First identification of a chemotactic receptor in an invertebrate species: Structural and functional characterization of Ciona intestinalis C3a receptor

In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98–102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process

Surviving Mousepox Infection Requires the Complement System

Poxviruses subvert the host immune response by producing immunomodulatory proteins, including a complement regulatory protein. Ectromelia virus provides a mouse model for smallpox where the virus and the host's immune response have co-evolved. Using this model, our study investigated the role of the complement system during a poxvirus infection. By multiple inoculation routes, ectromelia virus caused increased mortality by 7 to 10 days post-infection in C57BL/6 mice that lack C3, the central component of the complement cascade. In C3−/− mice, ectromelia virus disseminated earlier to target organs and generated higher peak titers compared to the congenic controls. Also, increased hepatic inflammation and necrosis correlated with these higher tissue titers and likely contributed to the morbidity in the C3−/− mice. In vitro, the complement system in naïve C57BL/6 mouse sera neutralized ectromelia virus, primarily through the recognition of the virion by natural antibody and activation of the classical and alternative pathways. Sera deficient in classical or alternative pathway components or antibody had reduced ability to neutralize viral particles, which likely contributed to increased viral dissemination and disease severity in vivo. The increased mortality of C4−/− or Factor B−/− mice also indicates that these two pathways of complement activation are required for survival. In summary, the complement system acts in the first few minutes, hours, and days to control this poxviral infection until the adaptive immune response can react, and loss of this system results in lethal infection

Elucidating the Role of the Complement Control Protein in Monkeypox Pathogenicity

Monkeypox virus (MPXV) causes a smallpox-like disease in humans. Clinical and epidemiological studies provide evidence of pathogenicity differences between two geographically distinct monkeypox virus clades: the West African and Congo Basin. Genomic analysis of strains from both clades identified a ∼10 kbp deletion in the less virulent West African isolates sequenced to date. One absent open reading frame encodes the monkeypox virus homologue of the complement control protein (CCP). This modulatory protein prevents the initiation of both the classical and alternative pathways of complement activation. In monkeypox virus, CCP, also known as MOPICE, is a ∼24 kDa secretory protein with sequence homology to this superfamily of proteins. Here we investigate CCP expression and its role in monkeypox virulence and pathogenesis. CCP was incorporated into the West African strain and removed from the Congo Basin strain by homologous recombination. CCP expression phenotypes were confirmed for both wild type and recombinant monkeypox viruses and CCP activity was confirmed using a C4b binding assay. To characterize the disease, prairie dogs were intranasally infected and disease progression was monitored for 30 days. Removal of CCP from the Congo Basin strain reduced monkeypox disease morbidity and mortality, but did not significantly decrease viral load. The inclusion of CCP in the West African strain produced changes in disease manifestation, but had no apparent effect on disease-associated mortality. This study identifies CCP as an important immuno-modulatory protein in monkeypox pathogenesis but not solely responsible for the increased virulence seen within the Congo Basin clade of monkeypox virus

Cloning and purification of the Rainbow Trout fifth component of complement (C5).

To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifthcomponent of complement from a rainbow trout (Oncorhynchus mykiss) liver cDNA library; this is the ®rst report of C5 cloningin a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25 kb), representingapproximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validatethe molecular information derived from the cloning we developed an improved puri®cation protocol. Mass spectrometricanalysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partialcDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liverand Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence ofC5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish

Characterization of factor H-like molecules in Rainbow Trout

Characterization of factor H-like molecules in Rainbow Trou

Attenuation of Staphylococcus aureus-induced bacteremia by human mini-antibodies targeting the complement inhibitory protein Efb

