polymerase chain reaction (RT-PCR) and Western Blot (WB). We identified 129 differentially expressed genes (DEGs) in EMs, of which 78 were up-regulated and 51 were down-regulated. Through GO functional enrichment analysis, we found that the DEGs are mainly enriched in cell adhesion, extracellular matrix remodeling, chemokine regulation, angiogenesis regulation, epithelial cell proliferation, et al. In Hallmark pathway enrichment analysis, coagulation pathway showed great significance and the terms in which included the central complement factors. Moreover, the genes were dominating in PPI network. Combined co-expression analysis with experimental verification, we found that the up-regulated expression of complement (C1S, C1QA, C1R, and C3) was positively related to tissue factor (TF) in EMs. In this study, we discovered the over expression complement and the positive correlation between complement and TF in EMs, which suggested that interaction of complement and coagulation system may play a role within the pathophysiology of EMS.

Periodontal (formerly type VIII) Ehlers-Danlos syndrome: Description of 13 novel cases and expansion of the clinical phenotype.

Periodontal Ehlers-Danlos syndrome (pEDS) is a rare condition caused by pathogenic variants in the C1R and C1S genes, encoding subunits C1r and C1s of the first component of the classical complement pathway. It is characterized by early-onset periodontitis with premature tooth loss, pretibial hyperpigmentation and skin fragility. Rare arterial complications have been reported, but venous insufficiency is rarely described. Here we report 13 novel patients carrying heterozygous pathogenic variants in C1R and C1S including three novel C1S variants (c.962G > C, c.961 T > G and c.961 T > A). In addition to the pEDS phenotype, three patients and one relative displayed widespread venous insufficiency leading to persistent varicose leg ulcers. One patient suffered an intracranial aneurysm with familial vascular complications including thoracic and abdominal aortic aneurysm and dissection and intracranial aneurysm rupture. This work confirms that vascular complications can occur, although they are not frequent, which leads us to propose to carry out a first complete non-invasive vascular evaluation at the time of the diagnosis in pEDS patients. However, larger case series are needed to improve our understanding of the link between complement pathway activation and connective tissue alterations observed in these patients, and to better assess the frequency, type and consequences of the vascular complications.

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury-Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Complement component C1q is produced by isolated articular chondrocytes.

OBJECTIVE: Inflammation and innate immune responses may contribute to development and progression of Osteoarthritis (OA). Chondrocytes are the sole cell type of the articular cartilage and produce extracellular-matrix molecules. How inflammatory mediators reach chondrocytes is incompletely understood. Previous studies have shown that chondrocytes express mRNA encoding complement proteins such as C1q, suggesting local protein production, which has not been demonstrated conclusively. The aim of this study is to explore C1q production at the protein level by chondrocytes. DESIGN: We analysed protein expression of C1q in freshly isolated and cultured human articular chondrocytes using Western blot, ELISA and flow cytometry. We examined changes in mRNA expression of collagen, MMP-1 and various complement genes upon stimulation with pro-inflammatory cytokines or C1q. mRNA expression of C1 genes was determined in articular mouse chondrocytes. RESULTS: Primary human articular chondrocytes express genes encoding C1q, C1QA, C1QB, C1QC, and secrete C1q to the extracellular medium. Stimulation of chondrocytes with pro-inflammatory cytokines upregulated C1QA, C1QB, C1QC mRNA expression, although this was not confirmed at the protein level. Extracellular C1q bound to the chondrocyte surface dose dependently. In a pilot study, binding of C1q to chondrocytes resulted in changes in the expression of collagens with a decrease in collagen type 2 and an increase in type 10. Mouse articular chondrocytes also expressed C1QA, C1QB, C1QC, C1R and C1S at the mRNA level. CONCLUSIONS: C1q protein can be expressed and secreted by human articular chondrocytes and is able to bind to chondrocytes influencing the relative collagen expression.

C1R Mutations Trigger Constitutive Complement 1 Activation in Periodontal Ehlers-Danlos Syndrome.

Heterozygous missense or in-frame insertion/deletion mutations in complement 1 subunits C1r and C1s cause periodontal Ehlers-Danlos Syndrome (pEDS), a specific EDS subtype characterized by early severe periodontal destruction and connective tissue abnormalities like easy bruising, pretibial haemosiderotic plaques, and joint hypermobility. We report extensive functional studies of 16 C1R variants associated with pEDS by in-vitro overexpression studies in HEK293T cells followed by western blot, size exclusion chromatography and surface plasmon resonance analyses. Patient-derived skin fibroblasts were analyzed by western blot and Enzyme-linked Immunosorbent Assay (ELISA). Overexpression of C1R variants in HEK293T cells revealed that none of the pEDS variants was integrated into the C1 complex but cause extracellular presence of catalytic C1r/C1s activities. Variants showed domain-specific abnormalities of intracellular processing and secretion with preservation of serine protease function in the supernatant. In contrast to C1r wild type, and with the exception of a C1R missense variant disabling a C1q binding site, pEDS variants had different impact on the cell: retention of C1r fragments inside the cell, secretion of aggregates, or a new C1r cleavage site. Overexpression of C1R variants in HEK293T as well as western blot analyses of patient fibroblasts showed decreased levels of secreted C1r. Importantly, all available patient fibroblasts exhibited activated C1s and activation of externally added C4 in the supernatant while control cell lines secreted proenzyme C1s and showed no increase in C4 activation. The central elements in the pathogenesis of pEDS seem to be the intracellular activation of C1r and/or C1s, and extracellular presence of activated C1s that independently of microbial triggers can activate the classical complement cascade.

