C4d Staining Is Present in Normal Placentas From Pregnancies Prior to Pregnancy Loss Associated With Chronic Histiocytic Intervillositis and Is Reduced by Immunomodulatory Therapy in Subsequent Pregnancies.

INTRODUCTION: Chronic histiocytic intervillositis (CHI) is associated with adverse pregnancy outcomes and high recurrence risk. Recent studies suggest that CHI may represent a host-vs-graft rejection, and that C4d immunostain can be used as a marker for complement activation and antibody-mediated rejection in the CHI. MATERIALS AND METHODS: This retrospective cohort study focused on 5 fetal autopsy cases associated with CHI (5 index cases) from 5 women. We analyzed placentas from the index cases (fetal autopsy cases associated with CHI) and placentas from the women's previous and subsequent pregnancies. We assessed the presence and extent of CHI and C4d immunostaining in these placentas. We evaluated each available placenta and graded the severity of CHI as either <50% or ≥50%. Additionally, we conducted C4d immunostaining on one representative section from each placenta and graded the staining levels as follows: 0+ for staining <5%; 1+ for staining between 5% and <25%; 2+ for staining between 25% and <75%; and 3+ for staining ≥75%. RESULTS: Three of the 5 women had pregnancies prior to their index cases (fetal autopsy cases associated with CHI). Despite the absence of CHI in their initial pregnancies, the placentas displayed positive C4d staining with grades of 1+, 3+, and 3+, respectively. These results suggest the presence of complement activation and antibody-mediated rejection in placentas from their prior pregnancies without CHI. Three of the 5 women received immunomodulatory therapy after experiencing pregnancy losses associated with CHI. After treatment, 2 of these women had live births at 35 and 37 gestational weeks, respectively, while the third had a stillbirth at 25 gestational weeks. The severity of CHI and the degree of C4d staining in the placentas decreased in all 3 cases following immunomodulatory therapies. Specifically, the level of C4d staining decreased from 3+ to 2+, 2+ to 0+, and 3+ to 1+ in these 3 cases, respectively. DISCUSSION: In women with a history of recurrent pregnancy loss associated with CHI, C4d immunostaining was present in the placentas from their previous non-CHI pregnancies, suggesting activation of the classical complement pathway and antibody-mediated reaction in their prior non-CHI pregnancies before the development of CHI in subsequent pregnancies. Immunomodulatory therapy may improve pregnancy outcomes by reducing complement activation, as shown by the reduction of C4d immunopositivity in the placentas after immunomodulatory treatment. Although we believe that the study provides valuable insights, we acknowledge that there are limitations to the findings. Therefore, to further elucidate the pathogenesis of CHI, additional research efforts with a collaborative and multidisciplinary approach are necessary.

Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P.

Lack of simple and robust methods to determine complement activation in human serum induced by antigen-antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen-antibody complexes.

Contribution of classical complement activation and IgM to the control of Rickettsia infection.

Pathogenic Rickettsia are obligate intracellular bacteria and the etiologic agents of many life-threatening infectious diseases. Due to the serious nature of these infections, it is imperative to both identify the responsive immune sensory pathways and understand the associated immune mechanisms that restrict Rickettsia proliferation. Previous studies have demonstrated that the mammalian complement system is both activated during Rickettsia infection and contributes to the immune response to infection. To further define this component of the mammalian anti-Rickettsia immune response, we sought to identify the mechanism(s) of complement activation during Rickettsia infection. We have employed a series of in vitro and in vivo models of infection to investigate the role of the classical complement activation pathway during Rickettsia infection. Depletion or elimination of complement activity demonstrates that both C1q and pre-existing IgM contribute to complement activation; thus implicating the classical complement system in Rickettsia-mediated complement activation. Elimination of the classical complement pathway from mice increases susceptibility to R. australis infection with both increased bacterial loads in multiple tissues and decreased immune activation markers. This study highlights the role of the classical complement pathway in immunity against Rickettsia and implicates resident Rickettsia-responsive IgM in the response to infection.

