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1.  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.
Summary
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.
doi:10.1002/eji.201243196
PMCID: PMC3760018  PMID: 23508668
complement system; complement inhibitor; mutation; reproductive immunology
2.  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.
doi:10.1038/ni.1917
PMCID: PMC4011020  PMID: 20694009
3.  Autoimmunity: homeostasis of innate immunity gone awry 
Journal of clinical immunology  2012;32(6):1148-1152.
doi:10.1007/s10875-012-9815-8
PMCID: PMC3529793  PMID: 23054347
4.  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.
doi:10.1038/nri3459
PMCID: PMC3941009  PMID: 23702979
5.  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.
doi:10.1128/JVI.00917-13
PMCID: PMC3700207  PMID: 23658447
6.  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.
doi:10.1016/j.clim.2012.09.007
PMCID: PMC3501611  PMID: 23117396
C3 deficiency; Neisserial infection; C4 nephritic factor; autoimmunity
7.  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.
doi:10.1155/2013/348092
PMCID: PMC3676958  PMID: 23762772
8.  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.
doi:10.1371/journal.ppat.1004086
PMCID: PMC3990718  PMID: 24743949
9.  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.
doi:10.1016/j.ymgme.2011.08.018
PMCID: PMC3283036  PMID: 21944884
Mucopolysaccharidosis VII; Cathepsin S; Matrix metalloproteinase 12; Complement system; Aortic dilatation; Gene therapy
10.  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.
doi:10.4049/jimmunol.0901366
PMCID: PMC2899487  PMID: 19667083
11.  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
12.  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.
doi:10.1016/j.vaccine.2008.11.001
PMCID: PMC2768381  PMID: 19388160
13.  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
14.  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
15.  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.
doi:10.4049/jimmunol.1100750
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
16.  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.
doi:10.1016/j.virol.2011.02.022
PMCID: PMC3089955  PMID: 21429549
Dengue virus; Flavivirus; non-structural protein NS1; N-linked glycosylation; complement
17.  Complement-Mediated Neutralization of Dengue Virus Requires Mannose-Binding Lectin 
mBio  2011;2(6):e00276-11.
ABSTRACT
Mannose-binding lectin (MBL) is a key soluble pathogen recognition protein of the innate immune system that binds specific mannose-containing glycans on the surfaces of microbial agents and initiates complement activation via the lectin pathway. Prior studies showed that MBL-dependent activation of the complement cascade neutralized insect cell-derived West Nile virus (WNV) in cell culture and restricted pathogenesis in mice. Here, we investigated the antiviral activity of MBL in infection by dengue virus (DENV), a related flavivirus. Using a panel of naïve sera from mouse strains deficient in different complement components, we showed that inhibition of infection by insect cell- and mammalian cell-derived DENV was primarily dependent on the lectin pathway. Human MBL also bound to DENV and neutralized infection of all four DENV serotypes through complement activation-dependent and -independent pathways. Experiments with human serum from naïve individuals with inherent variation in the levels of MBL in blood showed a direct correlation between the concentration of MBL and neutralization of DENV; samples with high levels of MBL in blood neutralized DENV more efficiently than those with lower levels. Our studies suggest that allelic variation of MBL in humans may impact complement-dependent control of DENV pathogenesis.
IMPORTANCE
Dengue virus (DENV) is a mosquito-transmitted virus that causes a spectrum of clinical disease in humans ranging from subclinical infection to dengue hemorrhagic fever and dengue shock syndrome. Four serotypes of DENV exist, and severe illness is usually associated with secondary infection by a different serotype. Here, we show that mannose-binding lectin (MBL), a pattern recognition molecule that initiates the lectin pathway of complement activation, neutralized infection of all four DENV serotypes through complement activation-dependent and -independent pathways. Moreover, we observed a direct correlation with the concentration of MBL in human serum and neutralization of DENV infection. Our studies suggest that common genetic polymorphisms that result in disparate levels and function of MBL in humans may impact DENV infection, pathogenesis, and disease severity.
doi:10.1128/mBio.00276-11
PMCID: PMC3236064  PMID: 22167226
18.  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.
doi:10.1172/JCI28622
PMCID: PMC1451225  PMID: 16670764
19.  Hepatic IL-17 Responses in Human and Murine Primary Biliary Cirrhosis 
Journal of autoimmunity  2008;32(1):43-51.