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.

C1r/C1s deficiency is insufficient to induce murine systemic lupus erythematosus.

C1s deficiency is strongly associated with the development of human systemic lupus erythematosus (SLE); however, the mechanisms by which C1s deficiency contributes to the development of SLE have not yet been elucidated in detail. Using ICR-derived-glomerulonephritis (ICGN) mouse strain that develops SLE and very weakly expresses C1s in the liver, we investigated the protective roles of C1s against SLE. A genetic sequence analysis revealed complete deletion of the C1s1 gene, a mouse homolog of the human C1s gene, with partial deletion of the C1ra and C1rb genes in the ICGN strain. This deletion led to the absence of C1r/C1s and a low level of C1q in the circulation. In order to investigate whether the C1r/C1s deficiency induces SLE, we produced a congenic mouse strain by introducing the deletion region of ICGN into the C57BL/6 strain. Congenic mice exhibited no C1r/C1s and a low level of C1q in the circulation, but did not have any autoimmune defects. These results suggest that C1r/C1s deficiency is not sufficient to drive murine SLE and also that other predisposing genes exist in ICGN mice.

Periodontal Ehlers-Danlos syndrome is associated with leukoencephalopathy.

Here, we report brain white matter alterations in individuals clinically and genetically diagnosed with periodontal Ehlers-Danlos syndrome, a rare disease characterized by premature loss of teeth and connective tissue abnormalities. Eight individuals of two families clinically diagnosed with periodontal Ehlers-Danlos syndrome were included in the present study and underwent general physical, dental, and neurological examination. Whole exome sequencing was performed, and all patients included in the study underwent MRI of the brain. Whole exome sequencing revealed heterozygous C1R mutations c.926G>T (p.Cys309Phe, Family A) and c.149_150TC>AT (p.Val50Asp, Family B). All adult individuals (n = 7; age range 31 to 68 years) investigated by MRI had brain white matter abnormalities. The MRI of one investigated child aged 8 years was normal. The MRI pattern was suggestive of an underlying small vessel disease that is progressive with age. As observed in other leukoencephalopathies related to microangiopathies, the extent of the white matter changes was disproportionate to the neurologic features. Medical history revealed recurrent headaches or depression in some cases. Neurological examination was unremarkable in all individuals but one had mild cognitive decline and ataxia and experienced a seizure. The observation that periodontal Ehlers-Danlos syndrome caused by missense mutations in C1R is consistently associated with a leukoencephalopathy opens a new pathogenic link between the classical complement pathway, connective tissue, brain small vessels, and brain white matter abnormalities.

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.

Molecular features and the transcriptional and functional delineation of complement system activators C1r and C1s from Sebastes schlegelii.

C1r and C1s are serine proteases responsible for activating the classical complement pathway to initiate the complement cascade, which plays a crucial role in eliminating invading pathogenic microbes. In this study, cDNA sequences of C1r and C1s were identified from black rockfish and designated as SsC1r and SsC1s, respectively. In both sequences, two CUB domains, an EGF-like domain, two CCP domains, and a trypsin-like serine protease domain were identified. Multiple sequence alignments with known vertebrate homologs demonstrated that both sequences were highly conserved and, especially, the catalytic and substrate binding residues were completely conserved. In the constructed phylogenetic tree, C1r and C1s formed two separate clusters, which further branched into groups of related organisms. SsC1r and SsC1s joined with their respective teleostean clusters. Transcriptional analysis showed that the highest mRNA expression level was in the liver under normal physiological conditions. Significantly upregulated expression of both mRNAs in spleen and liver after pathologic stress, by intraperitoneal injection with different stimuli, suggested their vital role in immunity. The serine protease domains of SsC1r and SsC1s were cloned and the recombinant proteins were expressed and purified. A protease assay, conducted to confirm their functionality, indicated that both recombinant proteins had proteolytic activity. Taken together, these results indicate that SsC1r and SsC1s have significant properties to aid in the immunity of black rockfish by activating the complement system by proteolytic cleavage.

C1r and C1s from Nile tilapia (Oreochromis niloticus): Molecular characterization, transcriptional profiling upon bacterial and IFN-γ inductions and potential role in response to bacterial infection.

