Complement activation in wasp venom-induced acute kidney injury.

Previous studies have highlighted the significant role of complement activation in kidney injuries induced by rhabdomyolysis, intravascular hemolysis, sepsis, and ischemia-reperfusion. Nevertheless, the specific role and mechanism of complement activation in acute kidney injury (AKI) caused by wasp venom remain unclear. The aim of this study was to elucidate the specific complement pathway activated and investigate complement activation in AKI induced by wasp venom. In this study, a complement-depleted mouse model was used to investigate the role of complement in wasp venom-induced AKI. Mice were randomly categorized into control, cobra venom factor (CVF), AKI, and CVF + AKI groups. Compared to the AKI group, the CVF + AKI group showed improved pathological changes in kidneys and reduced blood urea nitrogen (BUN) levels. The expression levels of renal complement 3 (C3), complement 5 (C5), complement 1q (C1q), factor B (FB), mannose-binding lectin (MBL), and C5b-9 in AKI group were upregulated compared with the control group. Conversely, the renal tissue expression levels of C3, C5, C1q, FB, MBL, and C5b-9 were decreased in the CVF + AKI group compared to those in the AKI group. Complement activation occurs through all three pathways in AKI induced by wasp venom. Furthermore, complement depletion by CVF attenuates wasp venom-induced nephrotoxicity, suggesting that complement activation plays a primary role in the pathogenesis of wasp venom-induced AKI.

Factor H-related protein 1 promotes complement-mediated opsonization of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for a wide range of infections. The complement system is the main early host defense mechanism to control these infections. P. aeruginosa counteracts complement attack by binding Factor H (FH), a complement regulator that inactivates C3b, preventing the formation of the C3-convertase and complement amplification on the bacterial surface. Factor H-related proteins (FHRs) are a group of plasma proteins evolutionarily related to FH that have been postulated to interfere in this bacterial mechanism of resisting complement. Here, we show that FHR-1 binds to P. aeruginosa via the outer membrane protein OprG in a lipopolysaccharide (LPS) O antigen-dependent manner. Binding assays with purified components or with FHR-1-deficient serum supplemented with FHR-1 show that FHR-1 competes with FH for binding to P. aeruginosa. Blockage of FH binding to C3b deposited on the bacteria reduces FH-mediated cofactor activity of C3b degradation, increasing the opsonization of the bacteria and the formation of the potent chemoattractant C5a. Overall, our findings indicate that FHR-1 is a host factor that promotes complement activation, facilitating clearance of P. aeruginosa by opsonophagocytosis.

Complement dysregulation is a prevalent and therapeutically amenable feature of long COVID.

BACKGROUND: Long COVID encompasses a heterogeneous set of ongoing symptoms that affect many individuals after recovery from infection with SARS-CoV-2. The underlying biological mechanisms nonetheless remain obscure, precluding accurate diagnosis and effective intervention. Complement dysregulation is a hallmark of acute COVID-19 but has not been investigated as a potential determinant of long COVID. METHODS: We quantified a series of complement proteins, including markers of activation and regulation, in plasma samples from healthy convalescent individuals with a confirmed history of infection with SARS-CoV-2 and age/ethnicity/sex/infection/vaccine-matched patients with long COVID. FINDINGS: Markers of classical (C1s-C1INH complex), alternative (Ba, iC3b), and terminal pathway (C5a, TCC) activation were significantly elevated in patients with long COVID. These markers in combination had a receiver operating characteristic predictive power of 0.794. Other complement proteins and regulators were also quantitatively different between healthy convalescent individuals and patients with long COVID. Generalized linear modeling further revealed that a clinically tractable combination of just four of these markers, namely the activation fragments iC3b, TCC, Ba, and C5a, had a predictive power of 0.785. CONCLUSIONS: These findings suggest that complement biomarkers could facilitate the diagnosis of long COVID and further suggest that currently available inhibitors of complement activation could be used to treat long COVID. FUNDING: This work was funded by the National Institute for Health Research (COV-LT2-0041), the PolyBio Research Foundation, and the UK Dementia Research Institute.

