Expression Of Complement Genes Research Articles

Hepatocyte nuclear factor 4α is a critical factor for the production of complement components in the liver.

The complement system plays an important role in biological defense as an effector to eliminate microorganisms that invade an organism and it is composed of more than 50 proteins, most of which are produced in the liver. Of these proteins, the mRNA expression of C3 and Cfb is known to be positively regulated by the nuclear receptor HNF4α. To investigate whether HNF4α regulates the complement system, we analyzed the hepatic expression of genes involved in the complement activation pathway and membrane attack complex (MAC) formation within the complement system using liver-specific Hnf4a-null mice (Hnf4aΔHep mice) and tamoxifen-induced liver-specific Hnf4a-null mice (Hnf4af/f;AlbERT2cre mice). We found that hepatic expression of many complement genes including C8a, C8b, C8g, and C9 that are involved in formation of the MAC was markedly decreased in Hnf4aΔHep mice and Hnf4af/f;AlbERT2cre mice. Furthermore, expression of C8A, C8B, and C8G was also decreased in human hepatoma cell lines in which the expression of HNF4α was suppressed, and expression of C8G and C9 was induced in a human immortalized hepatocyte cell line with forced expression of HNF4α. Transactivation of C8g and C9 was dependent on HNF4α expression of HNF4α binding sites, indicating that C8g and C9 are novel target genes of HNF4α. The results suggest that hepatic HNF4α plays an important role in regulation of the complement system, mainly MAC formation.

Effects of dietary supplementation of lignocellulose-derived cello-oligosaccharides on growth performance, antioxidant capacity, immune response, and intestinal microbiota in rainbow trout (Oncorhynchus mykiss)

This study evaluated the prebiotic potential of cello-oligosaccharides (COS) produced from birch (Betula pendula), an under-utilised lignocellulosic source from the forestry industry, on growth performance, mucosal immunity, gut microbiota composition, and antioxidant capacity of juvenile rainbow trout (Oncorhynchus mykiss). In a 45-day trial, the fish were fed with diets containing 0%, 0.1%, 0.5% and 1.5% COS, while a diet containing fructo-oligosaccharides (0.5% FOS) was used as a positive control. Fish fed with the 0.5% and 1.5% COS diets showed significantly (P < 0.05) higher abundance of Ruminococcaceae, Bacillaceae and Lactobacillaceae, in the faecal microbiota. The COS diets also induced higher antioxidant capacity in the gut and serum, but there were no treatment effects (P > 0.05) on growth of rainbow trout. Gene expression analysis of the intestine showed significant elevation (P < 0.05) in expression of complement (c3 and c-type lectin) and receptor (tlr2) genes of the innate immune system in COS-fed fish. However, for cytokine and adaptive immune genes, no significant differences (P > 0.05) in gene transcripts were observed between the COS/FOS diets with the control diet. These results suggest that dietary cello-oligosaccharides can be a useful feed supplement for rainbow trout, which can modulate intestinal microbial communities, innate immune response and antioxidant capacity of the host.

Intraocular complement activation is related to retinal vascular and neuronal degeneration in myopic retinopathy.

To investigate the relationship between the intraocular levels of complement proteins and myopia-related retinal neuronal and vascular degeneration. Aqueous humour from 147 myopic patients, including 60 low-myopia and 87 high-myopia were collected during Implantable Collamer Lens implantation surgery. All participants received comprehensive ophthalmic examinations, including logMAR best corrected visual acuity, axial length measurement, fundus photography and ocular B-scan ultrasonography. The myopic eyes were further classified into simple myopia (SM, n = 78), myopic posterior staphyloma (PS, n = 39) and PS with myopic chorioretinal atrophy (PS + CA, n = 30). Retinal thickness and vascular density in the macula (6 mm × 6 mm) and optic nerve head (4.5 mm × 4.5 mm) were measured using Optical Coherence Tomography (OCT) and OCT angiography (OCTA). The levels of complement proteins including C1q, C3, C3b/iC3b, C4, CFB, CFH, C2, C4b, C5, C5a, CFD, MBL and CFI in the aqueous humour were measured using the Luminex Multiplexing system. The real-time RT-PCR was conducted to examine the expression of complement genes (C1q, C2, C3, C4, CFI and CFD) in the guinea pig model of long-term form deprivation-induced myopic retinal degeneration. OCTA showed that retinal neuronal thickness and vascular density in superficial and deep layers of the macular zone as well as vascular density in the optic nerve head were progressively decreased from SM to PS and PS + CA (p < 0.05). The aqueous humour levels of C1q, C3, C3b/iC3b, C4, CFB, CFH, C2, C4b, C5 and CFI were significantly higher in high-myopic eyes compared to those in low-myopic eyes. Further subgroup analysis revealed the highest levels of complement components/fragments in the PS + CA group. The intraocular levels of complement factors particularly C3b/iC3b and C4 were negatively correlated with macular zone deep layer retinal thickness and vascular density and optic nerve head vascular density. The expression of C2, C3 and C4 genes was significantly higher in guinea pig eyes with myopic retinal degeneration compared to control eyes. The intraocular classical pathway and alternative pathway of the complement system are partially activated in pathological myopia. Their activation is related to the degeneration of retinal neurons and the vasculature in the macula and the vasculature in the optic nerve head.