The immune system of jawless vertebrates: insights into the prototype of the adaptive immune system.

Jawless vertebrates diverged from an ancestor of jawed vertebrates approximately 550 million years ago. They mount adaptive immune responses to repetitive antigenic challenges, despite lacking major histocompatibility complex molecules, immunoglobulins, T cell receptors, and recombination-activating genes. Instead of B cell and T cell receptors, agnathan lymphocytes express unique antigen receptors named variable lymphocyte receptors (VLRs), which generate diversity through a gene conversion-like mechanism. Although gnathostome antigen receptors and VLRs are structurally unrelated, jawed and jawless vertebrates share essential features of lymphocyte-based adaptive immunity, including the expression of a single type of receptor on each lymphocyte, clonal expansion of antigen-stimulated lymphocytes, and the dichotomy of cellular and humoral immunity, indicating that the backbone of the adaptive immune system was established in a common ancestor of all vertebrates. Furthermore, recent evidence indicates that, unlike previously thought, agnathans have a unique classical pathway of complement activation where VLRB molecules act as antibodies instead of immunoglobulins. It seems likely that the last common ancestor of all vertebrates had an adaptive immune system resembling that of jawless vertebrates, suggesting that, as opposed to jawed vertebrates, agnathans have retained the prototype of vertebrate adaptive immunity.

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces.

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.

Status epilepticus triggers long-lasting activation of complement C1q-C3 signaling in the hippocampus that correlates with seizure frequency in experimental epilepsy.

Status epilepticus (SE) triggers a myriad of neurological alterations that include unprovoked seizures, temporal lobe epilepsy (TLE), and cognitive deficits. Although SE-induced loss of hippocampal dendritic structures and synaptic remodeling are often associated with this pathophysiology, the underlying mechanisms remain elusive. Recent evidence points to the classical complement pathway as a potential mechanism. Signaling through the complement protein C1q to C3, which is cleaved into smaller biologically active fragments including C3b and iC3b, contributes to the elimination of synaptic structures in the normal developing brain and in models of neurodegenerative disorders. We recently found increased protein levels of C1q and iC3b fragments in human drug-resistant epilepsy. Thus, to identify a potential role for C1q-C3 in SE-induced epilepsy, we performed a temporal analysis of C1q protein levels and C3 cleavage in the hippocampus along with their association to seizures and hippocampal-dependent cognitive functions in a rat model of SE and acquired TLE. We found significant increases in the levels of C1q, C3, and iC3b in the hippocampus at 2-, 3- and 5-weeks after SE relative to controls (p<0.05). In the SE group, greater iC3b levels were significantly correlated with higher seizure frequency (p<0.05). Together, these data support that hyperactivation of the classical complement pathway after SE parallels the progression of epilepsy. Future studies will determine whether C1q-C3 signaling contributes to epileptogenic synaptic remodeling in the hippocampus.

Classical complement pathway activation in the nasal tissue of patients with chronic rhinosinusitis.

BACKGROUND: Complement plays a major role in inflammatory diseases, but its involvement and mechanisms of activation in patients with chronic rhinosinusitis (CRS) are not known. OBJECTIVES: After earlier studies discovering autoantibodies in patients with CRS, we sought to investigate the nature, extent, and location of complement activation in nasal tissue of patients with CRS. Specifically, we were interested in whether antibody-mediated activation through the classical pathway was a major mechanism for complement activation in patients with CRS. METHODS: Nasal tissue was obtained from patients with CRS and control subjects. Tissue homogenates were analyzed for complement activation products (ELISA-C5b-9, C4d, activated C1, and C5a) and major complement-fixing antibodies (Luminex). Tissue sections were stained for C5b-9, C4d, and laminin. Antibodies were purified with protein A/G columns from nasal polyps (NP), matching patient serum, and control serum and assayed for basement membrane binding by means of ELISA. RESULTS: C5b-9 levels were significantly increased in NP tissue compared with uncinate tissue (UT) of patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and those with chronic rhinosinusitis without nasal polyps (CRSsNP; P < .01). Similarly, C4d levels were increased in NPs compared with UT of patients with CRSwNP, patients with CRSsNP, and control subjects (P < .05). Activated C1 levels were also increased in NP tissue compared with UT of patients with CRSsNP and control subjects (P < .05) and correlated with levels of C5a (P < .01), local immunoglobulins (especially IgM, P < .0001), and anti-double-stranded DNA IgG (P < .05). Immunofluorescence showed that C5b-9 and C4d deposition occurred linearly along the epithelial basement membrane. NP tissue extracts had significantly more anti-basement membrane antibodies than sera from patients with CRSwNP and control subjects (P < .0001). CONCLUSION: Levels of C5b-9, C4d, and activated C1 were significantly increased locally in NP tissue. C5b-9 and C4d were almost universally deposited linearly along the basement membrane of NP tissue. Furthermore, activated C1 levels were best correlated with local immunoglobulin and C5a levels. Together, these data suggest that the classical pathway plays a major role in complement activation in patients with CRS.

