A non-synonymous variant rs12614 of complement factor B associated with risk of chronic hepatitis B in a Korean population.

BACKGROUND: Hepatitis B is known to cause several forms of liver diseases including chronic hepatitis B (CHB), and hepatocellular carcinoma. Previous genome-wide association study of CHB risk has demonstrated that rs12614 of complement factor B (CFB) was significantly associated with CHB risk. In this study, fine-mapping study of previously reported GWAS single nucleotide polymorphism (SNP; CFB rs12614) was performed to validate genetic effect of rs12614 on CHB susceptibility and identify possible additional causal variants around rs12614 in a Korean population. This association study was conducted in order to identify genetic effects of CFB single nucleotide polymorphisms (SNPs) and to identify additional independent CHB susceptible causal markers within a Korean population. METHODS: A total of 10 CFB genetic polymorphisms were selected and genotyped in 1716 study subjects comprised of 955 CHB patients and 761 population controls. RESULTS: A non-synonymous variant, rs12614 (Arg32Trp) in exon2 of CFB, had significant associations with risk of CHB (odds ratio = 0.43, P = 5.91 × 10- 10). Additional linkage disequilibrium and conditional analysis confirmed that rs12614 had independent genetic effect on CHB susceptibility with previously identified CHB markers. The genetic risk scores (GRSs) were calculated and the CHB patients had higher GRSs than the population controls. Moreover, OR was found to increase significantly with cumulative GRS. CONCLUSIONS: rs12614 showed significant genetic effect on CHB risk within the Korean population. As such rs12614 may be used as a possible causal genetic variant for CHB susceptibility.

A previously unrecognized role of C3a in proteinuric progressive nephropathy.

Podocyte loss is the initial event in the development of glomerulosclerosis, the structural hallmark of progressive proteinuric nephropathies. Understanding mechanisms underlying glomerular injury is the key challenge for identifying novel therapeutic targets. In mice with protein-overload induced by bovine serum albumin (BSA), we evaluated whether the alternative pathway (AP) of complement mediated podocyte depletion and podocyte-dependent parietal epithelial cell (PEC) activation causing glomerulosclerosis. Factor H (Cfh(-/-)) or factor B-deficient mice were studied in comparison with wild-type (WT) littermates. WT+BSA mice showed podocyte depletion accompanied by glomerular complement C3 and C3a deposits, PEC migration to capillary tuft, proliferation, and glomerulosclerosis. These changes were more prominent in Cfh(-/-) +BSA mice. The pathogenic role of AP was documented by data that factor B deficiency preserved glomerular integrity. In protein-overload mice, PEC dysregulation was associated with upregulation of CXCR4 and GDNF/c-Ret axis. In vitro studies provided additional evidence of a direct action of C3a on proliferation and CXCR4-related migration of PECs. These effects were enhanced by podocyte-derived GDNF. In patients with proteinuric nephropathy, glomerular C3/C3a paralleled PEC activation, CXCR4 and GDNF upregulation. These results indicate that mechanistically uncontrolled AP complement activation is not dispensable for podocyte-dependent PEC activation resulting in glomerulosclerosis.

Complement activation and toll-like receptor-2 signaling contribute to cytokine production after renal ischemia/reperfusion.

The innate immune system causes tissue inflammation and injury after renal ischemia/reperfusion (I/R). The complement system is activated on ischemic tubular epithelial cells (TECs) and induces the cells to produce pro-inflammatory chemokines. TECs also express toll-like receptors (TLRs)-2 and -4. Signaling through the TLRs induces TECs to produce a variety of chemokines, some of which can also be induced by complement activation fragments. We sought to determine whether the effects of complement activation and TLR signaling in TECs are redundant, or whether additive protection can be achieved by blocking both of these innate immune systems. To confirm that the complement system, TLR-2 signaling, and TLR-4 signaling induce production of a similar repertoire of inflammatory chemokines, we stimulated TECs with complement sufficient serum or with TLR-2 and TLR-4 ligands in vitro. We found that all three of these stimuli induce TECs to produce KC, MIP-2, IL-6, and TNF-α, and that there was a trend toward greater production of KC in cells exposed to two stimuli. Based upon these results, we hypothesized that mice deficient in both complement activation and TLR-2 signaling would demonstrate greater protection from I/R than mice deficient only in the complement system. To test this hypothesis we induced ischemic acute kidney injury (AKI) in wild-type mice, mice with targeted deletion of complement factor B (fB(-/-) mice), or mice with targeted deletion of factor B and TLR-2 (fB(-/-)TLR2(-/-) mice). Surprisingly, we found that fB(-/-)TLR2(-/-) mice developed more severe injury than those with single deficiency of factor B. Our results indicate that blockade of the complement system may be more protective than simultaneous blockade of both the complement system and TLR-2 in ischemic AKI.