Plasmid-encoded toxin of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro.

Plasmid-encoded toxin (Pet) is an autotransporter protein of the serine protease autotransporters of Enterobacteriaceae (SPATE) family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the enteroaggregative E. coli (EAEC) virulence plasmid, pAA2. Although this virulence factor was initially described in EAEC, an intestinal E. coli pathotype, pet may also be present in other pathotypes, including extraintestinal pathogenic strains (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of E. coli to human serum.

The complement factor H-related protein-5 (CFHR5) exacerbates pathological bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease, characterized by excessive new bone formation. We previously reported that the complement factor H-related protein-5 (CFHR5), a member of the human factor H protein family, is significantly elevated in patients with AS compared to other rheumatic diseases. However, the pathophysiological mechanism underlying new bone formation by CFHR5 is not fully understood. In this study, we revealed that CFHR5 and proinflammatory cytokines (TNF, IL-6, IL-17A, and IL-23) were elevated in the AS group compared to the HC group. Correlation analysis revealed that CFHR5 levels were not significantly associated with proinflammatory cytokines, while CFHR5 levels in AS were only positively correlated with the high CRP group. Notably, treatment with soluble CFHR5 has no effect on clinical arthritis scores and thickness at hind paw in curdlan-injected SKG, but significantly increased the ectopic bone formation at the calcaneus and tibia bones of the ankle as revealed by micro-CT image and quantification. Basal CFHR5 expression was upregulated in AS-osteoprogenitors compared to control cells. Also, treatment with CFHR5 remarkedly induced bone mineralization status of AS-osteoprogenitors during osteogenic differentiation accompanied by MMP13 expression. We provide the first evidence demonstrating that CFHR5 can exacerbate the pathological bone formation of AS. Therapeutic modulation of CFHR5 could be promising for future treatment of AS. KEY MESSAGES: Serum level of CFHR5 is elevated and positively correlated with high CRP group of AS patients. Recombinant CFHR5 protein contributes to pathological bone formation in in vivo model of AS. CFHR5 is highly expressed in AS-osteoprogenitors compared to disease control. Recombinant CFHR5 protein increased bone mineralization accompanied by MMP13 in vitro model of AS.

Complement C7 and clusterin form a complex in circulation.

Introduction: The complement system is part of innate immunity and is comprised of an intricate network of proteins that are vital for host defense and host homeostasis. A distinct mechanism by which complement defends against invading pathogens is through the membrane attack complex (MAC), a lytic structure that forms on target surfaces. The MAC is made up of several complement components, and one indispensable component of the MAC is C7. The role of C7 in MAC assembly is well documented, however, inherent characteristics of C7 are yet to be investigated. Methods: To shed light on the molecular characteristics of C7, we examined the properties of serum-purified C7 acquired using polyclonal and novel monoclonal antibodies. The properties of serum­purified C7 were investigated through a series of proteolytic analyses, encompassing Western blot and mass spectrometry. The nature of C7 protein-protein interactions were further examined by a novel enzyme-linked immunosorbent assay (ELISA), as well as size­exclusion chromatography. Results: Protein analyses showcased an association between C7 and clusterin, an inhibitory complement regulator. The distinct association between C7 and clusterin was also demonstrated in serum-purified clusterin. Further assessment revealed that a complex between C7 and clusterin (C7-CLU) was detected. The C7-CLU complex was also identified in healthy serum and plasma donors, highlighting the presence of the complex in circulation. Discussion: Clusterin is known to dissociate the MAC structure by binding to polymerized C9, nevertheless, here we show clusterin binding to the native form of a terminal complement protein in vivo. The presented data reveal that C7 exhibits characteristics beyond that of MAC assembly, instigating further investigation of the effector role that the C7-CLU complex plays in the complement cascade.

