Complement Component C5a and Fungal Pathogen Induce Diverse Responses through Crosstalk between Transient Receptor Potential Channel (TRPs) Subtypes in Human Conjunctival Epithelial Cells.

The conjunctiva has immune-responsive properties to protect the eye from infections. Its innate immune system reacts against external pathogens, such as fungi. The complement factor C5a is an important contributor to the initial immune response. It is known that activation of transient-receptor-potential-vanilloid 1 (TRPV1) and TRP-melastatin 8 (TRPM8) channels is involved in different immune reactions and inflammation in the human body. The aim of this study was to determine if C5a and mucor racemosus e voluminae cellulae (MR) modulate Ca2+-signaling through changes in TRPs activity in human conjunctival epithelial cells (HCjECs). Furthermore, crosstalk was examined between C5a and MR in mediating calcium regulation. Intracellular Ca2+-concentration ([Ca2+]i) was measured by fluorescence calcium imaging, and whole-cell currents were recorded using the planar-patch-clamp technique. MR was used as a purified extract. Application of C5a (0.05-50 ng/mL) increased both [Ca2+]i and whole-cell currents, which were suppressed by either the TRPV1-blocker AMG 9810 or the TRPM8-blocker AMTB (both 20 µM). The N-terminal peptide C5L2p (20-50 ng/mL) blocked rises in [Ca2+]i induced by C5a. Moreover, the MR-induced rise in Ca2+-influx was suppressed by AMG 9810 and AMTB, as well as 0.05 ng/mL C5a. In conclusion, crosstalk between C5a and MR controls human conjunctival cell function through modulating interactions between TRPV1 and TRPM8 channel activity.

Different Complement Activation Patterns Following C5 Cleavage in MOGAD and AQP4-IgG+NMOSD.

OBJECTIVES: In myelin oligodendrocyte glycoprotein IgG-associated disease (MOGAD) and aquaporin-4 IgG+ neuromyelitis optica spectrum disorder (AQP4+NMOSD), the autoantibodies are mainly composed of IgG1, and complement-dependent cytotoxicity is a primary pathomechanism in AQP4+NMOSD. We aimed to evaluate the CSF complement activation in MOGAD. METHODS: CSF-C3a, CSF-C4a, CSF-C5a, and CSF-C5b-9 levels during the acute phase before treatment in patients with MOGAD (n = 12), AQP4+NMOSD (n = 11), multiple sclerosis (MS) (n = 5), and noninflammatory neurologic disease (n = 2) were measured. RESULTS: CSF-C3a and CSF-C5a levels were significantly higher in MOGAD (mean ± SD, 5,629 ± 1,079 pg/mL and 2,930 ± 435.8 pg/mL) and AQP4+NMOSD (6,017 ± 3,937 pg/mL and 2,544 ± 1,231 pg/mL) than in MS (1,507 ± 1,286 pg/mL and 193.8 ± 0.53 pg/mL). CSF-C3a, CSF-C4a, and CSF-C5a did not differ between MOGAD and AQP4+NMOSD while CSF-C5b-9 (membrane attack complex, MAC) levels were significantly lower in MOGAD (17.4 ± 27.9 ng/mL) than in AQP4+NMOSD (62.5 ± 45.1 ng/mL, p = 0.0019). Patients with MOGAD with severer attacks (Expanded Disability Status Scale [EDSS] ≥ 3.5) had higher C5b-9 levels (34.0 ± 38.4 ng/m) than those with milder attacks (EDSS ≤3.0, 0.9 ± 0.7 ng/mL, p = 0.044). DISCUSSION: The complement pathway is activated in both MOGAD and AQP4+NMOSD, but MAC formation is lower in MOGAD, particularly in those with mild attacks, than in AQP4+NMOSD. These findings may have pathogenetic and therapeutic implications in MOGAD.

Chemotaxis Assay of Bone Marrow-Derived Macrophages.

