Component 5 Research Articles

Identification of Loci Controlling Restriction of Parasite Growth in Experimental Taenia crassiceps Cysticercosis

Human neurocysticercosis (NC) caused by Taenia solium is a parasitic disease of the central nervous system that is endemic in many developing countries. In this study, a genetic approach using the murine intraperitoneal cysticercosis caused by the related cestode Taenia crassiceps was employed to identify host factors that regulate the establishment and proliferation of the parasite. A/J mice are permissive to T. crassiceps infection while C57BL/6J mice (B6) are comparatively restrictive, with a 10-fold difference in numbers of peritoneal cysticerci recovered 30 days after infection. The genetic basis of this inter-strain difference was explored using 34 AcB/BcA recombinant congenic strains derived from A/J and B6 progenitors, that were phenotyped for T. crassiceps replication. In agreement with their genetic background, most AcB strains (A/J-derived) were found to be permissive to infection while most BcA strains (B6-derived) were restrictive with the exception of a few discordant strains, together suggesting a possible simple genetic control. Initial haplotype association mapping using >1200 informative SNPs pointed to linkages on chromosomes 2 (proximal) and 6 as controlling parasite replication in the AcB/BcA panel. Additional linkage analysis by genome scan in informative [AcB55xDBA/2]F1 and F2 mice (derived from the discordant AcB55 strain), confirmed the effect of chromosome 2 on parasite replication, and further delineated a major locus (LOD = 4.76, p<0.01; peak marker D2Mit295, 29.7 Mb) that we designate Tccr1 (T. crassiceps cysticercosis restrictive locus 1). Resistance alleles at Tccr1 are derived from AcB55 and are inherited in a dominant fashion. Scrutiny of the minimal genetic interval reveals overlap of Tccr1 with other host resistance loci mapped to this region, most notably the defective Hc/C5 allele which segregates both in the AcB/BcA set and in the AcB55xDBA/2 cross. These results strongly suggest that the complement component 5 (C5) plays a critical role in early protective inflammatory response to infection with T. crassiceps.

Identification of a central role for complement in osteoarthritis

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis.

High Expression of Complement Component 5 (C5) at Tumor Site Associates with Superior Survival in Ewing's Sarcoma Family of Tumour Patients

Background. Unlike in most adult-onset cancers, an association between typical paediatric neoplasms and inflammatory triggers is rare. We studied whether immune system-related genes are activated and have prognostic significance in Ewing's sarcoma family of tumors (ESFTs). Method. Data analysis was performed on gene expression profiles of 44 ESFT patients, 11 ESFT cell lines, and 18 normal skeletal muscle samples. Differential expression of 238 inflammation and 299 macrophage-related genes was analysed by t-test, and survival analysis was performed according to gene expression. Results. Inflammatory genes are activated in ESFT patient samples, as 38 of 238 (16%) inflammatory genes were upregulated (P < 0.001) when compared to cell lines. This inflammatory gene activation was characterized by significant enrichment of macrophage-related gene expression with 58 of 299 (19%) of genes upregulated (P < 0.001). High expression of complement component 5 (C5) correlated with better event-free (P = 0.01) and overall survival (P = 0.004) in a dose-dependent manner. C5 and its receptor C5aR1 expression was verified at protein level by immunohistochemistry on an independent ESFT tumour tissue microarray. Conclusion. Immune system-related gene activation is observed in ESFT patient samples, and prognostically significant inflammatory genes (C5, JAK1, and IL8) for ESFT were identified.

Complement component 5 contributes to poor disease outcome in humans and mice with pneumococcal meningitis

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Disease outcome has been related to the severity of the proinflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies. Additionally, SNPs in genes encoding complement pathway proteins have been linked to susceptibility to pneumococcal infection, although no associations with disease severity or outcome have been established. Here, we have performed a robust prospective nationwide genetic association study in patients with bacterial meningitis and found that a common nonsynonymous complement component 5 (C5) SNP (rs17611) is associated with unfavorable disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients with bacterial meningitis correlated with several clinical indicators of poor prognosis. Consistent with these human data, C5a receptor-deficient mice with pneumococcal meningitis had lower CSF wbc counts and decreased brain damage compared with WT mice. Adjuvant treatment with C5-specific monoclonal antibodies prevented death in all mice with pneumococcal meningitis. Thus, our results suggest C5-specific monoclonal antibodies could be a promising new antiinflammatory adjuvant therapy for pneumococcal meningitis.