Complement Cascade Proteins Correlate with Fibrosis and Inflammation in Early-Stage Type 1 Diabetic Kidney Disease in the Ins2Akita Mouse Model.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses' results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = -0.58 to -0.842 and range for IGHM rs = -0.6 to -0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.

Anti-aquaporin-4 immune complex stimulates complement-dependent Th17 cytokine release in neuromyelitis optica spectrum disorders.

Proinflammatory cytokines, such as (IL: interleukin) IL-6 and IL-17A, and complement fixation are critical in the immunopathogenesis of neuromyelitis optica spectrum disorders (NMOSD). Blocking the IL-6 receptor or the C5 complement pathway reduces relapse risk. However, the role of interleukin (IL)-6 and complement in aquaporin-4 (AQP4) autoimmunity remains unclear. To investigate the role of the anti-AQP4 immunoglobulin (AQP4-IgG)/AQP4 immunocomplex on the induction and profile of ex vivo cytokine and surface marker expression in peripheral blood mononuclear cells (PBMC) culture. Isolated PBMCs obtained from 18 patients with AQP4-IgG-seropositive-NMOSD (8 treatment-naive, 10 rituximab-treated) or ten healthy controls were cultured with AQP4-immunocomplex with or without complement. Changes in PBMC surface markers and cytokine expression were profiled using flow cytometry and ELISA. PBMCs derived from treatment-naive NMOSD patients stimulated with a complex mixture of serum complement proteins produced significant elevations of IL-17A and IL-6. Rituximab-treated patients also exhibited higher IL-6 but not IL-17A release. IL-6 and IL-17A elevations are not observed without complement. Co-stimulation of PBMCs with AQP4-IgG/AQP4 immunocomplex and complement prompts a Th17-biased response consistent with the inflammatory paradigm observed in NMOSD. A possible inflammation model is proposed via antigen-specific autoreactive peripheral blood cells, including NK/NKT cells.

Borreliella burgdorferi factor H-binding proteins are not required for serum resistance and infection in mammals.

The causative agent of Lyme disease (LD), Borreliella burgdorferi, binds factor H (FH) and other complement regulatory proteins to its surface. B. burgdorferi B31 (type strain) encodes five FH-binding proteins (FHBPs): CspZ, CspA, and the OspE paralogs OspEBBN38, OspEBBL39, and OspEBBP38. This study assessed potential correlations between the production of individual FHBPs, FH-binding ability, and serum resistance using a panel of infectious B. burgdorferi clonal populations recovered from dogs. FHBP production was assessed in cultivated spirochetes and by antibody responses in naturally infected humans, dogs, and eastern coyotes (wild canids). FH binding specificity and sensitivity to dog and human serum were also assessed and compared. No correlation was observed between the production of individual FHBPs and FH binding with serum resistance, and CspA was determined to not be produced in animals. Notably, one or more clones isolated from dogs lacked CspZ or the OspE proteins (a finding confirmed by genome sequence determination) and did not bind FH derived from canines. The data presented do not support a correlation between FH binding and the production of individual FHBPs with serum resistance and infectivity. In addition, the limited number and polymorphic nature of cp32s in B. burgdorferi clone DRI85A that were identified through genome sequencing suggest no strict requirement for a defined set of these replicons for infectivity. This study reveals that the immune evasion mechanisms employed by B. burgdorferi are diverse, complex, and yet to be fully defined.

Clearance of ß-amyloid and synapses by the optogenetic depolarization of microglia is complement selective.