Immune responses rely on efficient and coordinated migration of immune cells to the site of infection or injury. To reach the site of immunological threat often requires long-range navigation of immune cells through complex tissue and vascular networks. Chemotaxis, cell migration steered by gradients of cell-attractive chemicals that bind sensory receptors, is central to this response. Chemoattractant receptors mostly belong to the G-protein-coupled receptor (GPCR) family, but the way attractant-receptor signaling directs cell migration is not fully understood. Direct-viewing chemotaxis chambers combined with time-lapse microscopy give a powerful tool to study the dynamic details of cells' responses to different attractant landscapes. Here, we describe the application of one such chamber (the Dunn chamber) to study bone marrow-derived macrophage chemotaxis to gradients of complement C5a.

Structure of Designer Antibody-like Peptides Binding to the Human C5a with Potential to Modulate the C5a Receptor Signaling.

C5a is an integral glycoprotein of the complement system that plays an important role in inflammation and immunity. The physiological concentration of C5a is observed to be elevated under various immunoinflammatory pathophysiological conditions in humans. The pathophysiology of C5a is linked to the "two-site" protein-protein interactions (PPIs) with two genomically related receptors, such as C5aR1 and C5aR2. Therefore, pharmacophores that can potentially block the PPIs between C5a-C5aR1 and C5a-C5aR2 have tremendous potential for development as future therapeutics. Notably, the FDA has already approved antibodies that target the precursors of C5a (Eculizumab, 148 kDa) and C5a (Vilobelimab, 149 kDa) for marketing as complement-targeted therapeutics. In this context, the current study reports the structural characterization of a pair of synthetic designer antibody-like peptides (DePA and DePA1; ≤3.8 kDa) that bind to hotspot regions on C5a and also demonstrates potential traits to neutralize the function of C5a under pathophysiological conditions.

C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages.

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 -/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 -/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 -/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 -/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 -/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 -/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 -/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 -/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

Dysregulated complement activation during acute myocardial infarction leads to endothelial glycocalyx degradation and endothelial dysfunction via the C5a:C5a-Receptor1 axis.

Introduction: Complement-mediated damage to the myocardium during acute myocardial infarction (AMI), particularly the late components of the terminal pathway (C5-convertase and C5b-9), have previously been characterized. Unfortunately, only few studies have reported a direct association between dysregulated complement activation and endothelial function. Hence, little attention has been paid to the role of the anaphylatoxin C5a. The endothelial glycocalyx (eGC) together with the cellular actin cortex provide a vasoprotective barrier against chronic vascular inflammation. Changes in their nanomechanical properties (stiffness and height) are recognized as hallmarks of endothelial dysfunction as they correlate with the bioavailability of vasoactive substances, such as nitric oxide (NO). Here, we determined how the C5a:C5aR1 axis affects the eGC and endothelial function in AMI. Methods: Samples of fifty-five patients with ST-elevation myocardial infarction (STEMI) vs. healthy controls were analyzed in this study. eGC components and C5a levels were determined via ELISA; NO levels were quantified chemiluminescence-based. Endothelial cells were stimulated with C5a or patient sera (with/without C5a-receptor1 antagonist "PMX53") and the nanomechanical properties of eGC quantified using the atomic force microscopy (AFM)-based nanoindentation technique. To measure actin cytoskeletal tension regulator activation (RhoA and Rac1) G-LISA assays were applied. Vascular inflammation was examined by quantifying monocyte-endothelium interaction via AFM-based single-cell-force spectroscopy. Results: Serum concentrations of eGC components and C5a were significantly increased during STEMI. Serum and solely C5a stimulation decreased eGC height and stiffness, indicating shedding of the eGC. C5a enhanced RhoA activation, resulting in increased cortical stiffness with subsequent reduction in NO concentrations. Monocyte adhesion to the endothelium was enhanced after both C5a and stimulation with STEMI serum. eGC degradation- and RhoA-induced cortical stiffening with subsequent endothelial dysfunction were attenuated after administering PMX53. Conclusion: This study demonstrates that dysregulated C5a activation during AMI results in eGC damage with subsequent endothelial dysfunction and reduced NO bioavailability, indicating progressively developing vascular inflammation. This could be prevented by antagonizing C5aR1, highlighting the role of the C5a:C5a-Receptor1 axis in vascular inflammation development and endothelial dysfunction in AMI, offering new therapeutic approaches for future investigations.

