Inhibition Of Complement Activation Research Articles

Subversion of the immune response of human pathogenic spirochetes.

Spirochetes are a large group of prokaryotes that originated from Gram‐negative bacteria and are capable of causing a variety of human and animal infections. However, the pathogenesis of spirochetes remains unclear, as different types of spirochetes play pathogenic roles through different pathogenic substances and mechanisms. To survive and spread in the host, spirochetes have evolved complicated strategies to evade host immune responses. In this review, we aimed to provide a comprehensive overview of immune evasion strategies in spirochetes infection. These strategies can be explained from the following points: (i) Antigenic variation: random, unidirectional, and segmental conversion of the gene to evade immune surveillance; (ii) Overcoming the attack of the complement system: recruitment of host complement regulators, cleavage of complement components and inhibition of complement activation to evade immune defenses; (iii) Interfering with immune cells to regulating the immune system; (iv) Persistent infection: invading and colonizing the host cell to escape immune damage.

Host Immune Responses to Salivary Components - A Critical Facet of Tick-Host Interactions.

Tick sialome is comprised of a rich cocktail of bioactive molecules that function as a tool to disarm host immunity, assist blood-feeding, and play a vibrant role in pathogen transmission. The adaptation of the tick’s blood-feeding behavior has lead to the evolution of bioactive molecules in its saliva to assist them to overwhelm hosts’ defense mechanisms. During a blood meal, a tick secretes different salivary molecules including vasodilators, platelet aggregation inhibitors, anticoagulants, anti-inflammatory proteins, and inhibitors of complement activation; the salivary repertoire changes to meet various needs such as tick attachment, feeding, and modulation or impairment of the local dynamic and vigorous host responses. For instance, the tick’s salivary immunomodulatory and cement proteins facilitate the tick’s attachment to the host to enhance prolonged blood-feeding and to modulate the host’s innate and adaptive immune responses. Recent advances implemented in the field of “omics” have substantially assisted our understanding of host immune modulation and immune inhibition against the molecular dynamics of tick salivary molecules in a crosstalk between the tick–host interface. A deep understanding of the tick salivary molecules, their substantial roles in multifactorial immunological cascades, variations in secretion, and host immune responses against these molecules is necessary to control these parasites. In this article, we reviewed updated knowledge about the molecular mechanisms underlying host responses to diverse elements in tick saliva throughout tick invasion, as well as host defense strategies. In conclusion, understanding the mechanisms involved in the complex interactions between the tick salivary components and host responses is essential to decipher the host defense mechanisms against the tick evasion strategies at tick-host interface which is promising in the development of effective anti-tick vaccines and drug therapeutics.

Discovery of APL-1030, a Novel, High-Affinity Nanofitin Inhibitor of C3-Mediated Complement Activation.

Uncontrolled complement activation contributes to multiple immune pathologies. Although synthetic compstatin derivatives targeting C3 and C3b are robust inhibitors of complement activation, their physicochemical and molecular properties may limit access to specific organs, development of bifunctional moieties, and therapeutic applications requiring transgenic expression. Complement-targeting therapeutics containing only natural amino acids could enable multifunctional pharmacology, gene therapies, and targeted delivery for underserved diseases. A Nanofitin library of hyperthermophilic protein scaffolds was screened using ribosome display for C3/C3b-targeting clones mimicking compstatin pharmacology. APL-1030, a recombinant 64-residue Nanofitin, emerged as the lead candidate. APL-1030 is thermostable, binds C3 (KD, 1.59 nM) and C3b (KD, 1.11 nM), and inhibits complement activation via classical (IC50 = 110.8 nM) and alternative (IC50 = 291.3 nM) pathways in Wieslab assays. Pharmacologic activity (determined by alternative pathway inhibition) was limited to primate species of tested sera. C3b-binding sites of APL-1030 and compstatin were shown to overlap by X-ray crystallography of C3b-bound APL-1030. APL-1030 is a novel, high-affinity inhibitor of primate C3-mediated complement activation developed from natural amino acids on the hyperthermophilic Nanofitin platform. Its properties may support novel drug candidates, enabling bifunctional moieties, gene therapy, and tissue-targeted C3 pharmacologics for diseases with high unmet need.