An amphioxus gC1q protein binds human IgG and initiates the classical pathway: Implications for a C1q-mediated complement system in the basal chordate.

The origin of the classical complement pathway remains open during chordate evolution. A C1q-like member, BjC1q, was identified in the basal chordate amphioxus. It is predominantly expressed in the hepatic caecum, hindgut, and notochord, and is significantly upregulated following challenge with bacteria or lipoteichoic acid and LPS. Recombinant BjC1q and its globular head domain specifically interact with lipoteichoic acid and LPS, but BjC1q displays little lectin activity. Moreover, rBjC1q can assemble to form the high molecular weight oligomers necessary for binding to proteases C1r/C1s and for complement activation, and binds human C1r/C1s/mannan-binding lectin-associated serine protease-2 as well as amphioxus serine proteases involved in the cleavage of C4/C2, and C3 activation. Importantly, rBjC1q binds with human IgG as well as an amphioxus Ig domain containing protein, resulting in the activation of the classical complement pathway. This is the first report showing that a C1q-like protein in invertebrates is able to initiate classical pathway, raising the possibility that amphioxus possesses a C1q-mediated complement system. It also suggests a new scenario for the emergence of the classical complement pathway, in contrast to the proposal that the lectin pathway evolved into the classical pathway.

Collagen-binding microbial surface components recognizing adhesive matrix molecule (MSCRAMM) of Gram-positive bacteria inhibit complement activation via the classical pathway.

Members of a family of collagen-binding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) from Gram-positive bacteria are established virulence factors in several infectious diseases models. Here, we report that these adhesins also can bind C1q and act as inhibitors of the classical complement pathway. Molecular analyses of Cna from Staphylococcus aureus suggested that this prototype MSCRAMM bound to the collagenous domain of C1q and interfered with the interactions of C1r with C1q. As a result, C1r2C1s2 was displaced from C1q, and the C1 complex was deactivated. This novel function of the Cna-like MSCRAMMs represents a potential immune evasion strategy that could be used by numerous Gram-positive pathogens.

Long-term efficacy and safety of continued complement C1s inhibition with sutimlimab in cold agglutinin disease: CADENZA study Part B.