Spatial determination and prognostic impact of the fibroblast transcriptome in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease.

Single substitution in H3.3G34 alters DNMT3A recruitment to cause progressive neurodegeneration

Germline histone H3.3 amino acid substitutions, including H3.3G34R/V, cause severe neurodevelopmental syndromes. To understand how these mutations impact brain development, we generated H3.3G34R/V/W knock-in mice and identified strikingly distinct developmental defects for each mutation. H3.3G34R-mutants exhibited progressive microcephaly and neurodegeneration, with abnormal accumulation of disease-associated microglia and concurrent neuronal depletion. G34R severely decreased H3K36me2 on the mutant H3.3 tail, impairing recruitment of DNA methyltransferase DNMT3A and its redistribution on chromatin. These changes were concurrent with sustained expression of complement and other innate immune genes possibly through loss of non-CG (CH) methylation and silencing of neuronal gene promoters through aberrant CG methylation. Complement expression in G34R brains may lead to neuroinflammation possibly accounting for progressive neurodegeneration. Our study reveals that H3.3G34-substitutions have differential impact on the epigenome, which underlie the diverse phenotypes observed, and uncovers potential roles for H3K36me2 and DNMT3A-dependent CH-methylation in modulating synaptic pruning and neuroinflammation in post-natal brains.

Coordinated local RNA overexpression of complement induced by interferon gamma in myositis

Complement proteins are deposited in the muscles of patients with myositis. However, the local expression and regulation of complement genes within myositis muscle have not been well characterized. In this study, bulk RNA sequencing (RNAseq) analyses of muscle biopsy specimens revealed that complement genes are locally overexpressed and correlate with markers of myositis disease activity, including the expression of interferon-gamma (IFNγ)-induced genes. Single cell and single nuclei RNAseq analyses showed that most local expression of complement genes occurs in macrophages, fibroblasts, and satellite cells, with each cell type expressing different sets of complement genes. Biopsies from immune-mediated necrotizing myopathy patients, who have the lowest levels of IFNγ-induced genes, also had the lowest complement gene expression levels. Furthermore, data from cultured human cells showed that IFNγ upregulates complement expression in macrophages, fibroblasts, and muscle cells. Taken together, our results suggest that in myositis muscle, IFNγ coordinates the local overexpression of complement genes that occurs in several cell types.

Novel prognostic implications of complement activation in the tumour microenvironment for de novo metastatic BRAF V600E mutant colorectal cancer.

Prognosis of metastatic BRAF V600E mutant colorectal cancer (CRC) is poor, and the prognostic implications of immune contextures in the tumour microenvironment (TME) for CRC remain elusive. We collected the primary tumour specimens and clinicopathological characteristics of patients with de novo metastatic microsatellite-stable BRAF V600E mutant CRC from two medical centres. Gene expression analysis was performed using the nCounterⓇ PanCancer Immune Profiling Panel. The Cox proportional hazards regression model was used for analysing survival outcomes in association with immune gene expression and immune cells. Our complement score was defined on the basis of the average gene expression in the selected co-expression module. High expression of classical and regulatory complement genes was significantly associated with poor prognosis (N = 54). A high complement score (defined as a score above the median value) indicated significantly shorter survival. The overall survival (OS) impact of the high score remained significant in multivariate analyses. Additionally, our complement score was strongly correlated with C4d expression in immunohistochemical staining and tumour-associated macrophage (TAM) M2 signatures. Complement activation in the TME was significantly associated with poor OS and was correlated with TAM M2 in patients with de novo metastatic BRAF V600E mutant CRC.

