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1.  Whole-exome sequencing identifies rare, functional CFH variants in families with macular degeneration 
Human Molecular Genetics  2014;23(19):5283-5293.
We sequenced the whole exome of 35 cases and 7 controls from 9 age-related macular degeneration (AMD) families in whom known common genetic risk alleles could not explain their high disease burden and/or their early-onset advanced disease. Two families harbored novel rare mutations in CFH (R53C and D90G). R53C segregates perfectly with AMD in 11 cases (heterozygous) and 1 elderly control (reference allele) (LOD = 5.07, P = 6.7 × 10−7). In an independent cohort, 4 out of 1676 cases but none of the 745 examined controls or 4300 NHBLI Exome Sequencing Project (ESP) samples carried the R53C mutation (P = 0.0039). In another family of six siblings, D90G similarly segregated with AMD in five cases and one control (LOD = 1.22, P = 0.009). No other sample in our large cohort or the ESP had this mutation. Functional studies demonstrated that R53C decreased the ability of FH to perform decay accelerating activity. D90G exhibited a decrease in cofactor-mediated inactivation. Both of these changes would lead to a loss of regulatory activity, resulting in excessive alternative pathway activation. This study represents an initial application of the whole-exome strategy to families with early-onset AMD. It successfully identified high impact alleles leading to clearer functional insight into AMD etiopathogenesis.
PMCID: PMC4159152  PMID: 24847005
2.  Complement regulator CD46: genetic variants and disease associations 
Human Genomics  2015;9(1):7.
Membrane cofactor protein (MCP; CD46) is an ubiquitously expressed complement regulatory protein that protects host cells from injury by complement. This type-I membrane glycoprotein serves as a cofactor for the serine protease factor I to mediate inactivation of C3b and C4b deposited on host cells. More than 60 disease-associated mutations in MCP have now been identified. The majority of the mutations are linked to a rare thrombotic microangiopathic-based disease, atypical hemolytic uremic syndrome (aHUS), but new putative links to systemic lupus erythematosus, glomerulonephritis, and pregnancy-related disorders among others have also been identified. This review summarizes our current knowledge of disease-associated mutations in this complement inhibitor.
PMCID: PMC4469999  PMID: 26054645
CD46; Membrane cofactor protein; Complement; Complement regulation; Atypical hemolytic uremic syndrome
3.  Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS) 
Molecular immunology  2014;63(2):287-296.
Autoantibody formation against Factor H (FH) is found in 7–10% of patients who are diagnosed with atypical haemolytic uraemic syndrome (aHUS). These autoantibodies predominately target the C-terminal cell binding recognition domain of FH and are associated with absence of FHR1. Additional autoantibodies have also been identified in association with aHUS, for example autoantibodies to Factor I. Based on this, and that there are genetic mutations in other complement regulators and activators associated with aHUS, we hypothesised that other complement regulator proteins, particularly surface bound regulators in the kidney, might be the target for autoantibody formation in aHUS. Therefore, we assayed serum derived from 89 patients in the Newcastle aHUS cohort for the presence of autoantibodies to CD46 (membrane cofactor protein, MCP), CD55 (decay accelerating factor, DAF), CD35 (complement receptor type 1, CR1; TP10) and CD59. We also assayed 100 healthy blood donors to establish the normal levels of reactivity towards these proteins in the general population. Recombinant proteins CD46 and CD55 (purified from Escherichia coli) as well as soluble CR1 (CD35) and oligomeric C4BP-CD59 (purified from eukaryotic cell media) were used in ELISA to detect high responders. False positive results were established though Western blot and flow cytometric analysis. After excluding false positive responders to bacterial proteins in the CD46 and CD55 preparations, and responses to blood group antigens in CD35, we found no significant level of patient serum IgG reactivity with CD46, CD55, CD35 or CD59 above that detected in the normal population. These results suggest that membrane anchored complement regulators are not a target for autoantibody generation in aHUS.
PMCID: PMC4452024  PMID: 25150608
Autoantibodies; Complement; Surface regulators; Ahus
4.  Rare genetic variants in the CFI gene are associated with advanced age-related macular degeneration and commonly result in reduced serum factor I levels 
Human Molecular Genetics  2015;24(13):3861-3870.