The complement components C1r and C1s play a vital role in immunity with the activation of C1 complex in the classical complement pathway against pathogen infection. In this study, Nile tilapia (Oreochromis niloticus) C1r and C1s orthologs (OnC1r and OnC1s) were identified and characterized. The cDNA of OnC1r and OnC1s ORFs consisted of 1902 bp and 2100 bp of nucleotide sequence encoding polypeptides of 633 and 699 amino acids, respectively. The deduced OnC1r and OnC1s proteins both possessed CUB, EGF, CCP and SP domains, which were significantly homology to teleost. Spatial mRNA expression analysis revealed that the OnC1r and OnC1s were highly expressed in liver. After the in vivo challenges of Streptococcus agalactiae (S. agalactiae) and lipopolysaccharide (LPS), the mRNA expressions of OnC1r and OnC1s were significantly up-regulated in liver and spleen, which were consistent with immunohistochemical detection at the protein level. The up-regulation of OnC1r and OnC1s expressions were also demonstrated in head kidney monocytes/macrophages in vitro stimulated with LPS, S. agalactiae, and recombinant OnIFN-γ. Taken together, the results of this study indicated that OnC1r and OnC1s were likely to get involved in the immune response of Nile tilapia against bacterial infection.

Brief Report: Deficiency of Complement 1r Subcomponent in Early-Onset Systemic Lupus Erythematosus: The Role of Disease-Modifying Alleles in a Monogenic Disease.

OBJECTIVE: To identify a genetic cause of early-onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease. METHODS: We performed whole-exome sequencing and single-nucleotide polymorphism array genotyping in family members with and without SLE. Protein and gene expression, cytokine profile, neutrophil extracellular trap (NET) formation, and presence of low-density granulocytes were evaluated in patient primary cells and serum samples. RESULTS: We identified a novel, homozygous, loss-of-function mutation (p.Pro445Leufs*11) in the C1R gene. Using the Sanger method of DNA sequencing in 14 family members, we confirmed the presence of the mutation in 4 patients with SLE and in an asymptomatic 9-year-old girl. Complement levels were low in sera from patients with truncated C1r protein. Two siblings with SLE who were available for detailed evaluation exhibited strong type I interferon (IFN) inflammatory signatures despite their disease being clinically inactive at the time of sampling. The type I IFN transcriptional signature in the patients' blood correlated with disease expressivity, whereas the neutrophil signature in peripheral blood mononuclear cells was likely associated with disease severity. The female patient with SLE with the most severe phenotype presented with a stronger neutrophil signature, defined by enhanced NET formation and the presence of low-density granulocytes. Analysis of exome data for modifying alleles suggested enrichment of common SLE-associated variants in the more severely affected patients. Lupus-associated HLA alleles or HLA haplotypes were not shared among the 4 affected subjects. CONCLUSION: Our findings revealed a novel high-penetrance mutation in C1R as the cause of monogenic SLE. Disease expressivity in this family appears to be influenced by additional common and rare genetic variants.

Structure and activation of C1, the complex initiating the classical pathway of the complement cascade.

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.

PAR1 activation affects the neurotrophic properties of Schwann cells.

Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR.

Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Crucial genes associated with diabetic nephropathy explored by microarray analysis.

BACKGROUND: This study sought to investigate crucial genes correlated with diabetic nephropathy (DN), and their potential functions, which might contribute to a better understanding of DN pathogenesis. METHODS: The microarray dataset GSE1009 was downloaded from Gene Expression Omnibus, including 3 diabetic glomeruli samples and 3 healthy glomeruli samples. The differentially expressed genes (DEGs) were identified by LIMMA package. Their potential functions were then analyzed by the GO and KEGG pathway enrichment analyses using the DAVID database. Furthermore, miRNAs and transcription factors (TFs) regulating DEGs were predicted by the GeneCoDis tool, and miRNA-DEG-TF regulatory network was visualized by Cytoscape. Additionally, the expression of DEGs was validated using another microarray dataset GSE30528. RESULTS: Totally, 14 up-regulated DEGs and 430 down-regulated ones were identified. Some DEGs (e.g. MTSS1, CALD1 and ACTN4) were markedly relative to cytoskeleton organization. Besides, some other ones were correlated with arrhythmogenic right ventricular cardiomyopathy (e.g. ACTN4, CTNNA1 and ITGB5), as well as complement and coagulation cascades (e.g. C1R and C1S). Furthermore, a series of miRNAs and TFs modulating DEGs were identified. The transcription factor LEF1 regulated the majority of DEGs, such as ITGB5, CALD1 and C1S. Hsa-miR-33a modulated 28 genes, such as C1S. Additionally, 143 DEGs (one upregulated gene and 142 downregulated genes) were also differentially expressed in another dataset GSE30528. CONCLUSIONS: The genes involved in cytoskeleton organization, cardiomyopathy, as well as complement and coagulation cascades may be closely implicated in the progression of DN, via the regulation of miRNAs and TFs.