No prominent role for complement C1-esterase inhibitor in Marfan syndrome mice.

Marfan syndrome (MFS) is a connective tissue disorder causing aortic aneurysm formation. Currently, only prophylactic aortic surgery and blood pressure lowering drugs are available to reduce the risk for aortic rupture. Upon whole genome sequencing (WGS) of a Marfan family, we identified a complement gene C1R variant (p.Ser152Leu), which associated with the severe aortic patients. Therefore, we assessed the role of complement activation in MFS aortic tissue. Expression of various complement genes and proteins was detected in human and murine MFS aneurysm tissue, which prompted us to study complement inhibition in MFS mice. Treatment of the Fbn1C1041G/+ MFS mice with human plasma-derived C1-esterase inhibitor Cetor® resulted in reduced complement deposition, decreased macrophage influx in the aorta and lower circulating TNFα levels. However, in line with previous anti-inflammatory treatments, complement inhibition did not change aortic dilatation rate in this MFS mouse model. Thus, while complement factors/C3 activation were detected in human/murine MFS aorta, Cetor® had no effect on aortic dilatation in MFS mice, indicating that complement inhibition is not a suitable treatment strategy in MFS.

415.4: Ischemia Minimization Improves Cardiac Function in an Ex Vivo Xeno Working Heart Model

Introduction: Previous studies showed that ischemia minimization (IM), accomplished by perfusing the heart xenograft during storage, prevents ‘initial cardiac xenograft dysfunction’ (ICXD) and enables prolonged survival following orthotopic cardiac xenotransplantation. Here we report initial observations in a model designed to evaluate effects on heart performance associated with IM and genetic modifications targeted to address xeno-injury mechanisms in a working ex vivo model of ICXD. Method: Hearts from genetically modified and wildtype (WT) pigs were procured after flushing with cold preservation solution (UW, 4oC) and stored for 3 hours either in cold saline (0.9%, 4oC: cold storage (CS)) or were perfused with oxygenated STEEN Solution with RBCs (IM). IM perfusion at 40 mmHg was initiated at room temperature for 20 minutes to facilitate homogeneous graft perfusion before cooling to 4oC for the remainder of the storage period. The genetically modified hearts either had combined knockouts of three specific xenogeneic carbohydrate genes (GTKO, CMAHKO, b4GALNT2KO: TKO) with variable expression of human transgenes (hTG) including complement and thrombo-regulatory proteins (n=29); or GTKO with additional expression of hCD55 (GTKO.hCD55; n=3). Heart function and laboratory parameters were assessed at specific timepoints on a working heart rig while perfused with freshly collected heparinized whole human blood. Troponin I was used as a marker for myocardial injury. Results: In total, 41 hearts were perfused ex vivo, 23 after CS (TKO + hTG n=14, GTKO.hCD55 n=2, WT n=7), and 18 after IM (TKO + hTG n=15, GTKO.hCD55 n=1, WT n=2). Cardiac output (mL/min) was significantly improved for the IM group at all loading conditions relative to CS, (see figure 1). There was a strong trend toward reduced troponin I release, particularly within the first 60 minutes of perfusion (see figure 2). Conclusion: IM is protective with respect to cardiac function following pig heart ischemia and subsequent reperfusion with human blood in an ex vivo model of heart xenotransplantation. The hypothesis that variation in physiologic and biochemical outcomes within groups is influenced by variable expression of complement and coagulation pathway regulatory genes is being investigated.

A single-cell lung atlas of complement genes identifies the mesothelium and epithelium as prominent sources of extrahepatic complement proteins.

To understand functional duality of the complement system in host defense and lung injury, a more comprehensive view of its localized production in the lung, and the impact of age on complement production are essential. Here, we explored the expression of complement genes through computational analysis of preexisting single cell RNA sequencing data from lung transcriptomes of healthy young (3 months) and old C57BL/6 mice (24 months), and humans. We characterized the distribution of 48 complement genes. Across 28 distinct immune and non-immune cell types in mice, mesothelial cells expressed the greatest number of complement genes (e.g., C1ra, C2, C3), and regulators (e.g., Serping1, Cfh). C5 was abundant in type II alveolar epithelial cells and C1q in interstitial lung macrophages. There were only moderate differences in gene expression between young and old mice. Among 57 human lung cell types, mesothelial cells showed abundant complement expression. A few differences in gene expression (e.g., FCN1, CFI, C6, C7) were also evident between mice and human lung cells. Our findings present a novel perspective on the expression patterns of complement genes in normal lungs. These findings highlight the potential functions of complement in tissue-specific homeostasis and immunity and may foster a mechanistic understanding of its role in lung health and disease.