Emerging role of C5aR2: novel insights into the regulation of uterine immune cells during pregnancy.

Pregnancy is a fascinating immunological phenomenon because it allows allogeneic fetal and placental tissues to survive inside the mother. As a component of innate immunity with high inflammatory potential, the complement system must be tightly regulated during pregnancy. Dysregulation of the complement system plays a role in pregnancy complications including pre-eclampsia and intrauterine growth restriction. Complement components are also used as biomarkers for pregnancy complications. However, the mechanisms of detrimental role of complement in pregnancy is poorly understood. C5a is the most potent anaphylatoxin and generates multiple immune reactions via two transmembrane receptors, C5aR1 and C5aR2. C5aR1 is pro-inflammatory, but the role of C5aR2 remains largely elusive. Interestingly, murine NK cells have been shown to express C5aR2 without the usual co-expression of C5aR1. Furthermore, C5aR2 appears to regulate IFN-γ production by NK cells in vitro. As IFN-γ produced by uterine NK cells is one of the major factors for the successful development of a vital pregnancy, we investigated the role anaphylatoxin C5a and its receptors in the establishment of pregnancy and the regulation of uterine NK cells by examinations of murine C5ar2-/- pregnancies and human placental samples. C5ar2-/- mice have significantly reduced numbers of implantation sites and a maternal C5aR2 deficiency results in increased IL-12, IL-18 and IFN-γ mRNA expression as well as reduced uNK cell infiltration at the maternal-fetal interface. Human decidual leukocytes have similar C5a receptor expression patterns showing clinical relevance. In conclusion, this study identifies C5aR2 as a key contributor to dNK infiltration and pregnancy success.

An herbal formula Shenlian decoction upregulates M1/M2 macrophage proportion in hepatocellular carcinoma by suppressing complement cascade.

The immunosuppressive microenvironment is a vital factor for the hepatocellular carcinoma (HCC) progression. However, effective treatment is lacking at current. Shenlian decoction (SLD) is a registered herbal therapy for the HCC treatment, but the underlying mechanism of SLD remains largely elusive. Here, we aimed to explore the anti-tumor effect of SLD in the treatment of HCC. SLD was intragastrically given after the tumor initiation in ß-catenin/C-Met or DEN and CCl4 induced HCC mouse model. The tumor growth levels were evaluated by liver weight and histological staining. The tumor-infiltrating immune cells were detected by immunological staining and flow cytometry. The mechanism of the SLD was detected by non-targeted proteomics and verified by a cell co-culture system. The result showed that SLD significantly attenuated HCC progression. SLD promoted macrophage infiltration and increased the M1/M2 macrophage ratio within the tumor tissues. Non-targeted proteomics showed the inhibition of complement C5/C5a signaling is the key mechanism of SLD. Immunological staining showed SLD inhibited C5/C5a expression and C5aR1+ macrophage infiltration. The suggested mechanism was demonstrated by application of C5aR1 inhibitor, PMX-53 in mouse HCC model. Hepatoma cell-macrophage co-culture showed SLD targeted hepatoma cells and inhibited the supernatant-induced macrophage M2 polarization. SLD inhibited AMPK/p38 signaling which is an upstream mechanism of C5 transcription. In conclusion, we found SLD relieved immune-suppressive environment by inhibiting C5 expression. SLD could suppress the C5 secretion in hepatoma cells via inhibition of AMPK/p38 signaling. We suggested that SLD is a potential herbal therapy for the treatment of HCC by alleviating immune-suppressive status.