Background: Cold agglutinin disease (CAD) is a rare autoimmune haemolytic anaemia mediated by the classical complement pathway (CP). Sutimlimab selectively targets complement C1s inhibiting classical CP activation. In CADENZA Part A (26-weeks), a placebo-controlled study in patients without recent transfusion history, sutimlimab reduced haemolysis, anaemia, and fatigue, and was generally well tolerated. Methods: The CADENZA study (NCT03347422) started in March 2018 (Part A) and completed in December 2021 (Part B). All patients in Part B were eligible to receive sutimlimab for up to 1 year after the last patient completed Part A. Efficacy and safety was assessed throughout Part B, until the last on-treatment visit with available assessment (LV), and after a 9-week washout. Findings: In total, 32/39 patients completed Part B; median treatment duration: 99 weeks. Similar sustained improvements in haemolysis, anaemia, and quality of life were observed in patients switching to sutimlimab and those continuing sutimlimab. Mean LV values for the combined group (ie, placebo-to-sutimlimab group and sutimlimab-to-sutimlimab group) improved from baseline for haemoglobin (≥11.0 g/dL on-treatment vs 9.3 g/dL at baseline), bilirubin (≤20.0 µmol/L on-treatment vs 35.0 µmol/L at baseline), and FACIT-Fatigue scores. Following a 9-week washout, inhibition of CP activity was reversed, and haemolytic markers approached baseline levels. Overall, sutimlimab was generally well tolerated throughout the study. No patients developed systemic lupus erythematosus or meningococcal infections. During the 9-week washout, most adverse events could be attributed to recurrence of underlying CAD. Interpretation: The CADENZA Part B results support the sustained efficacy and safety of sutimlimab for treatment of CAD; however, upon discontinuation disease activity reoccurs. Funding: Sanofi.

Walnut-Derived Peptides Alleviate Learning and Memory Impairments in a Mice Model via Inhibition of Microglia Phagocytose Synapses.

Microglia phagocytose synapses have an important effect on the pathogenesis of neurological disorders. Here, we investigated the neuroprotective effects of the walnut-derived peptide, TWLPLPR(TW-7), against LPS-induced cognitive deficits in mice and explored the underlying C1q-mediated microglia phagocytose synapses mechanisms in LPS-treated HT22 cells. The MWM showed that TW-7 improved the learning and memory capacity of the LPS-injured mice. Both transmission electron microscopy and immunofluorescence analysis illustrated that synaptic density and morphology were increased while associated with the decreased colocalized synapses with C1q. Immunohistochemistry and immunofluorescence demonstrated that TW-7 effectively reduced the microglia phagocytosis of synapses. Subsequently, overexpression of C1q gene plasmid was used to verify the contribution of the TW-7 via the classical complement pathway-regulated mitochondrial function-mediated microglia phagocytose synapses in LPS-treated HT22 cells. These data suggested that TW-7 improved the learning and memory capability of LPS-induced cognitively impaired mice through a mechanism associated with the classical complement pathway-mediated microglia phagocytose synapse.

Pharmacokinetics, pharmacodynamics, safety, and tolerability of a single-dose riliprubart, an anti-C1s humanized monoclonal antibody in East-Asian adults: results from a Phase 1, randomized, open-label trial.

OBJECTIVES: This Phase 1 trial was planned to investigate the pharmacokinetics (PK), pharmacodynamics (PD), safety, and tolerability of a single dose of riliprubart in healthy East-Asian adult participants. METHODS: A single-center, parallel-group, randomized, open-label, single-dose study was performed to evaluate the PK, PD, safety, and tolerability of riliprubart (50 mg/kg intravenous [IV] or 600 mg subcutaneous [SC]) in 37 healthy East-Asian (Chinese, Japanese, and Korean) participants. RESULTS: Riliprubart was slowly absorbed after SC administration (median tmax: 7.01-10.48 days) and showed a long half-life after IV or SC administration (mean: 9.52-11.0 weeks), with a bioavailability of 74.6% after SC administration. The PD profiles, which are evaluated by classical complement pathway activity or CH50, were similar and largely overlapped across East-Asian participants after a single IV or SC dose. Riliprubart was safe and well tolerated in participants following a single IV or SC dose. CONCLUSIONS: Riliprubart was safe and well tolerated and demonstrated favorable PK and PD profiles in healthy East-Asian participants following a single IV or SC dose. These results are comparable to first-in-human study results from non-East-Asian participants and support the same dosing regimen of riliprubart for global simultaneous clinical development. CLINICAL TRIAL REGISTRATION: This trial is registered at https://cris.nih.go.kr (identifier: KCT0006571).

Anti-C1q antibodies: a biomarker for diagnosis and management of lupus nephritis. A narrative review.