Mechanism for the attenuation of neutrophil and complement hyperactivity by MSC exosomes

Complements and neutrophils are two key players of the innate immune system that are widely implicated as drivers of severe COVID-19 pathogenesis, as evident by the direct correlation of respiratory failure and mortality with elevated levels of terminal complement complex C5b-9 and neutrophils. In this study, we identified a feed-forward loop between complements and neutrophils that could amplify and perpetuate the cytokine storm seen in severe SARS-CoV-2–infected patients. We observed for the first time that the terminal complement activation complex C5b-9 directly triggered neutrophil extracellular trap (NET) release and interleukin (IL)-17 production by neutrophils. This is also the first report that the production of NETs and IL-17 induced by C5b-9 assembly on neutrophils could be abrogated by mesenchymal stem cell (MSC) exosomes. Neutralizing anti-CD59 antibodies abolished this abrogation. Based on our findings, we hypothesize that MSC exosomes could alleviate the immune dysregulation in acute respiratory failure, such as that observed in severe COVID-19 patients, by inhibiting complement activation through exosomal CD59, thereby disrupting the feed-forward loop between complements and neutrophils to inhibit the amplification and perpetuation of inflammation during SARS-CoV-2 infection.

An Excretory Protein of Echinococcus multilocularis Inhibits Complement Classical Pathway Activation.

IntroductionAlveolar echinococcosis is a lethal zoonosis caused by Echinococcus multilocularis (E.m) larvae. The mechanism by which E.m evades host immune attacks and ensures long-term survival remains unexplained. The complement system is a cascade of sequentially activated complement proteins that results in opsonization-related phagocytosis or membrane lysis of invading organisms. Excretory/secretory proteins (ESPs) of parasites are the main antigens that induce the immune response and play important roles in the long-term survival.MethodsWe investigated the possibility that E.m inhibits complement activation through ESPs and examined the potential related mechanism. A haemolysis assay was used to determine if and how in vitro culture medium of E.m containing ESPs can inhibit complement activation. Potential ESPs were annotated using bioinformatics methods, and one ESP was subsequently expressed as a recombinant protein with a eukaryotic expression system. The ability of this protein to inhibit complement activation was also tested by haemolysis assay.ResultsThese assays showed that in vitro culture medium of E.m inhibited activation of the complement classical pathway. EmuJ_000439500 encodes a protein containing seven Sushi domains, which was the only potential E.m-derived complement inhibitor (Em-CI, UniProt: A0A068Y4F2) annotated among the 653 ESPs. Recombinant Em-CI also displayed the ability to inhibit activation of the complement classical pathway.DiscussionThe discovery of Em-CI sheds light on the mechanism by which E.m escapes killing by the complement system and provides potential targets for immunotherapy for parasitic diseases.

Esculetin alleviates murine lupus nephritis by inhibiting complement activation and enhancing Nrf2 signaling pathway

Ethnopharmacological relevanceEsculetin is a bioactive compound of medicinal herb Hydrangea paniculata, and has showed anti-oxidation and anti-inflammation bioactivities. Renal local oxidative stress and inflammation are import contributors for progression of lupus nephritis (LN). Aim of the studyIn the present study, the renal protective effect of esculetin against LN was evaluated using MRL/lpr mice. Materials and methodsMRL/lpr mice were orally administrated with esculetin (20 mg/kg and 40 mg/kg) from 10 to 20 weeks and then renal function and kidney pathology were analyzed. ResultsEsculetin significantly attenuated renal impairment in MRL/lpr mice by reducing blood urea nitrogen (BUN), serum creatinine (Scr) and albuminuria, and ameliorated the glomerular hypertrophy, tubular interstitial fibrosis and mononuclear cell infiltration into interstitium. mRNA microarray suggested that esculetin could significantly down-regulate complement cascade, inflammation and fibrosis pathway, and up-regulate Nrf2-related anti-oxidation genes. Most surprising finding in the current study was that esculetin could inhibit the complement activation both in classical and alternative pathway using in vitro hemolysis assay, further enzyme assay suggested that esculetin blocked the C3 convertase (C4b2a) to exert this inhibitory capability. Molecular docking predicted that esculetin had four conventional hydrogen bonds interacting with C4b2a, and CDOCKER energy is relatively lower. Luciferase reporter gene demonstrated that esculetin could activate Nrf2 signaling pathway, and further flow cytometry confirmed that anti-oxidation bioactivity of esculetin was dependent on Nrf2 activation. On the other hand, esculetin could inhibit NFκB nuclear translocation and TGFβ-smad3 profibrosis pathway. ConclusionEsculetin shows beneficial effect on LN progression, and it may be a good natural leading compound for design of chemical compounds to treat LN.