C4BPA: A Novel Co-Regulator of Immunity and Fat Metabolism in the Bovine Mammary Epithelial Cells.

The C4b binding protein alpha (C4BPA) chain primarily engages in critical inflammatory and coagulation processes. The previous transcriptomic analysis showed that C4BPA is a differentially expressed gene in lower and higher fat content mammary gland cell lines from Chinese Holstein. This study aimed to investigate the effects of C4BPA on the inflammation and milk fat synthesis in bMECs by C4BPA knockdown and overexpression. The results highlighted that knockdown of C4BPA in bMECs could suppress the mRNA and protein expression of IL-6, IL-8, IL-12, and the TLR-4/NF-κB pathway-related genes and promote the expression of complement and coagulation cascade pathways related genes as well as TNF-α. Moreover, knockdown of C4BPA expression in bMECs reduced the content of triglyceride (TG) and cholesterol (CHOL) in bMECs, increased NEFA content, reduced mRNA and protein expression of ACSL1 and PPARA, and increased the mRNA and protein expression of ELOVL6, FADS1, and LPL. The bMECs, with the overexpression of C4BPA, showed the enhanced expression of TLR-4/NF-κB linked genes, IL-6, IL-8, IL-12, and mRNA and protein level while reduced mRNA expression of TNF-α, compliment, and coagulation cascade related genes was observed. In bMECs, overexpression of C4BPA enhanced the content of TG and CHOL while reducing NEFA and stimulated the mRNA and protein expression of ACSL1, PPARA, and PPARG genes while inhibiting the mRNA and protein expression of FADS1 and LPL genes. Our results show that C4BPA not only regulates the lipid metabolism through the PPAR signaling pathway in bMECs but also contributes to the inflammatory response through TLR-4/NF-κB and the complement and coagulation cascade pathways. This study, for the first time, provides the primary basis for understanding the role of C4BPA in immunity and fat metabolism, which enables the researchers for innovative direction to investigate genes associated with fat metabolism and immunity. This study also advocates that the breeders must pay attention to such type of genes with multiple functions during animal breeding.

Altered cell and RNA isoform diversity in aging Down syndrome brains

Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.

Chemical complementarity between immune receptors and cancer mutants, independent of antigen presentation protein binding, is associated with increased survival rates

Uterine cancer has been associated with a T-cell immune response that leads to increased survival. Therefore, we used several bioinformatics approaches to explore specific interactions between T-cell receptor (TCR) and tumor mutant peptide sequences. Using endometrioid uterine cancer exome files from the The Cancer Genome Atlas database, we obtained tumor resident V-J recombinations for the T-Cell Receptor alpha gene (TRA). The charged-based, chemical complementarity for each patient's LRP2 or TTN mutant amino acids (AAs) and the recovered, TRA complementarity determining region-3 (CDR3) sequences was calculated, allowing a division of patients into complementary and noncomplementary groups. Complementary groups with TTN mutants had increased disease-free survival and increased expression of complement genes. Furthermore, the survival distinction based on CDR3-mutant peptide complementarity was independent of programmatically assessed HLA class II binding and was not observable based on the CDR3 AA chemical features alone. The above approach provides a potential, highly efficient method for identifying TCR targets in uterine cancer and may aid in the development of novel prognostic tools.

REV-ERBα mediates complement expression and diurnal regulation of microglial synaptic phagocytosis.

The circadian clock regulates various aspects of brain health including microglial and astrocyte activation. Here, we report that deletion of the master clock protein BMAL1 in mice robustly increases expression of complement genes, including C4b and C3, in the hippocampus. BMAL1 regulates expression of the transcriptional repressor REV-ERBα, and deletion of REV-ERBα causes increased expression of C4b transcript in neurons and astrocytes as well as C3 protein primarily in astrocytes. REV-ERBα deletion increased microglial phagocytosis of synapses and synapse loss in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis which was antiphase to REV-ERBα expression. This daily variation in microglial synaptic phagocytosis was abrogated by global REV-ERBα deletion, which caused persistently elevated synaptic phagocytosis. This work uncovers the BMAL1-REV-ERBα axis as a regulator of complement expression and synaptic phagocytosis in the brain, linking circadian proteins to synaptic regulation.