The emergence of new regulatory and pro-inflammatory immune cell subsets and cytokines dictates the need to re-examine the role of these subsets in various diseases involving the immune system. IL-17 has been recently identified as a key cytokine involved in numerous autoimmune processes. However, its role in liver autoimmune diseases remains unclear. Primary biliary cirrhosis (PBC) is characterized histologically by autoreactive CD4 and CD8 T cells surrounding damaged bile ducts. CD4+ T cells are a major source of IL-17, which compose a distinct T helper subset (Th17). Thus we set out determine the role of IL-17 in both human and a murine model of PBC in a liver-targeted manner. Our data demonstrate an increase in the frequency of IL-17+ lymphocytic infiltration in liver tissues from PBC patients and those with other liver dysfunctions as compared to healthy livers. IL-2 receptor α knockout mice, a recently identified murine model of human PBC, also demonstrate marked aggregations of IL-17 positive cells within portal tracts and increased frequencies of Th17 cells in the liver compared to the periphery. Interestingly, CD4+ T cells from livers of normal C57BL/6J mice also secreted higher levels of IL-17 relative to those from spleens, indicating a preferential induction of Th17 cells in liver tissues. Importantly, C57BL/6J cocultures of splenic CD4+ T cells and liver non-parenchymal cells increased IL-17 production approximately 10 fold compared to T cells alone, suggesting a role of the liver microenvironment in Th17 induction in cases of liver autoimmunity and other liver inflammatory diseases.
doi:10.1016/j.jaut.2008.11.001
PMCID: PMC3225053  PMID: 19101114
IL-17; primary biliary cirrhosis; IL-2 receptor alpha; liver; CD46; Tr1; microenvironment; CD4+ T cells
20.  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.
doi:10.1172/JCI27759
PMCID: PMC1359066  PMID: 16453017
21.  Targeted and restricted complement activation on acrosome-reacted spermatozoa 
Journal of Clinical Investigation  2005;115(5):1241-1249.
A specific hypoglycosylated isoform of the complement regulator membrane cofactor protein (MCP; CD46) is expressed on the inner acrosomal membrane (IAM) of spermatozoa. This membrane is exposed after the acrosome reaction, an exocytosis event that occurs upon contact with the zona pellucida. We initiated this investigation to assess MCP’s regulatory function in situ on spermatozoa. Upon exposure of human spermatozoa to autologous serum or follicular fluid, we unexpectedly observed that acrosome-reacted spermatozoa activated the complement cascade efficiently through C3 but not beyond. Using FACS to simultaneously evaluate viability, acrosomal status, and complement deposition, we found that complement activation was initiated by C-reactive protein (CRP) and was C1q, C2, and factor B dependent. This pattern is consistent with engagement of the classical pathway followed by amplification through the alternative pathway. C3b deposition was targeted to the IAM, where it was cleaved to C3bi. Factor H, and not MCP, was the cofactor responsible for C3b cleavage. We propose that this localized deposition of complement fragments aids in the fusion process between the spermatozoa and egg, in a role akin to that of complement in immune adherence. In addition, we speculate that this “targeted and restricted” form of complement activation on host cells is a common strategy to handle modified self.
doi:10.1172/JCI200523213
PMCID: PMC1077172  PMID: 15849610
22.  Down-regulation of CD46 by Piliated Neisseria gonorrhoeae 
The Journal of Experimental Medicine  2003;198(9):1313-1322.