Complement activation during cardiopulmonary bypass and association with clinical outcomes.

In this prospective, single‐centre observational study of 30 patients undergoing cardiopulmonary bypass (CPB), the effect of unfractionated heparin (UFH), CPB surgery and protamine sulphate on complement and on post‐operative blood loss were assessed. Although C3 and C4 levels decreased significantly immediately following the administration of UFH, C3a, C5a, Bb fragment and SC5b‐9 remained unchanged. During CPB, C3 and C4 continued to fall whilst both alternative and classical pathways activation markers, Bb, C3a, C5a and SC5b‐9 increased significantly. Protamine sulphate had no effect on classical pathway components or activation markers but decreased alternative pathway activation marker Bb. Over the 12–24 h post‐surgery, both classical and alternative pathway activation markers returned to baseline, whilst C3 and C4 levels increased significantly but not to baseline values. Total drain volume 24 h after the surgery showed a moderate inverse correlation with post‐protamine C3 (r = −0.46, p = 0.01) and C4 (r = −0.57, p = 0.0009) levels, whilst a moderate positive correlation was observed with post‐protamine C3a (r = 0.46, p = 0.009), C5a (r = 0.37, p = 0.04) and SC5b‐9 (r = 0.56, p = 0.001) levels but not with Bb fragment (r = 0.25, p = 0.17). Thus, inhibition of complement activation may be a therapeutic intervention to reduce post‐operative blood in patients undergoing CPB.

N-Acetyl-L-Cysteine Potentially Inhibits Complement Activation in Transplantation-Associated Thrombotic Microangiopathy

Transplant-associated thrombotic microangiopathy (TA-TMA) has a high mortality rate and lacks effective treatments. We searched the GEO database and analyzed RNA-seq data and whole-genome sequencing data from patients' blood samples. We identified N-acetyl-L-cysteine (NAC) as a possible therapeutic target for TA-TMA. In vitro experiments showed that NAC reduced complement activation and VWF multimerization in HUVECs. We also treated a 36-year-old female TA-TMA patient with NAC. Hemoglobin, platelet counts, lactate dehydrogenase levels, and sC5b-9 levels and schistocytes were normalized after using NAC. It shows that NAC may be an effective drug to improve TA-TMA symptoms by inhibiting complement activation.

Total coumarin derivates from Hydrangea paniculata attenuate renal injuries in cationized-BSA induced membranous nephropathy by inhibiting complement activation and interleukin 10-mediated interstitial fibrosis

BackgroundTotal coumarins extracted from Hydrangea. Paniculata, Sieb (HP) have showed renal protective effect in several experimental acute and chronic kidney diseases. PurposeThe aim of current study is to evaluate renal protective effect of HP against cationized-BSA (c-BSA) induced experimental membranous nephritis (MN), and further investigate its underlying mechanisms. MethodsRat MN model was established by intravenous injection of 5 mg c-BSA for consecutive 14 days, and after albuminuria confirmed, HP was orally administrated with 7.5, 15, 30 mg/kg for nine weeks. The renal function was measured and histopathological injuries were observed. RNA sequencing was used to analyze the altered signaling pathways in kidneys. Pharmacokinetics was performed to investigate the pharmacodynamics of major ingredients in HP and possible metabolites. Discover X platform helped to clarify the possible molecular mechanisms of major compound in HP. ResultsHP administration could significantly improve the renal function, and ameliorate the dyslipidemia and histopathological injuries. mRNA sequencing demonstrated that HP had anti-inflammation and anti-fibrosis effects possible through down-regulating the complement activation and PI3K-AKT pathways. Pharmacokinetics demonstrated that skimmin and 7-hydoxycoumarin (7-HC) were major compound or metabolite in plasma after oral administration. Based on Discover X platform, we confirmed that skimmin and 7-HC inhibited the IL10 production by inflammatory macrophages through blocking PI3K-AKT and NFκB signaling pathways. Finally, we demonstrated that HP protected tubulointerstitium from complement attack by reducing the C3 self-production and auto-cleavage in tubular cells. ConclusionsHP has a renal protective effect, and its drug development may provide one alternative strategy to treat immune-mediated nephropathy.

Anti-C2 Antibody ARGX-117 Inhibits Complement in a Disease Model for Multifocal Motor Neuropathy.