Effects of the sulfated polysaccharide ulvan from Ulva ohnoi on the modulation of the immune response in Senegalese sole (Solea senegalensis)

Sulfated polysaccharides derived from green seaweeds exhibit many beneficial biological activities and have great potential to be used as nutraceutical in aquaculture. In this work, we evaluated the effects of the sulfated polysaccharide ulvan from Ulva ohnoi on Senegalese sole (Solea senegalensis) juveniles at the transcriptomic level. Cytotoxicity assay performed in liver primary cell cultures from sole determined that the different ulvan concentrations assayed did not impair cell viability. Juveniles were intraperitoneally (IP) injected with ulvan (0.5 mg/fish) followed by a challenge with Photobacterium damselae subsp. piscicida (Phdp) at 7 days. RNASeq analyses at 2 days post injection (dpi) revealed that 402 transcripts were differentially expressed in liver between ulvan IP injected and control groups before the challenge. Genes related to bacterial and antiviral defence, complement system, chemokines, proteasomes and antigen presentation were upregulated in ulvan treated groups. A detailed expression analysis of sixteen genes related to innate and adaptive immune system was performed in two systemic tissues: liver and spleen. Ulvan injection provoked the upregulation of tlr22 and a transient inflammatory response was initiated in both liver and spleen at 2 dpi. As consequence, expression of acute phase proteins, antimicrobial peptides and complement genes was induced. Moreover, expression of mhcI, mhcII, psmb10 and bcl6 was also induced 2 dpi. At 2 dpi with Phdp, inflammatory cytokines and genes related to bacterial and antiviral defense, iron metabolism, complement system and antigen presentation were differentially modulated in survival juveniles previously IP injected with ulvan. Moreover, mortality was retarded in ulvan treated juveniles. These results provide new evidence about the role of ulvan as a bioactive compound with immunomodulatory activity in Senegalese sole as well as its possible use as vaccine adjuvant against Phdp. This is the first published study that evaluates the transcriptomic response of Senegalese sole IP injected with ulvan.

Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interactions.

Modeling age‐related macular degeneration (AMD) is challenging, because it is a multifactorial disease. To focus on interactions between the retinal pigment epithelium (RPE) and Bruch's membrane, we generated RPE from AMD patients and used an altered extracellular matrix (ECM) that models aged Bruch's membrane. Induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from AMD patients or age‐matched (normal) controls. RPE derived from iPSCs were analyzed by morphology, marker expression, transepithelial electrical resistance (TER), and phagocytosis of rod photoreceptor outer segments. Cell attachment and viability was tested on nitrite‐modified ECM, a typical modification of aged Bruch's membrane. DNA microarrays with hierarchical clustering and analysis of mitochondrial function were used to elucidate possible mechanisms for the observed phenotypes. Differentiated RPE displayed cell‐specific morphology and markers. The TER and phagocytic capacity were similar among iPSC‐derived RPE cultures. However, distinct clusters were found for the transcriptomes of AMD and control iPSC‐derived RPE. AMD‐derived iPSC‐RPE downregulated genes responsible for metabolic‐related pathways and cell attachment. AMD‐derived iPSC‐RPE exhibited reduced mitochondrial respiration and ability to attach and survive on nitrite‐modified ECM. Cells that did attach induced the expression of complement genes. Despite reprogramming, iPSC derived from AMD patients yielded RPE with a transcriptome that is distinct from that of age‐matched controls. When challenged with an AMD‐like modification of Bruch's membrane, AMD‐derived iPSC‐RPE activated the complement immune system.

Genome-wide identification, evolution, and mRNA expression of complement genes in common carp (Cyprinus carpio)

Complement is a complex component of innate immune system, playing an important role in defense against pathogens and host homeostasis. The complement system has been comprehensively studied in mammals, however less is known about complement in teleost, especially in tetraploid common carp (Cyprinus carpio). In this study, a total of 110 complement genes were identified and characterized in common carp, which include almost all the homologs of mammalian complement genes. These genes were classified into three pathways (alternative pathways, lectin pathways and classical pathways), similar to those in mammals. Phylogenetic and selection pressure analysis showed that the complement genes were evolving-constrained and the function was conserved. Most of the complement genes were highly expressed in spleen, liver, brain and skin among the tested 12 health tissues of common carp. After Aeromonas hydrophila infection in the common carp, many members of complement genes were activated to bring about an immune response and expressed to against any pathogenic encroachment. Gene expression divergences which were found between two homoeologous genes suggested the functional divergences of the homoeologous genes after the 4R WGD event, revealing the evolutionary fate of the tetraploid common carp after the recent WGD.