Enhancing prognostic prediction of invasive candidiasis among cancer patients with a serum C5a-based scoring model.

PURPOSE: Invasive candidiasis poses a life-threatening risk, and early prognosis assessment is vital for timely interventions to reduce mortality. Serum C5a levels have recently been linked to prognosis, but confirmation in cancer patients is pending. METHODS: We detected the concentrations of serum C5a in hospitalized cancer patients with invasive candidiasis from 2020 to 2023, and retrospectively analyzed the clinical data. RESULTS: 372 cases were included in this study, with a 90-day mortality rate of 21.8%. Candida albicans (48.7%) remained the predominant pathogen, followed by Candida glabrata (25.5%), Candida tropicalis (12.4%), and Candida parapsilosis (8.3%). Gastrointestinal cancer was the most diagnosed pathology type (37.6%). Serum C5a demonstrated a noteworthy correlation with 90-day mortality, and employing a cutoff value of 36.7 ng/ml revealed significantly higher 90-day mortality in low-C5a patients (41.2%) compared to high-C5a patients (6.3%) (p < 0.001). We also identified no source control, no surgery, metastasis, or chronic renal failure independently correlated with the 90-day mortality. Based on this, a prognostic model combining C5a and clinical parameters was constructed, which performed better than models built solely on C5a or clinical parameters. Furthermore, we weighted scores to each parameter in the model and presented diagnostic sensitivity and specificity corresponding to different score points calculated by the model. CONCLUSION: We constructed a prognostic scoring model including serum C5a and clinical parameters, which would contribute to precise prognosis assessment and benefit the outcome among cancer patients.

Complement system is overactivated in patients with IgA nephropathy after COVID-19.

IgA nephropathy (IgAN), which has been confirmed as a complement mediated autoimmune disease, is also one form of glomerulonephritis associated with COVID-19. Here, we aim to investigate the clinical and immunological characteristics of patients with IgAN after COVID-19. The level of plasma level of C5a (p < 0.001), soluble C5b-9 (p = 0.018), FHR5 (p < 0.001) were all significantly higher in Group CoV (33 patients with renal biopsy-proven IgAN experienced COVID-19) compared with Group non-CoV (44 patients with IgAN without COVID-19), respectively. Compared with Group non-CoV, the intensity of glomerular C4d (p = 0.017) and MAC deposition (p < 0.001) and Gd-IgA1 deposition (p = 0.005) were much stronger in Group CoV. Our finding revealed that for IgAN after COVID-19, mucosal immune responses to SARS-CoV-2 infection may result in the overactivation of systemic and renal local complement system, and increased glomerular deposition of Gd-IgA1, which may lead to renal dysfunction and promote renal progression in IgAN patients.

Clinical values of serum C5a in Alzheimer's disease patients with different dementia stages.

Alzheimer's disease (AD) is characterized by abnormal inflammatory responses, and complement C5a (C5a) is known to initiate inflammation. This study aimed to investigate the associations between serum C5a, inflammatory responses, and cognitive function in AD patients. A total of 242 AD patients and 132 age-matched controls were included. Enzyme-linked immunosorbent assay revealed increased levels of C5a, interleukin (IL)-4, IL-6, IL-10, IL-1ß, and tumor necrosis factor (TNF)-α with advancing stages of AD. Pearson correlation coefficient and receiver operating characteristic curve revealed positive correlations between serum C5a levels, inflammatory cytokine levels, Neuropsychiatric Inventory (NPI) and Activities of Daily Living (ADL) scores, and negative correlations with Mini-mental State Examination (MMSE) and Montreal cognitive assessment (MoCA) scores. Serum C5a above 68.68 pg/mL could aid in the diagnosis of AD. Multivariable logistic analysis revealed that serum C5a was an independent risk factor for IL-1ß/IL-6/IL-10/TNF-α and an independent protective factor for IL-4. Higher serum C5a levels were associated with lower MMSE and MoCA scores. In conclusion, elevated serum C5a levels were beneficial for AD diagnosis and predictive of inflammation and cognitive dysfunction.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Molecular recognition and interaction between human plasminogen Kringle 5 and A2M domain in human complement C5 by biospecific methods coupled with molecular dynamics simulation.