Nephritis is a frequent and severe complication of Systemic Lupus Erythematous (SLE). The clinical course of lupus nephritis (LN) is usually characterized by alternating phases of remission and exacerbation. Flares of LN can lead to deterioration of kidney function, necessitating timely diagnosis and therapy. The presence of autoantibodies against C1q (anti-C1qAb) in the sera of SLE patients has been reported in various studies. Some research suggests that the presence and changes in the titer of anti-C1qAb may be associated with the development of LN, as well as with LN activity and renal flares. However, the exact role of anti-C1qAb in LN remains a subject of debate. Despite variability in the results of published studies, anti-C1qAb hold promise as noninvasive markers for assessing LN activity in SLE patients. Measuring anti-C1qAb levels could aid in diagnosing and managing LN during periods of both inactive disease and renal flares. Nevertheless, larger controlled trials with standardized laboratory assays are necessary to further establish the utility of anti-C1qAb in predicting the reactivation and remission of LN and guiding treatment strategies.

Investigation of Potential Drug Targets Involved in Inflammation Contributing to Alzheimer's Disease Progression.

Alzheimer's disease has become a major public health issue. While extensive research has been conducted in the last few decades, few drugs have been approved by the FDA to treat Alzheimer's disease. There is still an urgent need for understanding the disease pathogenesis, as well as identifying new drug targets for further drug discovery. Alzheimer's disease is known to arise from a build-up of amyloid beta (Aß) plaques as well as tangles of tau proteins. Along similar lines to Alzheimer's disease, inflammation in the brain is known to stem from the degeneration of tissue and build-up of insoluble materials. A minireview was conducted in this work assessing the genes, proteins, reactions, and pathways that link brain inflammation and Alzheimer's disease. Existing tools in Systems Biology were implemented to build protein interaction networks, mainly for the classical complement pathway and G protein-coupled receptors (GPCRs), to rank the protein targets according to their interactions. The top 10 protein targets were mainly from the classical complement pathway. With the consideration of existing clinical trials and crystal structures, proteins C5AR1 and GARBG1 were identified as the best targets for further drug discovery, through computational approaches like ligand-protein docking techniques.

Safety and Target Engagement of Complement C1q Inhibitor ANX007 in Neurodegenerative Eye Disease: Results from Phase I Studies in Glaucoma.

Purpose: Complement C1q, the initiating molecule of the classical complement cascade, is involved in synapse elimination and neuronal loss in neurodegenerative diseases including glaucoma. Here we report an evaluation of the safety, tolerability, and ocular pharmacokinetics (PK) and pharmacodynamics of intravitreal (IVT) injections of ANX007, an anti-C1q monoclonal antibody fragment that blocks activation of the classical complement cascade. Design: An open-label, single-dose-escalation phase Ia study followed by a double-masked, randomized, sham-controlled, repeat-injection phase Ib study. Participants: A total of 26 patients with primary open-angle glaucoma. Methods: Nine patients with primary open-angle glaucoma (mean Humphrey visual field deviation between -3 and -18 decibels [dB]) were enrolled in phase Ia and received single doses of ANX007 (1.0 mg, n = 3; 2.5 mg, n = 3; or 5.0 mg, n = 3). Seventeen patients (mean Humphrey visual field deviation between -3 and -24 dB) were enrolled in phase Ib and randomized to 2 monthly doses of ANX007 (sham, n = 6; 2.5 mg ANX007, n = 6; or 5 mg ANX007, n = 5). Main Outcome Measures: Safety and tolerability (including laboratory evaluation of urinalysis, complete blood count, and serum chemistries), ANX007 PK, target engagement, and immunogenicity. Results: The mean age overall was 70 years in phase Ia and 68 years in phase Ib. In both studies, no serious adverse events were observed, no non-ocular treatment-emergent adverse events (TEAEs) attributable to study drug were reported, and ocular TEAEs were mild. Intraocular pressure returned to normal levels for all patients within 45 minutes of IVT injection. No clinically significant deviations in laboratory results were observed. In the phase Ib study, C1q in the aqueous humor was reduced to undetectable levels in both the 2.5 mg and 5 mg cohorts 4 weeks after the first ANX007 dose. Conclusions: In these studies, single and repeat IVT ANX007 injections were well tolerated and demonstrated full target engagement 4 weeks after dosing with both low and high doses, supporting monthly or less-frequent dosing. Further investigation in neurodegenerative ocular diseases is warranted. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Long term follow-up of complement parameters to improve the management of acquired angioedema due to C1-inhibitor deficiency.