The Complement Component 5 Gene and Age-Related Macular Degeneration

To investigate the association between variants in the complement component 5 (C5) gene and age-related macular degeneration (AMD). Separate and combined data from 3 large AMD case-control studies and a prospective population-based study (The Rotterdam Study). A total of 2599 AMD cases and 3458 ethnically matched controls. Fifteen single nucleotide polymorphisms (SNPs) spanning the C5 gene were initially genotyped in 375 cases and 199 controls from The Netherlands (The Amsterdam/Rotterdam-Netherlands [AMRO-NL] study population). Replication testing of selected SNPs was performed in the Rotterdam Study (NL) and study populations from Southampton, United Kingdom (UK), and New York, United States (US). Early and late stages of prevalent and incident AMD, graded according to (a modification of) the international grading and classification system of AMD. Significant allelic or genotypic associations between 8 C5 SNPs and AMD were found in the AMRO-NL study and this risk seemed to be independent of CFH Y402H, LOC387715 A69S, age, and gender. None of these findings could be confirmed consistently in 3 replication populations. Although the complement pathway, including C5, plays a crucial role in AMD, and the C5 protein is present in drusen, no consistent significant associations between C5 SNPs and AMD were found in any of these studies. The implications for genetic screening of AMD are discussed.

Association of TNFAIP3 polymorphism with rheumatic heart disease in Chinese Han population

In a pair-matched case-control study (239 versus 478) conducted in Chinese Han population, we investigated the association between tumor necrosis factor-alpha-induced protein 3 (TNFAIP3) gene, tumor necrosis factor receptor-associated factor 1 (TRAF1) gene, complement component 5 (C5) gene, and rheumatic heart disease (RHD). We observed no association with RHD for the five tagging single nucleotide polymorphisms (tSNP) in the C5 gene, the three tSNPs in the TNFAIP3 gene, or the two tSNPs in the TRAF1 gene. However, we determined that the tSNP, rs582757, located at intron_5 of the TNFAIP3 gene, associated with RHD in Chinese Han population. Both the distribution of genotype and allele frequencies differed significantly between case and control subjects (p = 0.001 and p = 0.0004, respectively). The minor C allele reduced the risk of RHD with a per-allele odds ratio of 0.57 (0.42-0.78) for the additive model in univariate analysis (p = 0.000). Under a dominant model, CC/CT carriers had a 0.54-fold reduced risk of RHD (95% confidence interval 0.38-0.75, p = 0.000) than TT carriers. Therefore, we report a new genetic variant (rs582757) in the TNFAIP3 gene that associated with the prevalence of RHD in Chinese Han population. Further genetic and functional studies are required to identify the etiological variants in linkage disequilibrium with this polymorphism.

Single nucleotide polymorphisms of complement component 5 and periodontitis

Polymorphisms of host defence genes might increase one's risks for periodontitis. This study investigated whether tagging single nucleotide polymorphisms (SNPs) of the gene encoding complement component 5 (C5) are associated with periodontitis in a Hong Kong Chinese population. Eleven tagging SNPs of 229 patients with at least moderate periodontitis and 207 control subjects without periodontitis were genotyped using an i-plexGOLD MassARRAY mass-spectrometry system. Genotype AG of SNP rs17611 was more prevalent in the group of periodontitis patients than in the controls (54.6% vs. 41.7%, p = 0.007). The haplotype CGCA of the haplotype block consisting of rs1035029, rs17611, rs25681 and rs992670 was significantly associated with periodontitis in a dominant model (p = 0.001). The SNP rs17611 showed high linkage disequilibrium with rs1035029, rs25681 and rs992670. Smoking was also significantly associated with periodontitis (p = 0.006). The tagging SNP rs17611 of the C5 gene and smoking may be associated with periodontitis among the Hong Kong Chinese population.