IMMU-35. COMPLEMENT SYSTEM AND GLIOBLASTOMA: AN INTRICATE RELATIONSHIP

Abstract The complement system is a vital part of the innate immune system which plays a critical role in immune surveillance and inflammatory processes. Malignant and host cells express various complement inhibitory proteins on their surface to protect against complement mediated cytotoxicity. Imbalanced complement activation triggers inflammation and alters the tumor microenvironment. Complement activation has been shown to induce proliferation and migration of breast, ovarian and lung cancer cells. At this time, the expression and functional role of the complement cascade in glioblastoma (GBM) remain elusive. Here, we investigated the role of complement proteins and their receptor expression in human primary glioma stem-like cells (GSCs) and human GBM tissues. Western blot data demonstrated a high level of expression of central complement components and their receptors in GSCs. RT-PCR data further confirmed the high expression of complement genes which was similar or higher to normal human astrocytes (HA), a cell with high baseline expression levels of complement genes. Flow cytometry analysis revealed that almost 95% of GSCs expressed the anaphylatoxin complement receptors C3aR and C5aR on their cell surfaces which was consistent with our immunohistochemistry analysis of freshly resected GBM tissues. Furthermore, anaphylatoxin C5a exposure increased the proliferation of a subgroup of GSCs while reduced in another subset. Interestingly, C5a exposure was found to increase the expression of various pro-inflammatory markers in GSCs with reduced proliferation. Fluorescence-activated cell sorting (FACS) analysis of freshly isolated human GBM tissue revealed predominant expression of C5aR on cancer cells (CD11b-/CD45- cells) rather than on immune cells. RT-PCR analysis also demonstrated high expression levels of complement genes with concomitant decrease in complement inhibitory genes in human GBM tissue. Evaluation of the differential role of the complement system in GSCs along with their role in in vivo glioma models is ongoing.

Complement component C1q is produced by isolated articular chondrocytes

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. 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. 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. C1q protein can be expressed and secreted by human articular chondrocytes and is able to bind to chondrocytes influencing the relative collagen expression.

Studies on the Use of Flagellin as an Immunostimulant and Vaccine Adjuvant in Fish Aquaculture.

Immunostimulants and vaccines are important for controlling infectious diseases in fish aquaculture. In this study we assess the potential of flagellin to be used for such purposes in rainbow trout (Oncorhynchus mykiss). A recombinant flagellin from the salmonid pathogen Yersinia ruckeri (YRF) has been produced previously by us and shown to be a potent activator of inflammatory cytokines, acute phase proteins and antimicrobial peptides in vitro. Here we show that YRF is the most potent inflammatory activator of three bacterial PAMPs (LPS, peptidoglycan and flagellin) tested. The host response to flagellin was next studied in vivo. The YRF modulated gene expression was examined in two systemic (spleen and liver) and two mucosa-associated (gills and skin) tissues. YRF injection initiated a transient systemic inflammatory response with key pro-inflammatory cytokines (IL-1β, TNFα, IL-6, and IL-11 etc.) and chemokines (CXCL_F4 and CXCL-8) induced rapidly (by 6 h) but subsiding quickly (by 24 h) in multiple tissues. Consequently, a variety of anti-microbial pathways were activated systemically with heightened expression of acute phase proteins, antimicrobial peptides and complement genes in multiple tissues, which was sustained to 24 h in the liver and mucosal tissues. The Th17 cytokine IL-17A/F1 was also induced in the spleen and liver, and Th2 cytokine IL-4/13 was induced in the liver. However, the anti-inflammatory IL-10 and the Th1 cytokine IFNγ were refractory. A secreted form of TLR5 (TLR5s) was induced by flagellin in all tissues examined whilst the membrane form was refractory, suggesting that TLR5s may function as a negative feedback regulator. Trout liver appeared to be an important organ responding to flagellin stimulation, with marked induction of IL-11, IL-23P19, IL-17C1, SAA, and cathelicidin-2. YRF induced a strong antibody response. These antibodies reacted against the middle domain of YRF and were able to decrease YRF bioactivity. Intact YRF was necessary for its bioactivity, as deletion of the N-terminal, C terminal or middle domain of YRF led to functional loss. This study suggests that flagellin could be a potent immunostimulant and vaccine adjuvant for fish aquaculture.