Acquired angioedema due to C1-inhibitor deficiency (C1-INH-AAE) is a rare disease that can be diagnosed via complement testing. It often accompanies lymphoproliferative underlying diseases. Our study aimed to examine if there is a connection between complement parameters and the clinical symptoms of C1-INH-AAE, and, in case of a known underlying disease, its activity. The other question is how a connection, if proven, could help in the development of the therapeutic strategy of C1-INH-AAE patients. In the past 30 years, out of the 3938 patients sent to the Angioedema Center with angioedema symptoms, we have diagnosed C1-INH-AAE in 19 cases. An underlying disease was diagnosed in 15 patients. Most often lymphoma (6/19 patients) and monoclonal gammopathy of undetermined significance (6/19 patients) were found. Angioedema specific long-term prophylaxis did not result in an improvement in neither the frequency of the attacks nor in the complement parameters. A connection has been found between the presence and activity of any underlying disease, the frequency of the angioedema attacks and the decreased level of proteins of the complement system. Decreasing complement parameters warn about the appearance or the worsening of the underlying disease. The treatment of the underlying disease brings improvement in the complement parameters. Rituximab treatment reduced the number of attacks or completely made them disappear, and we experienced positive changes in complement parameters. Complement parameters supported the long-term efficacy of rituximab treatment for C1-INH-AAE. The change in complement parameters predict the relapse of the underlying disease, and it is a good indicator for the prediction of angioedematous attacks. In C1-INH-AAE, it is essential to examine the patients for underlying diseases, and to regularly follow up the patient's complement parameters.

Inflammasome-Dependent Peroxiredoxin 2 Secretion Induces the Classical Complement Pathway Activation.

Peroxiredoxins (Prxs) are ubiquitously expressed peroxidases that reduce hydrogen peroxide or alkyl peroxide production in cells. Prxs are released from cells in response to various stress conditions, and they function as damage-associated molecular pattern molecules. However, the secretory mechanism of Prxs and their roles have not been elucidated. Thus, we aimed to determine whether inflammasome activation is a secretory mechanism of Prxs and subsequently identify the effect of the secreted Prxs on activation of the classical complement pathway. Using J774A.1, a murine macrophage cell line, we demonstrated that NLRP3 inflammasome activation induces Prx1, Prx2, Prx5, and Prx6 secretion in a caspase-1 dependent manner. Using HEK293T cells with a transfection system, we revealed that the release of Prx1 and Prx2 relies on gasdermin-D (GSDMD)-mediated secretion. Next, we confirmed the binding of both Prx1 and Prx2 to C1q; however, only Prx2 could induce the C1q-mediated classical complement pathway activation. Collectively, our results suggest that inflammasome activation is a secretory mechanism of Prxs and that GSDMD is a mediator of their secretion. Moreover, secreted Prx1 and Prx2 bind with C1q, but only Prx2 mediates the classical complement pathway activation.

C1qa deficiency in mice increases susceptibility to mouse hepatitis virus A59 infection.