The potent angiogenesis inhibitor known as human plasminogen Kringle 5 has shown promise in the treatment of vascular disorders and malignancies. The study aimed to investigate the recognition and interaction between Kringle 5 and the A2M domain of human complement component C5 using bio-specific methodologies and molecular dynamics (MD) simulation. Initially, the specific interaction between Kringle 5 and A2M was confirmed and characterized through Ligand Blot and ELISA, yielding the dissociation constant (Kd) of 1.70 × 10-7 mol/L. Then, Kringle 5 showcased a dose-dependent inhibition of the production of C5a in lung cancer A549 cells, consequently impeding their proliferation and migration. Following the utilization of frontal affinity chromatography (FAC), it was revealed that there exists a singular binding site with the binding constant (Ka) of 3.79 × 105 L/mol. Following the implementation of homology modeling and MD optimization, the detailed results indicate that only a specific segment of the N-terminal structure of the A2M molecule engages in interaction with Kringle 5 throughout the binding process and the principal driving forces encompass electrostatic force, hydrogen bonding, and van der Waals force. In conclusion, the A2M domain of human complement C5 emerges as a plausible binding target for Kringle 5 in vivo.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes.

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3-4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Validation of dot blot immunoassay for measurement of complement opsonization of nanoparticles.

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field.

Icariin ameliorates LPS-induced acute lung injury in mice via complement C5a-C5aR1 and TLR4 signaling pathways.

Acute lung injury (ALI) is an acute respiratory-related progressive disorder, which lacks specific pharmacotherapy. Icariin (ICA) has been shown to be effective in treating ALI. However, the targets and pharmacological mechanisms underlying the effects of ICA in the treatment of ALI are relatively lacking. Based on network pharmacology and molecular docking analyses, the gene functions and potential target pathways of ICA in the treatment of ALI were determined. In addition, the underlying mechanisms of ICA were verified by immunohistochemistry, immunofluorescence, quantitative Real-time PCR, and Western blot in LPS-induced ALI mice. The biological processes targeted by ICA in the treatment of ALI included the pathological changes, inflammatory response, and cell signal transduction. Network pharmacology, molecular docking, and in vivo experimental results revealed that ICA inhibited the complement C5a-C5aR1 axis, TLR4 mediated NF-κB, MAPK, and JAK2-STAT3 signaling pathways related gene and protein expressions, and decreased inflammatory cytokine, chemokine, adhesion molecule expressions, and mitochondrial apoptosis in LPS-induced ALI.

Discovery and Characterization of a New Class of C5aR1 Antagonists Showing In Vivo Activity.

C5a is an anaphylatoxin protein produced by the cleavage of the complement system's component C5 protein. It signals through the G-protein-coupled receptor C5a receptor 1 (C5aR1) to induce the chemotaxis of primarily neutrophils and monocytes and the release of inflammatory molecules. A large body of evidence linking C5aR1 signaling to acute and chronic inflammatory disorders has triggered interest in developing potent C5aR antagonists. Herein we report the discovery of new C5aR1 antagonistic chemical classes. Many representatives showed low nanomolar IC50 values in a C5aR1 ß-arrestin-2 recruitment assay, inhibiting the migration of human neutrophils toward C5a and the internalization of the receptor in human whole blood. Two leading compounds were characterized further in vivo. Target engagement of the receptor by these two C5aR1 antagonists was demonstrated in vivo. In particular, the inhibition of migration in vitro with the two compounds further translated in a dose-dependent efficacy in a rat model of C5a-induced neutrophilia.

Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers.

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. STATEMENT OF SIGNIFICANCE: Chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy. We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.