Structure of and influence of a tick complement inhibitor on human complement component 5

To provide insight into the structural and functional properties of human complement component 5 (C5), we determined its crystal structure at a resolution of 3.1 A. The core of C5 adopted a structure resembling that of C3, with the domain arrangement at the position corresponding to the C3 thioester being very well conserved. However, in contrast to C3, the convertase cleavage site in C5 was ordered and the C345C domain flexibly attached to the core of C5. Binding of the tick C5 inhibitor OmCI to C5 resulted in stabilization of the global conformation of C5 but did not block the convertase cleavage site. The structure of C5 may render possible a structure-based approach for the design of new selective complement inhibitors.

A Candidate Gene Approach Identifies the TRAF1/C5 Region as a Risk Factor for Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disorder affecting ∼1% of the population. The disease results from the interplay between an individual's genetic background and unknown environmental triggers. Although human leukocyte antigens (HLAs) account for ∼30% of the heritable risk, the identities of non-HLA genes explaining the remainder of the genetic component are largely unknown. Based on functional data in mice, we hypothesized that the immune-related genes complement component 5 (C5) and/or TNF receptor-associated factor 1 (TRAF1), located on Chromosome 9q33–34, would represent relevant candidate genes for RA. We therefore aimed to investigate whether this locus would play a role in RA.Methods and FindingsWe performed a multitiered case-control study using 40 single-nucleotide polymorphisms (SNPs) from the TRAF1 and C5 (TRAF1/C5) region in a set of 290 RA patients and 254 unaffected participants (controls) of Dutch origin. Stepwise replication of significant SNPs was performed in three independent sample sets from the Netherlands (n cases/controls = 454/270), Sweden (n cases/controls = 1,500/1,000) and US (n cases/controls = 475/475). We observed a significant association (p < 0.05) of SNPs located in a haplotype block that encompasses a 65 kb region including the 3′ end of C5 as well as TRAF1. A sliding window analysis revealed an association peak at an intergenic region located ∼10 kb from both C5 and TRAF1. This peak, defined by SNP14/rs10818488, was confirmed in a total of 2,719 RA patients and 1,999 controls (odds ratiocommon = 1.28, 95% confidence interval 1.17–1.39, p combined = 1.40 × 10−8) with a population-attributable risk of 6.1%. The A (minor susceptibility) allele of this SNP also significantly correlates with increased disease progression as determined by radiographic damage over time in RA patients (p = 0.008).ConclusionsUsing a candidate-gene approach we have identified a novel genetic risk factor for RA. Our findings indicate that a polymorphism in the TRAF1/C5 region increases the susceptibility to and severity of RA, possibly by influencing the structure, function, and/or expression levels of TRAF1 and/or C5.

A regulatory role for the C5a anaphylatoxin in type 2 immunity in asthma

Complement component 5 (C5) has been described as either promoting or protecting against airway hyperresponsiveness (AHR) in experimental allergic asthma, suggesting pleomorphic effects of C5. Here we report that local pharmacological targeting of the C5a receptor (C5aR) prior to initial allergen sensitization in murine models of inhalation tolerance or allergic asthma resulted in either induction or marked enhancement of Th2-polarized immune responses, airway inflammation, and AHR. Importantly, C5aR-deficient mice exhibited a similar, increased allergic phenotype. Pulmonary allergen exposure in C5aR-targeted mice resulted in increased sensitization and accumulation of CD4+ CD69+ T cells associated with a marked increase in pulmonary myeloid, but not plasmacytoid, DC numbers. Pulmonary DCs from C5aR-targeted mice produced large amounts of CC chemokine ligand 17 (CCL17) and CCL22 ex vivo, suggesting a negative impact of C5aR signaling on pulmonary homing of Th2 cells. In contrast, C5aR targeting in sensitized mice led to suppressed airway inflammation and AHR but was still associated with enhanced production of Th2 effector cytokines. These data suggest a dual role for C5a in allergic asthma, i.e., protection from the development of maladaptive type 2 immune responses during allergen sensitization at the DC/T cell interface but enhancement of airway inflammation and AHR in an established inflammatory environment.