BACKGROUND: Mouse hepatitis virus (MHV) A59 is a highly infectious pathogen and starts in the respiratory tract and progresses to systemic infection in laboratory mice. The complement system is an important part of the host immune response to viral infection. It is not clear the role of the classical complement pathway in MHV infection. OBJECTIVES: The purpose of this study was to determine the importance of the classical pathway in coronavirus pathogenesis by comparing C1qa KO mice and wild-type mice. METHODS: We generated a C1qa KO mouse using CRISPR/Cas9 technology and compared the susceptibility to MHV A59 infection between C1qa KO and wild-type mice. Histopathological and immunohistochemical changes, viral loads, and chemokine expressions in both mice were measured. RESULTS: MHV A59-infected C1qa KO mice showed severe histopathological changes, such as hepatocellular necrosis and interstitial pneumonia, compared to MHV A59-infected wild-type mice. Virus copy numbers in the olfactory bulb, liver, and lungs of C1qa KO mice were significantly higher than those of wild-type mice. The increase in viral copy numbers in C1qa KO mice was consistent with the histopathologic changes in organs. These results indicate that C1qa deficiency enhances susceptibility to MHV A59 systemic infection in mice. In addition, this enhanced susceptibility effect is associated with dramatic elevations in spleen IFN-γ, MIP-1 α, and MCP-1 in C1qa KO mice. CONCLUSIONS: These data suggest that C1qa deficiency enhances susceptibility to MHV A59 systemic infection, and activation of the classical complement pathway may be important for protecting the host against MHV A59 infection.

Beyond Binding: The Outcomes of Antibody-Dependent Complement Activation in Human Malaria.

Antibody immunity against malaria is effective but non-sterile. In addition to antibody-mediated inhibition, neutralisation or opsonisation of malaria parasites, antibody-mediated complement activation is also important in defense against infection. Antibodies form immune complexes with parasite-derived antigens that can activate the classical complement pathway. The complement system provides efficient surveillance for infection, and its activation leads to parasite lysis or parasite opsonisation for phagocytosis. The induction of complement-fixing antibodies contributes significantly to the development of protective immunity against clinical malaria. These complement-fixing antibodies can form immune complexes that are recognised by complement receptors on innate cells of the immune system. The efficient clearance of immune complexes is accompanied by complement receptor internalisation, abrogating the detrimental consequences of excess complement activation. Here, we review the mechanisms of activation of complement by alternative, classical, and lectin pathways in human malaria at different stages of the Plasmodium life cycle with special emphasis on how complement-fixing antibodies contribute to protective immunity. We briefly touch upon the action of anaphylatoxins, the assembly of membrane attack complex, and the possible reasons underlying the resistance of infected erythrocytes towards antibody-mediated complement lysis, relevant to their prolonged survival in the blood of the human host. We make suggestions for further research on effector functions of antibody-mediated complement activation that would guide future researchers in deploying complement-fixing antibodies in preventive or therapeutic strategies against malaria.

Different Aspects of Classical Pathway Overactivation in Patients With C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis.

The rare and heterogeneous kidney disorder C3 glomerulopathy (C3G) is characterized by dysregulation of the alternative pathway (AP) of the complement system. C3G is often associated with autoantibodies stabilizing the AP C3 convertase named C3 nephritic factors (C3NeF). The role of classical pathway (CP) convertase stabilization in C3G and related diseases such as immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) remains largely unknown. Here, we investigated the CP convertase activity in patients with C3G and IC-MPGN. Using a refined two-step hemolytic assay, we measured the stability of CP convertases directly in the serum of 52 patients and 17 healthy controls. In four patients, CP convertase activity was prolonged compared to healthy controls, i.e. the enzymatic complex was stabilized. In three patients (2 C3G, 1 IC-MPGN) the convertase stabilization was caused by immunoglobulins, indicating the presence of autoantibodies named C4 nephritic factors (C4NeFs). Importantly, the assay also enabled detection of non-immunoglobulin-mediated stabilization of the CP convertase in one patient with C3G. Prolonged CP convertase activity coincided with C3NeF activity in all patients and for up to 70 months of observation. Crucially, experiments with C3-depleted serum showed that C4NeFs stabilized the CP C3 convertase (C4bC2a), that does not contain C3NeF epitopes. All patients with prolonged CP convertase activity showed clear signs of complement activation, i.e. lowered C3 and C5 levels and elevated levels of C3d, C3bc, C3bBbP, and C5b-9. In conclusion, this work provides new insights into the diverse aspects and (non-)immunoglobulin nature of factors causing CP convertase overactivity in C3G/IC-MPGN.