To assess a potential diagnostic and therapeutic biomarker for age-related macular degeneration (AMD), we sequenced the complement factor I gene (CFI) in 2266 individuals with AMD and 1400 without, identifying 231 individuals with rare genetic variants. We evaluated the functional impact by measuring circulating serum factor I (FI) protein levels in individuals with and without rare CFI variants. The burden of very rare (frequency <1/1000) variants in CFI was strongly associated with disease (P = 1.1 × 10−8). In addition, we examined eight coding variants with counts ≥5 and saw evidence for association with AMD in three variants. Individuals with advanced AMD carrying a rare CFI variant had lower mean FI compared with non-AMD subjects carrying a variant (P < 0.001). Further new evidence that FI levels drive AMD risk comes from analyses showing individuals with a CFI rare variant and low FI were more likely to have advanced AMD (P = 5.6 × 10−5). Controlling for covariates, low FI increased the risk of advanced AMD among those with a variant compared with individuals without advanced AMD with a rare CFI variant (OR 13.6, P = 1.6 × 10−4), and also compared with control individuals without a rare CFI variant (OR 19.0, P = 1.1 × 10−5). Thus, low FI levels are strongly associated with rare CFI variants and AMD. Enhancing FI activity may be therapeutic and measuring FI provides a screening tool for identifying patients who are most likely to benefit from complement inhibitory therapy.
PMCID: PMC4459386  PMID: 25788521
5.  Autoimmunity: homeostasis of innate immunity gone awry 
Journal of clinical immunology  2012;32(6):1148-1152.
PMCID: PMC3529793  PMID: 23054347
6.  Complement Factor B is the Downstream Effector of Toll-Like Receptors and Plays an Important Role in a Mouse Model of Severe Sepsis¶ 
Severe sepsis involves massive activation of the innate immune system and leads to high mortality. Previous studies have demonstrated that various types of Toll-like receptors (TLRs) mediate a systemic inflammatory response and contribute to organ injury and mortality in animal models of severe sepsis. However, the downstream mechanisms responsible for TLR-mediated septic injury are poorly understood. Here, we show that activation of TLR2, TLR3 and TLR4 markedly enhanced complement factor B (cfB) synthesis and release by macrophages and cardiac cells. Polymicrobial sepsis, created by cecal ligation and puncture (CLP) in a mouse model, augmented cfB levels in the serum, peritoneal cavity and major organs including the kidney and heart. CLP also led to the alternative pathway (AP) activation, C3 fragment deposition in the kidney and heart, and cfB-dependent C3dg elevation. Bacteria isolated from septic mice activated the serum AP via a factor D-dependent manner. MyD88 deletion attenuated cfB/C3 up-regulation as well as cleavage induced by polymicrobial infection. Importantly, during sepsis, absence of cfB conferred a protective effect with improved survival and cardiac function, and markedly attenuated acute kidney injury. cfB deletion also led to increased neutrophil migratory function during the early phase of sepsis, decreased local and systemic bacterial load, attenuated cytokine production and reduced neutrophil reactive oxygen species production. Together, our data indicate that cfB acts as a downstream effector of TLR signaling and plays a critical role in the pathogenesis of severe bacterial sepsis.
PMCID: PMC3906719  PMID: 24154627
7.  Analysis of genes coding for CD46, CD55 and C4b-binding protein in patients with idiopathic, recurrent, spontaneous pregnancy loss 
European journal of immunology  2013;43(6):1617-1629.
Since a tightly regulated complement system is needed for a successful pregnancy, we hypothesized that alterations in complement inhibitors may be associated with idiopathic, recurrent miscarriage. We sequenced all exons coding for three complement inhibitors: C4b-binding protein (C4BP), CD46 and CD55 in 384 childless women with at least two miscarriages that could not be explained by known risk factors. Several alterations were found in C4BPA, of which the R120H, I126T, and the G423T mutations affected the expression level and/or the ability of recombinant C4BP to serve as cofactor for factor I. The only variant in C4BPB was located in the C-terminal part, and did not impair the polymerization of the molecule. Our results identify for the first time alterations in C4BP in women experiencing recurrent miscarriages. We also found four CD46 alterations in individual patients that were not found in healthy controls. One of the rare variants, P324L, showed decreased expression, whereas N213I resulted in deficient protein processing as well as an impaired cofactor activity in the degradation of both C4b and C3b. The identified alterations may result in in vivo consequences and contribute to the disorder but the degree of association must be evaluated in larger cohorts.