Ca2+/calmodulin-dependent kinase II triggers cell membrane injury by inducing complement factor B gene expression in the mouse heart.

Myocardial Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibition improves cardiac function following myocardial infarction (MI), but the CaMKII-dependent pathways that participate in myocardial stress responses are incompletely understood. To address this issue, we sought to determine the transcriptional consequences of myocardial CaMKII inhibition after MI. We performed gene expression profiling in mouse hearts with cardiomyocyte-delimited transgenic expression of either a CaMKII inhibitory peptide (AC3-I) or a scrambled control peptide (AC3-C) following MI. Of the 8,600 mRNAs examined, 156 were substantially modulated by MI, and nearly half of these showed markedly altered responses to MI with CaMKII inhibition. CaMKII inhibition substantially reduced the MI-triggered upregulation of a constellation of proinflammatory genes. We studied 1 of these proinflammatory genes, complement factor B (Cfb), in detail, because complement proteins secreted by cells other than cardiomyocytes can induce sarcolemmal injury during MI. CFB protein expression in cardiomyocytes was triggered by CaMKII activation of the NF-kappaB pathway during both MI and exposure to bacterial endotoxin. CaMKII inhibition suppressed NF-kappaB activity in vitro and in vivo and reduced Cfb expression and sarcolemmal injury. The Cfb-/- mice were partially protected from the adverse consequences of MI. Our findings demonstrate what we believe is a novel target for CaMKII in myocardial injury and suggest that CaMKII is broadly important for the genetic effects of MI in cardiomyocytes.

Role of complement in protection against Cryptococcus gattii infection.

Previous studies have shown that the alternative pathway of complement activation plays an important role in protection against infection with Cryptococcus neoformans. Cryptococcus gattii does not activate the alternative pathway as well as C. neoformans in vitro. The role of complement in C. gattii infection in vivo has not been reported. In this study, we used mice deficient in complement components to investigate the role of complement in protection against a C. gattii isolate from an ongoing outbreak in northwestern North America. While factor B-deficient mice showed an enhanced rate of death, complement component C3-deficient mice died even more rapidly, indicating that the alternative pathway was not the only complement pathway contributing to protection against disease. Both C3- and factor B-deficient mice had increased fungal burdens in comparison to wild-type mice. Histopathology revealed an overwhelming fungal burden in the lungs of these complement-deficient mice, which undoubtedly prevented efficient gas exchange, causing death. Following the fate of radiolabeled organisms showed that both factor B- and C3-deficient mice were less effective than wild-type mice in clearing organisms. However, opsonization of C. gattii with complement components was not sufficient to prolong life in mice deficient in complement. Killing of C. gattii by macrophages in vitro was decreased in the presence of serum from factor B- and C3-deficient versus wild-type mice. In conclusion, we have demonstrated that complement activation is crucial for survival in C. gattii infection. Additionally, we have shown that the alternative pathway of complement activation is not the only complement pathway contributing to protection.

Reduced neuronal cell death after experimental brain injury in mice lacking a functional alternative pathway of complement activation.

BACKGROUND: Neuroprotective strategies for prevention of the neuropathological sequelae of traumatic brain injury (TBI) have largely failed in translation to clinical treatment. Thus, there is a substantial need for further understanding the molecular mechanisms and pathways which lead to secondary neuronal cell death in the injured brain. The intracerebral activation of the complement cascade was shown to mediate inflammation and tissue destruction after TBI. However, the exact pathways of complement activation involved in the induction of posttraumatic neurodegeneration have not yet been assessed. In the present study, we investigated the role of the alternative complement activation pathway in contributing to neuronal cell death, based on a standardized TBI model in mice with targeted deletion of the factor B gene (fB-/-), a "key" component required for activation of the alternative complement pathway. RESULTS: After experimental TBI in wild-type (fB+/+) mice, there was a massive time-dependent systemic complement activation, as determined by enhanced C5a serum levels for up to 7 days. In contrast, the extent of systemic complement activation was significantly attenuated in fB-/- mice (P < 0.05,fB-/- vs. fB+/+; t = 4 h, 24 h, and 7 days after TBI). TUNEL histochemistry experiments revealed that posttraumatic neuronal cell death was clearly reduced for up to 7 days in the injured brain hemispheres of fB-/- mice, compared to fB+/+ littermates. Furthermore, a strong upregulation of the anti-apoptotic mediator Bcl-2 and downregulation of the pro-apoptotic Fas receptor was detected in brain homogenates of head-injured fB-/- vs. fB+/+ mice by Western blot analysis. CONCLUSION: The alternative pathway of complement activation appears to play a more crucial role in the pathophysiology of TBI than previously appreciated. This notion is based on the findings of (a) the significant attenuation of overall complement activation in head-injured fB-/- mice, as determined by a reduction of serum C5a concentrations to constitutive levels in normal mice, and (b) by a dramatic reduction of TUNEL-positive neurons in conjunction with an upregulation of Bcl-2 and downregulation of the Fas receptor in head-injured fB-/- mice, compared to fB+/+ littermates. Pharmacological targeting of the alternative complement pathway during the "time-window of opportunity" after TBI may represent a promising new strategy to be pursued in future studies.

