Guillain-Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the CH2 and CH3 domains. These data indicate that the IgG assembly is mediated through Fc-Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain-Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

The immune system homeostasis relies on a tight equilibrium of interconnected stimulatory and inhibitory signals. Disruption of this balance is characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE). Aside from activating the classical complement pathway and enhancing pathogens and apoptotic cells phagocytosis, C1q has been recently shown to play an important role in immune modulation and tolerance by interacting with several inhibitory and stimulatory immune receptors. Due to its functional organization into collagen-like (CLR) and globular (GR) regions and its multimeric nature, C1q is able to interact simultaneously with several of these receptors and locally congregate pro- and anti-inflammatory signals, thus modulating the immune response. Leukocyte associated immunoglobulin-like (Ig-like) receptor 1 (LAIR-1), a ubiquitous collagen receptor expressed in many immune cell types, has been reported to interact with the CLR of C1q. In this study, we provide new insights into the molecular and structural determinants underlying C1q/LAIR-1 interaction. Recombinant LAIR-1 extracellular Ig-like domain was produced and tested for its interaction with C1q. A molecular dissection of C1q combined with competition assays reveals that LAIR-1 interacts with C1q's CLR through a binding site close but different from the one of its associated C1r2s2 proteases tetramer. On the other side, we identified LAIR-1 residues involved in C1q interaction by site-directed mutational analysis. All together, these results lead to propose a possible model for C1q interaction with LAIR-1 and will contribute to the fundamental understanding of C1q-mediated immune tolerance.

The Influenza A virus (IAV) is a severe respiratory pathogen. C1q is the first subcomponent of the complement system's classical pathway. C1q is composed of 18 polypeptide chains. Each of these chains contains a collagen-like region located at the N terminus, and a C-terminal globular head region organized as a heterotrimeric structure (ghA, ghB and ghC). This study was aimed at investigating the complement activation-independent modulation by C1q and its individual recombinant globular heads against IAV infection. The interaction of C1q and its recombinant globular heads with IAV and its purified glycoproteins was examined using direct ELISA and far-Western blotting analysis. The effect of the complement proteins on IAV replication kinetics and immune modulation was assessed by qPCR. The IAV entry inhibitory properties of C1q and its recombinant globular heads were confirmed using cell binding and luciferase reporter assays. C1q bound IAV virions via HA, NA and M1 IAV proteins, and suppressed replication in H1N1, while promoting replication in H3N2-infected A549 cells. C1q treatment further triggered an anti-inflammatory response in H1N1 and pro-inflammatory response in H3N2-infected cells as evident from differential expression of TNF-α, NF-κB, IFN-α, IFN-β, IL-6, IL-12 and RANTES. Furthermore, C1q treatment was found to reduce luciferase reporter activity of MDCK cells transfected with H1N1 pseudotyped lentiviral particles, indicative of an entry inhibitory role of C1q against infectivity of IAV. These data appear to demonstrate the complement-independent subtype specific modulation of IAV infection by locally produced C1q.

Trigeminal nerve injury causes a distinct time window of glial activation in the trigeminal spinal subnucleus caudalis (Vc), which are involved in the initiation and maintenance phases of orofacial neuropathic pain. Microglia-derived factors enable the activation of astrocytes. The complement component C1q, which promotes the activation of astrocytes, is known to be synthesized in microglia. However, it is unclear whether microglia-astrocyte communication via C1q is involved in orofacial neuropathic pain. Here, we analyzed microglia-astrocyte communication in a rat model with infraorbital nerve injury (IONI). The orofacial mechanical hypersensitivity induced by IONI was significantly attenuated by preemptive treatment with minocycline. Immunohistochemical analyses revealed that minocycline inhibited the increase in c-Fos immune-reactive (IR) cells and the fluorescence intensity of both Iba1 and glial fibrillary acidic protein (GFAP) in the Vc following IONI. Intracisternal administration of C1q caused orofacial mechanical hypersensitivity and an increase in the number of c-Fos-IR cells and fluorescence intensity of GFAP. C1q-induced orofacial mechanical hypersensitivity was completely abrogated by intracisternal administration of fluorocitrate. The present findings suggest that the enhancement in the excitability of Vc nociceptive neurons is produced by astrocytic activation via the signaling of C1q released from activated microglia in the Vc following IONI, resulting in persistent orofacial neuropathic pain.

Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-β (TGF-β)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors.

Antibodies capable of activating the complement system (CS) when bound with antigen are referred to as "complement-fixing antibodies" and are involved in protection against Flaviviruses. A complement-fixing antibody test has been used in the past to measure the ability of dengue virus (DENV)-specific serum antibodies to activate the CS. As originally developed, the test is time-consuming, cumbersome, and has limited sensitivity for DENV diagnosis. Here, we developed and characterized a novel multiplex anti-DENV complement-fixing assay based on the Luminex platform to quantitate serum antibodies against all four serotypes (DENV1-4) that activate the CS based on their ability to fix the complement component 1q (C1q). The assay demonstrated good reproducibility and showed equivalent performance to a DENV microneutralization assay that has been used to determine DENV serostatus. In non-human primates, antibodies produced in response to primary DENV1-4 infection induced C1q fixation on homologous and heterologous serotypes. Inter-serotype cross-reactivity was associated with homology of the envelope protein. Interestingly, the antibodies produced following vaccination against Zika virus fixed C1q on DENV. The anti-DENV complement fixing antibody assay represents an alternative approach to determine the quality of functional antibodies produced following DENV natural infection or vaccination and a biomarker for dengue serostatus, while providing insights about immunological cross-reactivity among different Flaviviruses.

Traumatic brain injury (TBI) is of particular concern for the aging community since there is both increased incidence of TBI and decreased functional recovery in this population. In addition, TBI is the strongest environmental risk factor for development of Alzheimer's disease and other dementia-related neurodegenerative disorders. Critical changes that affect cognition take place over time following the initial insult. Our previous work identified immune system activation as a key contributor to cognitive deficits observed in aged animals. Using a focal contusion model in the current study, we demonstrate a brain lesion and cavitation formation, as well as prolonged blood⁻brain barrier breakdown. These changes were associated with a prolonged inflammatory response, characterized by increased microglial cell number and phagocytic activity 30 days post injury, corresponding to significant memory deficits. We next aimed to identify the injury-induced cellular and molecular changes that lead to chronic cognitive deficits in aged animals, and measured increases in complement initiation components C1q, C3, and CR3, which are known to regulate microglial⁻synapse interactions. Specifically, we found significant accumulation of C1q on synapses within the hippocampus, which was paralleled by synapse loss 30 days post injury. We used genetic and pharmacological approaches to determine the mechanistic role of complement initiation on cognitive loss in aging animals after TBI. Notably, both genetic and pharmacological blockade of the complement pathway prevented memory deficits in aged injured animals. Thus, therapeutically targeting early components of the complement cascade represents a significant avenue for possible clinical intervention following TBI in the aging population.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

An effective treatment and possible cure for type 1 diabetes is transplantation of pancreatic islets. Unfortunately, transplanted islets are rejected by the immune system with humoral-mediated responses being an important part of rejection. Sertoli cells (SC), an immune regulatory cell shown to survive as allografts long-term without immunosuppressants, have the potential to be used as a cell-based gene therapy vehicle to deliver endogenous insulin-a possible alternative to islets. Previously, we transduced a mouse SC line to produce human insulin. After transplantation into diabetic mice, these cells consistently produced low levels of insulin with graft survival of 75% at 50 days post-transplantation. The object of this study was to assess humoral immune regulation by these engineered SC. Both nontransduced and transduced SC survived exposure to human serum with complement in vitro. Analysis of allografts in vivo at 20 and 50 days post-transplantation revealed that despite IgG antibody detection, complement factor deposition was low and grafts survived through 50 days post-transplantation. Furthermore, the transduced SC secreted elevated levels of the complement inhibitor C1q binding protein. Overall, this suggests SC genetically engineered to express insulin maintain their ability to prevent complement-mediated killing. Since inhibiting complement-mediated rejection is important for graft survival, further studies of how SC modifies the immune response could be utilized to advance the use of genetically engineered SC or to prolong islet allograft survival to improve the treatment of diabetes.

Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.

Intrauterine infection is one of the most frequent causes of miscarriage. CpG oligodeoxynucleotide (CpG ODN) can mimic intrauterine infection. CpG ODN-induced embryo-resorption was observed consistently in the NK-cell deficient non-obese diabetic (NOD) mice but not in the wild-type (WT) mice. To elucidate the molecular mechanisms of differential pregnancy outcomes, differentially expressed genes (DEGs) in the placenta and decidua basalis was revealed by RNA-Seq with CpG ODN or control ODN treatment. Common DEGs in the WT and NOD mice were enriched in antimicrobial/antibacterial humoral responses that may be activated as a primary response to bacterial infection. The susceptibility to CpG ODN-induced embryo-resorption in the NOD mice might mainly be attributed to M1 macrophage polarization and the immunodeficient status, such as the down-regulation in antigen processing and presentation, allograft rejection, and natural killer cell mediated cytotoxicity. In contrast, the WT mice with normal immune systems could activate multiple immune responses and be resistant to CpG ODN-induced embryo-resorption, such as M2 macrophage differentiation and activation regulated by complement component C1q and peroxisome proliferation-activated receptor (PPAR) signaling pathways. Collectively, this study suggests that the immunodeficient status of NOD mice and the macrophage polarization regulated by C1q and PPAR signaling might be the basis for differential pregnancy outcomes between the NOD and WT mice.

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

3-Nitropropionic acid (NPA) administration to rodents produces degeneration of the striatum, accompanied by neurological disturbances that mimic Huntington's disease (HD) motor neurological dysfunctions. It has been shown that inflammation mediates NPA-induced brain degeneration, and activated microglia secreting cytokines interleukin-1α (IL-1α) and tumor necrosis factor α (TNFα) can induce a specific type of reactive neurotoxic astrocytes, named A1, which have been detected in post-mortem brain samples of Huntington's, Alzheimer's, and Parkinson's diseases. In this work we used an experimental model based on the intraperitoneal (i.p.) administration of NPA to adult Wistar rats at doses that can elicit extensive brain degeneration, and brain samples were taken before and after extensive brain damage monitored using 2,3,5-triphenyltetrazolium chloride (TTC) staining. Western blots and immunohistochemistry of brain slices show that i.p. NPA injections elicit significant increase in the expression levels of C3α subunit, a marker of generation of neurotoxic A1 astrocytes, and of cytokines IL-1α, TNFα, and C1q within the striatum, hippocampus, and cerebellum before the appearance of the HD-related neurological dysfunctions and neuronal death induced by NPA. Noteworthy, NPA administration primarily induces the generation of A1 astrocytes in the more recent phylogenetic area of the rat cerebellum. We conclude that the activation of complement C3 protein in the brain from Wistar rats is an early event in NPA-induced brain neurodegeneration.

Glaucoma is a leading cause of irreversible blindness worldwide, and increased intraocular pressure (IOP) is a major risk factor. We aimed to determine if early functional and molecular differences in the glaucomatous retina manifest before significant retinal ganglion cell (RGC) loss is apparent. Adenoviral vectors expressing a pathogenic form of myocilin (Ad5.MYOC) were used to induce IOP elevation in C57BL/6 mice. IOP and pattern electroretinograms (pERG) were recorded, and retinas were prepared for RNA sequencing, immunohistochemistry, or to determine RGC loss. Ocular injection of Ad5.MYOC leads to reliable IOP elevation, resulting in significant loss of RGC after nine weeks. A significant decrease in the pERG amplitude was evident in eyes three weeks after IOP elevation. Retinal gene expression analysis revealed increased expression for 291 genes related to complement cascade, inflammation, and antigen presentation in hypertensive eyes. Decreased expression was found for 378 genes associated with the γ-aminobutyric acid (GABA)ergic and glutamatergic systems and axon guidance. These data suggest that early functional changes in RGC might be due to reduced GABAA receptor signaling and neuroinflammation that precedes RGC loss in this glaucoma model. These initial changes may offer new targets for early detection of glaucoma and the development of new interventions.