Microglia actively monitor the neighboring brain microenvironments and constantly contact synapses with their unique ramified processes. In neurodegenerative diseases, including Alzheimer's disease (AD), microglia undergo morphological and functional alterations. Whether the direct manipulation of microglia can selectively or concurrently modulate synaptic function and the response to disease-associated factors remains elusive. Here, we employ optogenetic methods to stimulate microglia in vitro and in vivo. Membrane depolarization rapidly changes microglia morphology and leads to enhanced phagocytosis. We found that the optogenetic stimulation of microglia can efficiently promote ß-amyloid (Aß) clearance in the brain parenchyma, but it can also enhance synapse elimination. Importantly, the inhibition of C1q selectively prevents synapse loss induced by microglia depolarization but does not affect Aß clearance. Our data reveal independent microglia-mediated phagocytosis pathways toward Aß and synapses. Our results also shed light on a synergistic strategy of depolarizing microglia and inhibiting complement functions for the clearance of Aß while sparing synapses.

Persistent complement dysregulation with signs of thromboinflammation in active Long Covid.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

Characterization of the bispecific VHH antibody gefurulimab (ALXN1720) targeting complement component 5, and designed for low volume subcutaneous administration.

The bispecific antibody gefurulimab (also known as ALXN1720) was developed to provide patients with a subcutaneous treatment option for chronic disorders involving activation of the terminal complement pathway. Gefurulimab blocks the enzymatic cleavage of complement component 5 (C5) into the biologically active C5a and C5b fragments, which triggers activation of the terminal complement cascade. Heavy-chain variable region antigen-binding fragment (VHH) antibodies targeting C5 and human serum albumin (HSA) were isolated from llama immune-based libraries and humanized. Gefurulimab comprises an N-terminal albumin-binding VHH connected to a C-terminal C5-binding VHH via a flexible linker. The purified bispecific VHH antibody has the expected exact size by mass spectrometry and can be formulated at greater than 100 mg/mL. Gefurulimab binds tightly to human C5 and HSA with dissociation rate constants at pH 7.4 of 54 pM and 0.9 nM, respectively, and cross-reacts with C5 and serum albumin from cynomolgus monkeys. Gefurulimab can associate with C5 and albumin simultaneously, and potently inhibits the terminal complement activity from human serum initiated by any of the three complement pathways in Wieslab assays. Electron microscopy and X-ray crystallography revealed that the isolated C5-binding VHH recognizes the macroglobulin (MG) 4 and MG5 domains of the antigen and thereby is suggested to sterically prevent C5 binding to its activating convertase. Gefurulimab also inhibits complement activity supported by the rare C5 allelic variant featuring an R885H substitution in the MG7 domain. Taken together, these data suggest that gefurulimab may be a promising candidate for the potential treatment of complement-mediated disorders.

Genetic determinants of complement activation in the general population.

Complement is a fundamental innate immune response component. Its alterations are associated with severe systemic diseases. To illuminate the complement's genetic underpinnings, we conduct genome-wide association studies of the functional activity of the classical (CP), lectin (LP), and alternative (AP) complement pathways in the Cooperative Health Research in South Tyrol study (n = 4,990). We identify seven loci, encompassing 13 independent, pathway-specific variants located in or near complement genes (CFHR4, C7, C2, MBL2) and non-complement genes (PDE3A, TNXB, ABO), explaining up to 74% of complement pathways' genetic heritability and implicating long-range haplotypes associated with LP at MBL2. Two-sample Mendelian randomization analyses, supported by transcriptome- and proteome-wide colocalization, confirm known causal pathways, establish within-complement feedback loops, and implicate causality of ABO on LP and of CFHR2 and C7 on AP. LP causally influences collectin-11 and KAAG1 levels and the risk of mouth ulcers. These results build a comprehensive resource to investigate the role of complement in human health.

Artificial surface labelling of Escherichia coli with StrepTagII antigen to study how monoclonal antibodies drive complement-mediated killing.

Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen-antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM's enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

Complement System and the Kidney: Its Role in Renal Diseases, Kidney Transplantation and Renal Cell Carcinoma.