Human membrane cofactor protein (CD46) protects host cells against complement attack and may function as a receptor for pathogenic Neisseriae. We assessed CD46 expression in the human cervical cell line ME-180 after exposure to Neisseria gonorrhoeae. Piliated but not nonpiliated gonococci adhered to cells and produced up to an 80% reduction in CD46 surface expression by 6 h that persisted for at least 24 h. This response required a minimum multiplicity of infection of 10 and was not prevented by antibodies to CD46. CD46 down-regulation was not attributable to intracellular retention or a global or specific shutdown of mRNA or protein synthesis. Substantial quantities of CD46 were found in the supernatants, indicating a specific shedding of this protein. Adherent gonococci lacking the pilus retraction protein PilT did not down-regulate CD46 but de-repression of pilT expression restored CD46 down-regulation. After experimental infection of human volunteers with a gonococcal variant incapable of inducing CD46 down-regulation, variants of this strain were reisolated that exhibited CD46 down-regulation. Pilus-mediated interactions of gonococci with human epithelial cells results in a pathogen-induced manipulation of the host cell environment in which a membrane protein is removed from epithelial cells by liberation into the surrounding milieu.
doi:10.1084/jem.20031159
PMCID: PMC2194255  PMID: 14597734
Type IV pilus; PilT; pilus retraction; PilE; protein shedding
23.  CD46 Engagement on Human CD4+ T Cells Produces T Regulatory Type 1-Like Regulation of Antimycobacterial T Cell Responses ▿  
Infection and Immunity  2010;78(12):5295-5306.
Understanding the regulation of human immune responses is critical for vaccine development and treating infectious diseases. We have previously shown that simultaneous engagement of the T cell receptor (TCR) and complement regulator CD46 on human CD4+ T cells in the presence of interleukin-2 (IL-2) induces potent secretion of the immunomodulatory cytokine IL-10. These T cells mediate IL-10-dependent suppression of bystander CD4+ T cells activated in vitro with anti-CD3 and anti-CD28 costimulation, reflecting a T regulatory type 1 (Tr1)-like phenotype. However, CD46-mediated negative regulation of pathogen-specific T cells has not been described. Therefore, we studied the ability of CD46-activated human CD4+ T cells to suppress T cell responses to Mycobacterium bovis BCG, the live vaccine that provides infants protection against the major human pathogen Mycobacterium tuberculosis. Our results demonstrate that soluble factors secreted by CD46-activated human CD4+ T cells suppress mycobacterium-specific CD4+, CD8+, and γ9δ2 TCR+ T cells. Dendritic cell functions were not downregulated in our experiments, indicating that CD46-triggered factors directly suppress pathogen-specific T cells. Interestingly, IL-10 appeared to play a less pronounced role in our system, especially in the suppression of γ9δ2 TCR+ T cells, suggesting the presence of additional undiscovered soluble immunoregulatory factors. Blocking endogenous CD46 signaling 3 days after mycobacterial infection enhanced BCG-specific T cell responses in a subset of volunteers. Taken together, these results indicate that CD46-dependent negative regulatory mechanisms can impair T cell responses vital for immune defense against mycobacteria. Therefore, modulating CD46-induced immune regulation could be integral to the development of improved tuberculosis therapeutics or vaccines.
doi:10.1128/IAI.00513-10
PMCID: PMC2981321  PMID: 20921150
24.  Ectromelia Virus Inhibitor of Complement Enzymes Protects Intracellular Mature Virus and Infected Cells from Mouse Complement▿  
Journal of Virology  2010;84(18):9128-9139.
Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.
doi:10.1128/JVI.02677-09
PMCID: PMC2937632  PMID: 20610727
25.  Intracellular Complement Activation Sustains T Cell Homeostasis and Mediates Effector Differentiation 
Immunity  2013;39(6):1143-1157.
Summary
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
Highlights
•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
doi:10.1016/j.immuni.2013.10.018
PMCID: PMC3865363  PMID: 24315997

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