Background and ObjectivesTo determine the role of complement in the disease pathology of multifocal motor neuropathy (MMN), we investigated complement activation, and inhibition, on binding of MMN patient-derived immunoglobulin M (IgM) antibodies in an induced pluripotent stem cell (iPSC)-derived motor neuron (MN) model for MMN.MethodsiPSC-derived MNs were characterized for the expression of complement receptors and membrane-bound regulators, for the binding of circulating IgM anti-GM1 from patients with MMN, and for subsequent fixation of C4 and C3 on incubation with fresh serum. The potency of ARGX-117, a novel inhibitory monoclonal antibody targeting C2, to inhibit fixation of complement was assessed.ResultsiPSC-derived MNs moderately express the complement regulatory proteins CD46 and CD55 and strongly expressed CD59. Furthermore, MNs express C3aR, C5aR, and complement receptor 1. IgM anti-GM1 antibodies in serum from patients with MMN bind to MNs and induce C3 and C4 fixation on incubation with fresh serum. ARGX-117 inhibits complement activation downstream of C4 induced by patient-derived anti-GM1 antibodies bound to MNs.DiscussionBinding of IgM antibodies from patients with MMN to iPSC-derived MNs induces complement activation. By expressing complement regulatory proteins, particularly CD59, MNs are protected against complement-mediated lysis. Yet, because of expressing C3aR, the function of these cells may be affected by complement activation upstream of membrane attack complex formation. ARGX-117 inhibits complement activation upstream of C3 in this disease model for MMN and therefore represents an intervention strategy to prevent harmful effects of complement in MMN.

A potent truncated form of human soluble CR1 is protective in a mouse model of renal ischemia\u2013reperfusion injury

The complement system is a potent mediator of ischemia–reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

Use of Intravenous Immunoglobulin in the Treatment of Severe COVID-19 Disease – A Case Series

Objective: To study and document the outcomes of adjuvant use of high dose intravenous immunoglobulin (IVIg) therapy in patients with severe or critical corona virus disease 2019 (COVID-19). We report in a case series of five patients who were admitted with severe and critical COVID-19 disease and were treated with adjuvant IVIG along-with the institute's standard of care (SOC) treatment. Methods: It is a retrospective observational study. We retrospectively collected data on all patients with COVID-19 disease who were hospitalized in author’s unit. The severe and critical disease patients who received IVIg were shortlisted and are discussed. Results: Data from 101 patients were analyzed. Of them 5 patients were treated with IVIG along with institution’s SOC. 4 patients were male and 1 was female. Except one patient (P2) all were above 60 years of age and all had one or more co morbidities with Diabetes mellitus (DM) and Hypertension (HT) present all of them. 3 patients had past history of pulmonary tuberculosis (P1, P4 and P5). P2 had chronic kidney disease (CKD) and P4 had coronary artery disease (CAD) with cardiac resynchronization therapy (CRT) device in situ. Median length of stay was 13 days and 4 of them were discharged. Conclusions: This small case series demonstrates that administration of IVIg in patients with severe COVID-19 disease, who did not respond to usual standard of care treatment, could improve clinical outcome and reduce mortality rate. It should be especially considered in cases with severe critical COVID-19 disease along with evidence of hyper inflammation /cytokine storm. Clinical efficacy is possibly driven by its anti-cytokine effects, reduction of inflammation by inhibition of complement activation, and down-regulation of B and T cells’ functions. Among the various inflammatory markers IVIg reduced CRP and D Dimer levels. It did not show relevant effect on other inflammation markers. However, multicenter studies with large sample size are needed to substantiate these observations.

Role of uromodulin and complement activation in the progression of kidney disease.