PMCID: PMC3760018  PMID: 23508668
complement system; complement inhibitor; mutation; reproductive immunology
8.  Binding of Flavivirus Non-structural Protein NS1 to C4b Binding Protein Modulates Complement Activation 
The complement system plays a pivotal protective role in the innate immune response to many pathogens including Flaviviruses. Flavivirus NS1 is a secreted non-structural glycoprotein that accumulates in plasma to high levels and is displayed on the surface of infected cells but is absent from viral particles. Previous work has defined an immune evasion role of Flavivirus NS1 in limiting complement activation by forming a complex with C1s and C4 to promote cleavage of C4 to C4b. Here, we demonstrate a second mechanism, also involving C4 and its active fragment C4b, by which NS1 antagonizes complement activation. Dengue, West Nile or yellow fever virus NS1 directly associated with C4b binding protein (C4BP), a complement regulatory plasma protein that attenuates the classical and lectin pathways. Soluble NS1 recruited C4BP to inactivate C4b in solution and on the plasma membrane. Mapping studies revealed that the interaction sites of NS1 on C4BP partially overlap with the C4b binding sites. Together, these studies further define the immune evasion potential of NS1 in reducing the functional capacity of C4 in complement activation and control of Flavivirus infection.
PMCID: PMC3119735  PMID: 21642539
dengue virus (DENV); West Nile virus (WNV); yellow fever virus (YFV); non-structural protein NS1; immune evasion; pathogenesis; C4b binding protein (C4BP); C4; the fourth component of complement; complement
9.  Complement regulator CD46 temporally regulates cytokine production by conventional and unconventional T cells 
Nature immunology  2010;11(9):862-871.
In this study we demonstrate a new form of immunoregulation: engagement on CD4+ T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (TH1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4+ T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4+ T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-γ (IFN-γ). Finally, γδ T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) γδ and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.
PMCID: PMC4011020  PMID: 20694009
10.  Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis 
PLoS Pathogens  2014;10(4):e1004086.
The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar−/− mice completely lacking type I IFN signaling. In Mavs−/−×Ifnar−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar−/− and CD11c Cre+Ifnarf/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.
Author Summary
Although it is well established that the interferon (IFN) signaling pathway restricts infection by many viruses, the key cell types in vivo that contribute to this process remain poorly characterized. To address this question in the context of West Nile virus (WNV) pathogenesis, we infected mice that specifically delete the type I IFN receptor gene (Ifnar) in subsets of myeloid cells, including dendritic cells and macrophages. Remarkably, mice lacking Ifnar expression only in myeloid cell subsets rapidly developed a sepsis-like syndrome that was characterized by enhanced WNV infection and visceral organ injury and caused by massive proinflammatory cytokine production and complement activation. By using additional gene targeted deletion mice, we show that WNV infection triggered signaling through the RIG-I like receptor adaptor protein MAVS to cause complement activation, sepsis, and tissue damage. Indeed, liver damage was minimized in animals lacking specific complement components, or treated with neutralizing anti-complement or anti-TNF-α antibodies. Our results establish how type I IFN signaling in dendritic cells and macrophages restricts infection, controls inflammatory cascades, and prevents pathogenesis in vivo.
PMCID: PMC3990718  PMID: 24743949
11.  Immunology of age-related macular degeneration 
Nature reviews. Immunology  2013;13(6):438-451.
Age-related macular degeneration (AMD) is a leading cause of blindness in aged individuals. Recent advances have highlighted the essential role of immune processes in the development, progression and treatment of AMD. In this Review we discuss recent discoveries related to the immunological aspects of AMD pathogenesis. We outline the diverse immune cell types, inflammatory activators and pathways that are involved. Finally, we discuss the future of inflammation-directed therapeutics to treat AMD in the growing aged population.
PMCID: PMC3941009  PMID: 23702979
12.  Hepatitis C Virus Infection Upregulates CD55 Expression on the Hepatocyte Surface and Promotes Association with Virus Particles 
Journal of Virology  2013;87(14):7902-7910.
CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or full-length (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion.
PMCID: PMC3700207  PMID: 23658447
13.  Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation 
Immunity  2013;39(6):1143-1157.
Complement is viewed as a critical serum-operative component of innate immunity, with processing of its key component, C3, into activation fragments C3a and C3b confined to the extracellular space. We report here that C3 activation also occurred intracellularly. We found that the T cell-expressed protease cathepsin L (CTSL) processed C3 into biologically active C3a and C3b. Resting T cells contained stores of endosomal and lysosomal C3 and CTSL and substantial amounts of CTSL-generated C3a. While “tonic” intracellular C3a generation was required for homeostatic T cell survival, shuttling of this intracellular C3-activation-system to the cell surface upon T cell stimulation induced autocrine proinflammatory cytokine production. Furthermore, T cells from patients with autoimmune arthritis demonstrated hyperactive intracellular complement activation and interferon-γ production and CTSL inhibition corrected this deregulated phenotype. Importantly, intracellular C3a was observed in all examined cell populations, suggesting that intracellular complement activation might be of broad physiological significance.