Role for complement in mediating intestinal nitric oxide synthase-2 and superoxide dismutase expression.

Inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD) play an important role in the pathology of ischemia-reperfusion. This study sought to determine if the proinflammatory effects of complement modulate iNOS and SOD in the rat after gastrointestinal ischemia and reperfusion (GI/R). An inhibitory or noninhibitory anti-complement component 5 (C5) monoclonal antibody (18A or 16C, respectively) was administered before GI/R. RT-PCR revealed a significant increase in intestinal iNOS mRNA compared with sham after GI/R that was attenuated significantly by 18A. Immunohistochemistry demonstrated increased iNOS protein expression within the intestinal crypts after GI/R. Cu/Zn SOD (mRNA and protein) was unaffected by GI/R, whereas Cu/Zn SOD activity was reduced significantly. Mn SOD protein expression was decreased significantly by GI/R. Anti-C5 preserved Cu/Zn SOD activity and Mn SOD protein expression. Staining for nitrotyrosine showed that anti-C5 treatment reduced protein nitration in the reperfused intestine. Immunohistochemistry demonstrated prominent phosphorylated (p) inhibitory factor-kappaB (IkappaB)-alpha staining of intestinal tissue after GI/R, whereas anti-C5 reduced p-IkappaB-alpha expression. These data indicate that complement may mediate tissue damage during GI/R by increasing intestinal iNOS and decreasing the activity and protein levels of Cu/Zn SOD and Mn SOD, respectively.

Pathogenesis and genetic control of resistance to the Sterne strain of Bacillus anthracis

The pathogenesis of lethal infection by the nonecapsulated, toxigenic Sterne strain of Bacillus anthracis and the genetic basis of resistance were characterized in mice. Lethal doses of Sterne spores produced disease in susceptible mice similar to that caused by toxigenic and encapsulated B. anthracis. At the inoculation site, the mice developed an edematous exudate with large concentrations of bacilli and toxin. In the susceptible A/J strain, lethal infection was accompanied by systemic invasion and serum anthrax toxin levels increased in parallel with systemic bacterial concentrations and with the mortality rate. Host resistance to Sterne infection was associated with the ability to synthesize the complement component 5 (C5). All Sterne-resistant mouse strains had a functional gene ( Hc) encoding C5, whereas susceptible mice were deficient in C5. A/J mice could be passively protected from lethal challenge by C5-positive serum but not by serum from C5-negative congenic mice. Also resistance was linked to production of C5 in individual backcross (97%) and F2 (98%) mice. The distribution pattern for recombinant inbred mice was consistent with a major role in host resistance of Hc or a closely linked locus, although other genes probably contribute. This mouse model will be useful in characterizing the pathogenesis of anthrax and testing the safety and efficacy of new anthrax vaccines.

Sexual dimorphism of the fifth component of mouse complement.

Sexual dimorphism of mouse complement component 5 (C5) was detected by isoelectric focusing of desialated ethylenediamine tetraacetic acid (EDTA)-plasma on agarose gel, followed by immunofixation with anti-mouse C5. Male plasma displayed two C5 bands, basic and acidic, while female plasma had only a basic C5 band. In all mouse strains tested except C5-deficient strains, the identical patterns of the dimorphism were obtained. The basic and acidic C5 were antigenically and hemolytically indistinguishable from each other. In the neonatal mice of both sexes, the basic C5 was observed at the same levels, but the acidic C5 was only slightly detected. Injection of testosterone into mice resulted in an appearance of the acidic C5 in females and its increase in males. By contrast, injection of estradiol decreased the acidic C5. These results indicate that the expression of the acidic C5 is under the control of testosterone.