Uromodulin (UMOD) is a glycoprotein that is selectively expressed on the epithelial cells of the thick ascending limb of Henle's loop and the early distal renal tubule. The present study aimed to investigate whether UMOD was associated with complement activation in patients with renal diseases. In addition, its biological function was examined in vitro. The expression levels of UMOD and complement components, including C1q, C3, C4 and C3a, and membrane attack complex (MAC) in the plasma of patients with IgA nephropathy (IgAN; n=58) and lupus nephritis (LN; n=36) were detected using ELISA, which was used to determine the association between UMOD expression and complement components. In addition, a simulated hypoxia-reoxygenation (H/R) model was used to stimulate UMOD expression in mouse inner medullary collecting duct cells. Additionally, the association between UMOD expression and complement components C1q and C3d at the cellular level was identified using western blotting and immunofluorescence, respectively. It was revealed that the plasma UMOD concentration was significantly decreased in patients with IgAN and LN compared with in healthy controls, and the levels of C3a and MAC were significantly increased in the plasma of patients with IgAN and LN. Furthermore, the plasma levels of C1q, C3 and C4 in patients with LN, but not in patients with IgAN, were significantly decreased compared with in healthy controls. The plasma levels of UMOD were negatively correlated with the plasma C3a and MAC concentrations. However, the plasma levels of UMOD were significantly and positively correlated with the plasma C1q concentration, but not with that of C3 and C4. It was identified that UMOD expression started to increase after 1 h of simulated H/R, and continued to increase at 6 and 12 h. In addition, cells with lower UMOD expression had higher C3d expression in vitro. Collectively, the present results suggested that UMOD was associated with severe complement activation and may be involved in complement-mediated immune protection by inhibiting complement activation in renal disease.

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli.

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX−) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX− sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

Selective Binding of Heparin/Heparan Sulfate Oligosaccharides to Factor H and Factor H-Related Proteins: Therapeutic Potential for C3 Glomerulopathies.

Complement dysregulation is characteristic of the renal diseases atypical hemolytic uremic syndrome (aHUS) and complement component 3 glomerulopathy (C3G). Complement regulatory protein Factor H (FH) inhibits complement activity, whereas FH-related proteins (FHRs) lack a complement regulatory domain. FH and FHRs compete for binding to host cell glycans, in particular heparan sulfates (HS). HS is a glycosaminoglycan with an immense structural variability, where distinct sulfation patterns mediate specific binding of proteins. Mutations in FH, FHRs, or an altered glomerular HS structure may disturb the FH : FHRs balance on glomerular endothelial cells, thereby leading to complement activation and the subsequent development of aHUS/C3G. In this study, we aimed to identify specific HS structures that could specifically compete off FHRs from HS glycocalyx (HSGlx), without interfering with FH binding. FH/FHR binding to human conditionally immortalized glomerular endothelial cells (ciGEnCs) and HSGlx purified from ciGEnC glycocalyx was assessed. HS modifications important for FH/FHR binding to HSGlx were analyzed using selectively desulfated heparins in competition with purified HSGlx. We further assessed effects of heparinoids on FHR1- and FHR5-mediated C3b deposition on ciGEnCs. In the presence of C3b, binding of FH, FHR1 and FHR5 to ciGEnCs was significantly increased, whereas binding of FHR2 was minimal. FHR1 and 5 competitively inhibited FH binding to HSGlx, leading to alternative pathway dysregulation. FHR1 and FHR5 binding was primarily mediated by N-sulfation while FH binding depended on N-, 2-O- and 6-O-sulfation. Addition of 2-O-desulfated heparin significantly reduced FHR1- and FHR5-mediated C3b deposition on ciGEnCs. We identify 2-O-desulfated heparin derivatives as potential therapeutics for C3G and other diseases with dysregulated complement.

Lipoteichoic Acid from Staphylococcus aureus Activates the Complement System via C3 Induction and CD55 Inhibition.

Staphylococcus aureus inhibits complement activity by secreting a variety of toxins. However, the underlying mechanism of complement component regulation by lipoteichoic acid (LTA), a cell wall component of S. aureus, has not been elucidated. In this study, we observed that aLTA (LTA of S. aureus) increased C3 expression in THP-1 cells. The mechanism of aLTA-mediated C3 induction includes an aLTA-toll-like receptor (TLR) 2 interaction, interleukin 1 receptor associated kinase (IRAK) 2 recruitment, and nuclear factor kappa B (NF-kB) activation. In HepG2 cells, C3 protein production begins to increase from 3 h and increases steadily until 48 h. On the other hand, CD55 levels increased up to 6 h after aLTA treatment and started to decrease after 24 h and levels were decreased at 48 h by more than 50% compared to untreated cells. The expression of CD55 in HepG2 cells was shown to be regulated by IRAK-M induced by aLTA. Serum C3 levels increased in mice injected with aLTA, which resulted in an increase in the amount and activity of the membrane attack complex (MAC). We also observed that CD55 mRNA was increased in the liver 24 h after aLTA injection, but was decreased 48 h after injection. These results suggest that aLTA increases complement levels via induction of C3 and inhibition of CD55, which may cause associated MAC-mediated liver damage.