The crosstalk among the complement system, immune cells, and mediators of inflammation provides an efficient mechanism to protect the organism against infections and support the repair of damaged tissues. Alterations in this complex machinery play a role in the pathogenesis of different diseases. Core complement proteins C3 and C5, their activation fragments, their receptors, and their regulators have been shown to be active intracellularly as the complosome. The kidney is particularly vulnerable to complement-induced damage, and emerging findings have revealed the role of complement system dysregulation in a wide range of kidney disorders, including glomerulopathies and ischemia-reperfusion injury during kidney transplantation. Different studies have shown that activation of the complement system is an important component of tumorigenesis and its elements have been proved to be present in the TME of various human malignancies. The role of the complement system in renal cell carcinoma (RCC) has been recently explored. Clear cell and papillary RCC upregulate most of the complement genes relative to normal kidney tissue. The aim of this narrative review is to provide novel insights into the role of complement in kidney disorders.

Activation of the complement system by nanoparticles and strategies for complement inhibition.

The complement system is a multicomponent and multifunctional arm of the innate immune system. Complement contributes to non-specific host defence and maintains homeostasis through multifaceted processes and pathways, including crosstalk with the adaptive immune system, the contact (coagulation) and the kinin systems, and alarmin high-mobility group box 1. Complement is also present intracellularly, orchestrating a wide range of housekeeping and physiological processes in both immune and nonimmune cells, thus showing its more sophisticated roles beyond innate immunity, but its roles are still controversial. Particulate drug carriers and nanopharmaceuticals typically present architectures and surface patterns that trigger complement system in different ways, resulting in both beneficial and adverse responses depending on the extent of complement activation and regulation as well as pathophysiological circumstances. Here we consider the role of complement system and complement regulations in host defence and evaluate the mechanisms by which nanoparticles trigger and modulate complement responses. Effective strategies for the prevention of nanoparticle-mediated complement activation are introduced and discussed.

Diverse Functions of C4b-Binding Protein in Health and Disease.

C4b-binding protein (C4BP) is a fluid-phase complement inhibitor that prevents uncontrolled activation of the classical and lectin complement pathways. As a complement inhibitor, C4BP also promotes apoptotic cell death and is hijacked by microbes and tumors for complement evasion. Although initially characterized for its role in complement inhibition, there is an emerging recognition that C4BP functions in a complement-independent manner to promote cell survival, protect against autoimmune damage, and modulate the virulence of microbial pathogens. In this Brief Review, we summarize the structure and functions of human C4BP, with a special focus on activities that extend beyond the canonical role of C4BP in complement inhibition.

Modeling complement activation on human glomerular microvascular endothelial cells.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare kidney disease caused by dysregulation of the complement alternative pathway. The complement dysregulation specifically leads to damage to the glomerular endothelium. To further understand aHUS pathophysiology, we validated an ex vivo model for measuring complement deposition on both control and patient human glomerular microvascular endothelial cells (GMVECs). Methods: Endothelial cells were incubated with human test sera and stained with an anti-C5b-9 antibody to visualize and quantify complement depositions on the cells with immunofluorescence microscopy. Results: First, we showed that zymosan-activated sera resulted in increased endothelial C5b-9 depositions compared to normal human serum (NHS). The levels of C5b-9 depositions were similar between conditionally immortalized (ci)GMVECs and primary control GMVECs. The protocol with ciGMVECs was further validated and we additionally generated ciGMVECs from an aHUS patient. The increased C5b-9 deposition on control ciGMVECs by zymosan-activated serum could be dose-dependently inhibited by adding the C5 inhibitor eculizumab. Next, sera from five aHUS patients were tested on control ciGMVECs. Sera from acute disease phases of all patients showed increased endothelial C5b-9 deposition levels compared to NHS. The remission samples showed normalized C5b-9 depositions, whether remission was reached with or without complement blockage by eculizumab. We also monitored the glomerular endothelial complement deposition of an aHUS patient with a hybrid complement factor H (CFH)/CFH-related 1 gene during follow-up. This patient had already chronic kidney failure and an ongoing deterioration of kidney function despite absence of markers indicating an aHUS flare. Increased C5b-9 depositions on ciGMVECs were observed in all samples obtained throughout different diseases phases, except for the samples with eculizumab levels above target. We then tested the samples on the patient's own ciGMVECs. The C5b-9 deposition pattern was comparable and these aHUS patient ciGMVECs also responded similar to NHS as control ciGMVECs. Discussion: In conclusion, we demonstrate a robust and reliable model to adequately measure C5b-9-based complement deposition on human control and patient ciGMVECs. This model can be used to study the pathophysiological mechanisms of aHUS or other diseases associated with endothelial complement activation ex vivo.