Lack of complement factor C3, but not factor B, increases hyperlipidemia and atherosclerosis in apolipoprotein E-/- low-density lipoprotein receptor-/- mice.

OBJECTIVE: To investigate the effect of complement deficiency on atherogenesis and lipidemia, we used mice deficient in the third complement component (C3-/-) or factor B (FB-/-). METHODS AND RESULTS: Complement-deficient mice were crossed with mice deficient in both apolipoprotein E and the low-density lipoprotein receptor (Apoe-/- LDLR-/-). The percent lesion area in the aorta at 16 weeks, determined by en face analysis, was 84% higher in C3-/- mice than in controls (11.8%+/-0.4% versus 6.4%+/-0.8%, mean+/-SEM, P<0.00005). The C3-/- mice also had 58% higher serum triglyceride levels (P<0.05) and a more proatherogenic lipoprotein profile, with significantly more low-density lipoprotein cholesterol and very-low-density lipoprotein triglycerides than control mice. The C3-/- mice weighed 13% less (P<0.01) and had a lower body fat content (3.5%+/-1.0% versus 13.1%+/-3.0%, P<0.01). There were no differences between FB-/- mice and controls. CONCLUSIONS: Complement activation by the classical or lectin pathway exerts atheroprotective effects, possibly through the regulation of lipid metabolism.

The alternative activation pathway and complement component C3 are critical for a protective immune response against Pseudomonas aeruginosa in a murine model of pneumonia.

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia, and approximately 80% of patients with cystic fibrosis are infected with this bacterium. To investigate the overall role of complement and the complement activation pathways in the host defense against P. aeruginosa pulmonary infection, we challenged C3-, C4-, and factor B-deficient mice with P. aeruginosa via intranasal inoculation. In these studies, C3(-/-) mice had a higher mortality rate than C3(+/+) mice. Factor B(-/-) mice, but not C4(-/-) mice, infected with P. aeruginosa had a mortality rate similar to that of C3(-/-) mice, indicating that in this model the alternative pathway of complement activation is required for the host defense against Pseudomonas infection. C3(-/-) mice had 6- to 7-fold more bacteria in the lungs and 48-fold more bacteria in the blood than did C3(+/+) mice at 24 h postinfection. In vitro, phagocytic cells from C3(+/+) or C3(-/-) mice exhibited a decreased ability to bind and/or ingest P. aeruginosa in the presence of C3-deficient serum compared to phagocytic cells in the presence of serum with sufficient C3. C3(-/-) mice displayed a significant increase in neutrophils in the lungs and had higher levels of interleukin-1beta (IL-1beta), IL-6, IL-10, KC, and MIP-2 in the lungs at 24 h postinfection than did C3(+/+) mice. Collectively, these results indicate that complement activation by the alternative pathway is critical for the survival of mice infected with P. aeruginosa and that the protection provided by complement is at least in part due to C3-mediated opsonization and phagocytosis of P. aeruginosa.

The complement system in chronic lymphocytic leukemia: a possible role in autoimmune manifestations.

We describe the complement system of three CLL patients who developed autoimmune complications in the course of their disease. The complement profile was pathological in both CLL patients with active autoimmune diseases, while it showed no deficiency in the patient with quiescent autoimmunity. The complement profile could be an early marker for development of autoimmunity in CLL patients

Membrane-bound antibodies to bloodstream Trypanosoma cruzi in mice: strain differences in susceptibility to complement-mediated lysis.

The Y, CL and other strains of Trypanosoma cruzi display different morphological and immunological characteristics. Such observations are here extended to the interaction of bloodstream forms of different strains of T. cruzi with components of the complement system. We demonstrate that the bloodstream forms of the Y and B strains, but not those of the CL strain, are lysed by normal human serum. Lysis is mediated by combined activities of the alternative and classical complement pathways. These activities are triggered by antibodies on the surface of the parasites as shown by: (a) binding of fluorescein or radiolabelled anti-mouse immunoglobulin to the parasite's membrane and (b) the finding that bloodstream forms from lethally irradiated mice can be sensitized and rendered susceptible to complement-mediated lysis by incubation with sera from acutely infected animals. Bloodstream forms of the CL strain also bear surface immunoglobulin and sensitizing antibodies are present in the sera of mice infected with this strain. However, CL trypomastigotes from acutely infected mice fail to be lysed by human or mouse complement unless the parasites are pre-incubated with sera from chronically infected animals. The basis of the different interactions between CL and Y trypomastigotes with antibodies and the complement system, and their biological significance are discussed.