Graphical Abstract
•Complement C3 is activated intracellularly in human T cells by cathepsin L•Intracellular C3 activation mediates cell survival and Th1 induction•Increased intracellular C3 activation underlies T effector dysregulation in arthritis•Patients with serum C3-deficiency retain intracellular C3a generation
PMCID: PMC3865363  PMID: 24315997
14.  Autoantibody stabilization of the classical pathway C3 convertase leading to C3 deficiency and Neisserial sepsis: C4 nephritic factor revisited 
Clinical immunology (Orlando, Fla.)  2012;145(3):241-250.
C3 deficiency is a rare disorder that leads to recurrent pyogenic infections. Here we describe a previously healthy 18 y/o Caucasian male with severe meningococcal disease. Total hemolytic activity was zero secondary to an undetectable C3. The C3 gene was normal by sequencing. Mixing the patient’s serum with normal human serum led to C3 consumption. An IgG autoantibody in the patient’s serum was identified that stabilized the classical pathway C3 and C5 convertases, thus preventing decay of these enzyme complexes. This autoantibody is an example of a C4 nephritic factor, with an additional feature of stabilizing the C5 convertase. Previous patients with C4 nephritic factor had membranoproliferative glomerulonephritis. Two years after presentation, this patient’s C3 remains undetectable with no evidence of renal disease. We revisit the role of autoantibodies to classical pathway convertases in disease, reviews the literature on C4-NeF and we comment on its detection in the clinical laboratory.
PMCID: PMC3501611  PMID: 23117396
C3 deficiency; Neisserial infection; C4 nephritic factor; autoimmunity
15.  Antagonism of the complement component C4 by flavivirus nonstructural protein NS1 
The complement system plays an essential protective role in the initial defense against many microorganisms. Flavivirus NS1 is a secreted nonstructural glycoprotein that accumulates in blood, is displayed on the surface of infected cells, and has been hypothesized to have immune evasion functions. Herein, we demonstrate that dengue virus (DENV), West Nile virus (WNV), and yellow fever virus (YFV) NS1 attenuate classical and lectin pathway activation by directly interacting with C4. Binding of NS1 to C4 reduced C4b deposition and C3 convertase (C4b2a) activity. Although NS1 bound C4b, it lacked intrinsic cofactor activity to degrade C4b, and did not block C3 convertase formation or accelerate decay of the C3 and C5 convertases. Instead, NS1 enhanced C4 cleavage by recruiting and activating the complement-specific protease C1s. By binding C1s and C4 in a complex, NS1 promotes efficient degradation of C4 to C4b. Through this mechanism, NS1 protects DENV from complement-dependent neutralization in solution. These studies define a novel immune evasion mechanism for restricting complement control of microbial infection.
PMCID: PMC2856034  PMID: 20308361
16.  Smallpox Inhibitor of Complement Enzymes (SPICE): Dissecting Functional Sites and Abrogating Activity1 
Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host’s immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity ~200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent.
PMCID: PMC2899487  PMID: 19667083
17.  New roles for the major human 3'–5' exonuclease TREX1 in human disease 
Cell cycle (Georgetown, Tex.)  2008;7(12):1718-1725.
Aicardi-Goutières syndrome (AGS), Systemic Lupus Erythematosus (SLE), Familial Chilblain Lupus (FCL) and Retinal Vasculopathy and Cerebral Leukodystrophy (RVCL) {a new term encompassing three independently described conditions with a common etiology—Cerebroretinal Vasculopathy (CRV), Hereditary Vascular Retinopathy (HVR) and Hereditary Endotheliopathy, Retinopathy and Nephropathy (HERNS)}—have previously been regarded as distinct entities. However, recent genetic analysis has demonstrated that each of these diseases maps to chromosome 3p21 and can be caused by mutations in TREX1, the major human 3'–5' exonuclease. In this review, we discuss the putative functions of TREX1 in relationship to the clinical, genetic and functional characteristics of each of these conditions.
PMCID: PMC2825026  PMID: 18583934
TREX1; TREX2; DNase III; stroke; cerebrovascular disease
18.  Inhibiting Complement Activation on Cells at the Step of C3 Cleavage 
Vaccine  2008;26(Suppl 8):I22-I27.