Staphylococcus aureus can cause a broad range of potentially fatal inflammatory complications (e.g., sepsis and endocarditis). Its emerging antibiotic resistance and formidable immune evasion arsenal have emphasized the need for more effective antimicrobial approaches. Complement is an innate immune sensor that rapidly responds to bacterial infection eliciting C3-mediated opsonophagocytic and immunomodulatory responses. Extracellular fibrinogen-binding protein (Efb) is a key immune evasion protein of S. aureus that intercepts complement at the level of C3. To date, Efb has not been explored as a target for mAb-based antimicrobial therapeutics. In this study, we have isolated donor-derived anti-Efb IgGs that attenuate S. aureus survival through enhanced neutrophil killing. A phage library screen yielded mini-Abs that selectively inhibit the interaction of Efb with C3 partly by disrupting contacts essential for complex formation. Surface plasmon resonance-based kinetic analysis enabled the selection of mini-Abs with favorable Efb-binding profiles as therapeutic leads. Mini-Ab-mediated blockade of Efb attenuated S. aureus survival in a whole blood model of bacteremia. This neutralizing effect was associated with enhanced neutrophil-mediated killing of S. aureus, increased C5a release, and modulation of IL-6 secretion. Finally, these mini-Abs afforded protection from S. aureus-induced bacteremia in a murine renal abscess model, attenuating bacterial inflammation in kidneys. Overall, these findings are anticipated to pave the way toward novel Ab-based therapeutics for S. aureus-related diseases. Copyright © 2015 by The American Association of Immunologists, Inc

Attenuation of Staphylococcus aureus-induced bacteremia by human mini-antibodies targeting the complement inhibitory protein Efb

Staphylococcus aureus can cause a broad range of potentially fatal inflammatory complications (e.g., sepsis and endocarditis). Its emerging antibiotic resistance and formidable immune evasion arsenal have emphasized the need for more effective antimicrobial approaches. Complement is an innate immune sensor that rapidly responds to bacterial infection eliciting C3-mediated opsonophagocytic and immunomodulatory responses. Extracellular fibrinogen-binding protein (Efb) is a key immune evasion protein of S. aureus that intercepts complement at the level of C3. To date, Efb has not been explored as a target for mAb-based antimicrobial therapeutics. In this study, we have isolated donor-derived anti-Efb IgGs that attenuate S. aureus survival through enhanced neutrophil killing. A phage library screen yielded mini-Abs that selectively inhibit the interaction of Efb with C3 partly by disrupting contacts essential for complex formation. Surface plasmon resonance-based kinetic analysis enabled the selection of mini-Abs with favorable Efb-binding profiles as therapeutic leads. Mini-Ab-mediated blockade of Efb attenuated S. aureus survival in a whole blood model of bacteremia. This neutralizing effect was associated with enhanced neutrophil-mediated killing of S. aureus, increased C5a release, and modulation of IL-6 secretion. Finally, these mini-Abs afforded protection from S. aureus-induced bacteremia in a murine renal abscess model, attenuating bacterial inflammation in kidneys. Overall, these findings are anticipated to pave the way toward novel Ab-based therapeutics for S. aureus-related diseases. Copyright © 2015 by The American Association of Immunologists, Inc

Complement activation is critical to airway hyperresponsiveness after acute ozone exposure.

Ozone (O3) can induce airway hyperresponsiveness (AHR) and neutrophilic inflammation. We evaluated the role of complement in development of AHR and inflammation after acute O3 exposure in mice. Mice were exposed to O3 at 2 ppm for 3 hours, and airway responsiveness to methacholine was measured 8 hours after O3 exposure. Complement was depleted or inhibited by intraperitoneal injection of cobra venom factor (CVF) or complement receptor-related gene y (Crry)-Ig, a potent C3 convertase inhibitor; neutrophils were depleted using an antineutrophil monoclonal antibody. CVF attenuated the development of AHR by O3. Administration of Crry-Ig also prevented the development of AHR. Bronchoalveolar lavage (BAL) fluid neutrophilia after O3 exposure was significantly decreased by administration of either CVF or Crry-Ig. Increased BAL fluid total protein after O3 exposure was lowered by depletion or inhibition of complement. In contrast to the effects of complement inhibition or depletion, depletion of BAL neutrophil counts by more than 90% with the monoclonal antibody did not affect the development of AHR after O3 exposure. These data indicated that activation of the complement system follows acute O3 exposure and is important to the development of AHR and airway neutrophilia. However, this neutrophil response does not appear necessary for the development of AHR