Complement Receptor C3aR1 Contributes to Paclitaxel-Induced Peripheral Neuropathic Pain in Mice and Rats.

Cancer chemotherapy-induced neuropathic pain is a devastating pain syndrome without effective therapies. We previously reported that rats deficient in complement C3, the central component of complement activation cascade, showed a reduced degree of paclitaxel-induced mechanical allodynia (PIMA), suggesting that complement is integrally involved in the pathogenesis of this model. However, the underlying mechanism was unclear. Complement activation leads to the production of C3a, which mediates inflammation through its receptor C3aR1. In this article, we report that the administration of paclitaxel induced a significantly higher expression level of C3aR1 on dorsal root ganglion (DRG) macrophages and expansion of these macrophages in DRGs in wild-type (WT) compared with in C3aR1 knockout (KO) mice. We also found that paclitaxel induced less severe PIMA, along with a reduced DRG expression of transient receptor potential channels of the vanilloid subtype 4 (TRPV4), an essential mediator for PIMA, in C3aR1 KO than in WT mice. Treating WT mice or rats with a C3aR1 antagonist markedly attenuated PIMA in association with downregulated DRG TRPV4 expression, reduced DRG macrophages expansion, suppressed DRG neuron hyperexcitability, and alleviated peripheral intraepidermal nerve fiber loss. Administration of C3aR1 antagonist to TRPV4 KO mice further protected them from PIMA. These results suggest that complement regulates PIMA development through C3aR1 to upregulate TRPV4 on DRG neurons and promote DRG macrophage expansion. Targeting C3aR1 could be a novel therapeutic approach to alleviate this debilitating pain syndrome.

Proteomic analysis of lysine 2-hydroxyisobutyryl in SLE reveals protein modification alteration in complement and coagulation cascades and platelet activation Pathways.

BACKGROUND: Post-translational modifications (PTMs) are considered to be an important factor in the pathogenesis of Systemic lupus erythematosus (SLE). Lysine 2-hydroxyisobutyryl (Khib), as an emerging post-translational modification of proteins, is involved in some important biological metabolic activities. However, there are poor studies on its correlation with diseases, especially SLE. OBJECTIVE: We performed quantitative, comparative, and bioinformatic analysis of Khib proteins in Peripheral blood mononuclear cells (PBMCs) of SLE patients and PBMCs of healthy controls. Searching for pathways related to SLE disease progression and exploring the role of Khib in SLE. METHODS: Khib levels in SLE patients and healthy controls were compared based on liquid chromatography tandem mass spectrometry, then proteomic analysis was conducted. RESULTS: Compared with healthy controls, Khib in SLE patients was up-regulated at 865 sites of 416 proteins and down-regulated at 630 sites of 349 proteins. The site abundance, distribution and function of Khib protein were investigated further. Bioinformatics analysis showed that Complement and coagulation cascades and Platelet activation in immune-related pathways were significantly enriched, suggesting that differentially modified proteins among them may affect SLE. CONCLUSION: Khib in PBMCs of SLE patients was significantly up- or down-regulated compared with healthy controls. Khib modification of key proteins in the Complement and coagulation cascades and Platelet activation pathways affects platelet activation and aggregation, coagulation functions in SLE patients. This result provides a new direction for the possible significance of Khib in the pathogenesis of SLE patients.