Nearly half of the proteins in the complement system serve in regulation. Control at the central step of C3 activation is provided by an orchestrated interplay of membrane and plasma regulators. A model system employing Chinese hamster ovary (CHO) cells transfected with human regulators was employed to assist in making functional comparisons. Also, in this experimental setup, the pathway and magnitude of complement activation can be varied while monitoring C4b/C3b deposition and cleavage as well as cytotoxicity. This review describes lessons learned from a CHO model and the application of this model for functionally characterizing mutations in regulators associated with atypical hemolytic uremic syndrome.
PMCID: PMC2768381  PMID: 19388160
19.  Smallpox inhibitor of complement enzymes (SPICE): Regulation of complement activation on cells and mechanism of its cellular attachment 
Despite eradication of smallpox three decades ago, public health concerns remain due to its potential use as a bioterrorist weapon. Smallpox and other orthopoxviruses express virulence factors that inhibit the host’s complement system. In this study, our goals were to characterize the ability of the smallpox inhibitor of complement enzymes, SPICE, to regulate human complement on the cell surface. We demonstrate that SPICE binds to a variety of cell types and that the heparan sulfate and chondroitin sulfate glycosaminoglycans (GAGs) serve as attachment sites. A transmembrane engineered version as well as soluble recombinant SPICE inhibited complement activation at the C3 convertase step with equal or greater efficiency than that of the related host regulators. Moreover, SPICE attached to GAGs was more efficient than transmembrane SPICE. We also demonstrate that this virulence activity of SPICE on cells could be blocked by a mAb to SPICE. These results provide insights related to the complement inhibitory activities of poxviral inhibitors of complement and describe a mAb with therapeutic potential.
PMCID: PMC2774262  PMID: 18768877
complement regulation; poxviruses; glycosaminoglycans
20.  The Role of the Immune Response in Age-Related Macular Degeneration 
Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries; with the aging population, the negative health impacts and costs of the disease will increase dramatically over the next decade. Although the exact cause of AMD is unknown, genetic studies have implicated the complement system as well as other immune responses in disease pathogenesis and severity. Furthermore, histologic studies have shown the presence of macrophages, lymphocytes, and mast cells, as well as fibroblasts, in both atrophic lesions and with retinal neovascularization. This review summarizes discussions from the fifth annual conference of the Arnold and Mabel Beckman Initiative for Macular Research by the Inflammation and Immune Response Task Force. These deliberations focused on the role of inflammatory immune responses, including complement, inflammasomes, adaptive immune responses, and para-inflammation, unanswered questions and studies to address these questions, and potential immune-related therapeutic targets for AMD.
PMCID: PMC3676958  PMID: 23762772
21.  Membrane protein Crry maintains homeostasis of the complement system1 
Complement activation is tightly regulated to avoid excessive inflammatory and immune responses. Crry-/- is an embryonic lethal phenotype secondary to the maternal complement alternative pathway (AP) attacking a placenta deficient in this inhibitor. In this study, we demonstrate that Crry-/- mice could be rescued on a partial as well as on a complete factor B (fB)- or C3-deficient maternal background. The C3 and fB protein concentrations in Crry-/-C3+/- and Crry-/-fB+/- mice were substantially reduced for gene dosage secondary to enhanced AP turnover. Based on these observations, a breeding strategy featuring reduced maternal AP-activating capacity rescued the lethal phenotype. It led to a novel, stable line of Crry SKO mice carrying normal alleles for C3 and fB. Crry SKO mice also had accelerated C3 and fB turnover and therefore reduced AP-activating potential. These instructive results represent an example of a membrane regulatory protein being responsible for homeostasis of the complement system. They imply that there is constant turnover on cells of the AP pathway which functions as an immune surveillance system for pathogens and altered self.
PMCID: PMC2580744  PMID: 18684964
22.  Pathogenesis of aortic dilatation in mucopolysaccharidosis VII mice may involve complement activation 
Molecular Genetics and Metabolism  2011;104(4):608-619.
Mucopolysaccharidosis VII (MPS VII) is due to mutations within the gene encoding the lysosomal enzyme β-glucuronidase, and results in the accumulation of glycosaminoglycans. MPS VII causes aortic dilatation and elastin fragmentation, which is associated with upregulation of the elastases cathepsin S (CtsS) and matrix metalloproteinase 12 (MMP12). To test the role of these enzymes, MPS VII mice were crossed with mice deficient in CtsS or MMP12, and the effect upon aortic dilatation was determined. CtsS deficiency did not protect against aortic dilatation in MPS VII mice, but also failed to prevent an upregulation of cathepsin enzyme activity. Further analysis with substrates and inhibitors specific for particular cathepsins suggests that this enzyme activity was due to CtsB, which could contribute to elastin fragmentation. Similarly, MMP12 deficiency and deficiency of both MMP12 and CtsS could not prevent aortic dilatation in MPS VII mice. Microarray and reverse-transcriptase real-time PCR were performed to look for upregulation of other elastases. This demonstrated that mRNA for complement component D was elevated in MPS VII mice, while immunostaining demonstrated high levels of complement component C3 on surfaces within the aortic media. Finally, we demonstrate that neonatal intravenous injection of a retroviral vector encoding β-glucuronidase reduced aortic dilatation. We conclude that neither CtsS nor MMP12 are necessary for elastin fragmentation in MPS VII mouse aorta, and propose that CtsB and/or complement component D may be involved. Complement may be activated by the GAGs that accumulate, and may play a role in signal transduction pathways that upregulate elastases.
PMCID: PMC3283036  PMID: 21944884
Mucopolysaccharidosis VII; Cathepsin S; Matrix metalloproteinase 12; Complement system; Aortic dilatation; Gene therapy
23.  Bypassing complement: evolutionary lessons and future implications 
Journal of Clinical Investigation  2006;116(5):1215-1218.
Lectins like mannan-binding protein are part of the innate immune system. They circulate in association with serine proteases. Upon binding oligosaccharides, they activate the complement cascade analogous to the more familiar but evolutionarily more recent classical pathway, which is triggered by antibody binding to antigen. In this issue of the JCI, Selander et al. developed a sensitive and specific ELISA employing Salmonella-specific sugars to assess the activity of the lectin pathway of complement activation (see the related article beginning on page 1425). This more physiologic assay system allowed the investigators to rigorously define the requirements for lectin pathway activation. Furthermore, they uncovered an unsuspected means for this pathway to reach the desired critical step of activation of the opsonin C3. These types of functional assays will eventually replace the more laborious, less physiologic, and less informative approaches currently in use to monitor complement activation.
PMCID: PMC1451225  PMID: 16670764
24.  C5a and Fcγ receptors: a mutual admiration society 
Journal of Clinical Investigation  2006;116(2):304-306.
Phagocytosis is a key process in protection of the host against pathogens and in provision of antigens for the immune response. Synergism between C3b and IgG and their receptors in promoting adherence to and then ingestion of an antigen has been recognized for decades. Only more recently, however, has cross-talk between another complement activation fragment, the anaphylatoxin C5a, and Fcγ receptors (FcγRs) been defined. In this issue of the JCI, C5a is shown to signal, via its receptor, the upregulation of activating (proinflammatory-type) FcγRs. Moreover, engagement of FcγRs by the IgG-bearing immune complex instructs the cell to synthesize more C5, from which C5a is derived. Thus, this work establishes a feedback loop whereby FcγR expression and function are enhanced, a very desirable event in concert with an infection but potentially deleterious in autoimmunity.
PMCID: PMC1359066  PMID: 16453017
25.  N-linked glycosylation of Dengue virus NS1 protein modulates secretion, cell-surface expression, hexamer stability, and interactions with human complement 
Virology  2011;413(2):253-264.
Dengue virus (DENV) NS1 is a versatile non-structural glycoprotein that is secreted as a hexamer, binds to the cell surface of infected and uninfected cells, and has immune evasive functions. DENV NS1 displays two conserved N-linked glycans at N130 and N207. In this study, we examined the role of these two N-linked glycans on NS1 secretion, stability, and function. Because some groups have reported reduced yields of infectious DENV when N130 and N207 are changed, we analyzed glycosylation-deficient NS1 phenotypes using a transgenic expression system. We show that the N-linked glycan at position 130 is required for stabilization of the secreted hexamer whereas the N-linked glycan at residue 207 facilitates secretion and extracellular protein stability. Moreover, NS1 mutants lacking an N-linked glycan at N130 did not interact efficiently with complement components C1s and C4. In summary, our results elucidate the contribution of N-linked glycosylation to the function of DENV NS1.
PMCID: PMC3089955  PMID: 21429549
Dengue virus; Flavivirus; non-structural protein NS1; N-linked glycosylation; complement

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