Topical application of mesenchymal stem cell exosomes alleviates the imiquimod induced psoriasis-like inflammation

Severe psoriasis, a chronic inflammatory skin disease is increasingly being effectively managed by targeted immunotherapy but long-term immunotherapy poses health risk and loss of response. Therefore, there is a need for alternative therapy strategies. Mesenchymal stem/stromal cell (MSC) exosomes are widely known for their potent immunomodulatory properties. Here we investigated if topically applied MSC exosomes could alleviate psoriasis-associated inflammation. Topically applied fluorescent exosomes on human skin explants were confined primarily to the stratum corneum with <1% input fluorescence exiting the explant over a 24-h period. Nevertheless, topically applied MSC exosomes in a mouse model of imiquimod (IMQ) psoriasis significantly reduced IL-17 and terminal complement activation complex C5b-9 in the mouse skin. MSC exosomes were previously shown to inhibit complement activation, specifically C5b-9 complex formation through CD59. Infiltration of neutrophils into the stratum corneum is characteristic of psoriasis and neutrophils are a major cellular source of IL-17 in psoriasis through the release of neutrophil extracellular traps (NETs). We propose that topically applied MSC exosomes inhibit complement activation in the stratum corneum and this alleviates IL-17 release by NETS from neutrophils that accumulate in and beneath the stratum corneum.

Decreased Systemic Complement Activation Product C3a is Associated with a Reduction in Pancreatic β Cell Area in Islets of Female Rat Offspring following Chronic Placental Ischemia‐induced Hypertension

Preeclampsia is a pregnancy specific condition characterized by new-onset maternal hypertension and increased systemic inflammation, including increased complement activation. Our previous studies demonstrated that inhibition of complement activation attenuated placental ischemia-induced hypertension in the Reduced Uteroplacental Perfusion Pressure (RUPP) model of gestational hypertension in rat. Hypertensive pregnancy disorders can also result in increased risk of later-life cardiovascular and metabolic disease for the offspring. In fact, recent studies have demonstrated that gestational hypertension is associated with increased risk for Type 2 diabetes in offspring. Clearly, pancreatic β cell mass, established early in life, can influence whether an individual develops Type 2 diabetes. β cell area has also been linked to complement component C3 and activation product C3a, with increased C3a generation associated with prevention of human islet cell atrophy. Our previous studies demonstrated a significant reduction in β cell area in postnatal day (PD)13 female, but not male, offspring of rats subjected to chronic placental ischemia-induced hypertension in the RUPP model. Thus, we hypothesized that decreased C3a is associated with reductions in β cell area in the female offspring following placental insufficiency. The RUPP model was used to induce placental insufficiency by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation. Serum was collected via the abdominal aorta from PD13 pups born from RUPP and Sham pregnancies. Pancreatic islets of PD13 male and female offspring of RUPP and Sham dams were isolated by collagenase perfusion and handpicking. We previously demonstrated that the transcript message for membrane-bound complement regulator CD55 (negative regulator of C3 activation) was significantly increased in islets isolated from female, but not male, RUPP vs Sham offspring, with no change in acinar tissue. In addition, no significant change was observed in message for the parent molecule C3. The relative amount of circulating C3a in serum of PD13 offspring was determined by Western blot and expressed as C3a units/ul based on signal intensity of 1 ul of a standard pool of adult rat serum activated by yeast. Circulating C3a in female RUPP offspring (1.35 ± 0.37 units/ul) was significantly lower (p<0.05) than C3a in female Sham offspring (2.54 ± 0.44 units/ul). No difference was noted comparing C3a in serum of male offspring, RUPP vs Sham, where no change in β cell area has been observed. Thus, these data suggest an increase in CD55 may limit complement activation and C3a production in female offspring. This dysregulation of complement activation may contribute to reduction of β cell area and long-term susceptibility for Type 2 diabetes. Future studies are needed to determine if local or circulating C3a is instrumental in controlling pancreatic β cell area in offspring of hypertensive pregnancies.

Mini-Factor H Modulates Complement-Dependent IL-6 and IL-10 Release in an Immune Cell Culture (PBMC) Model: Potential Benefits Against Cytokine Storm.

Cytokine storm (CS), an excessive release of proinflammatory cytokines upon overactivation of the innate immune system, came recently to the focus of interest because of its role in the life-threatening consequences of certain immune therapies and viral diseases, including CAR-T cell therapy and Covid-19. Because complement activation with subsequent anaphylatoxin release is in the core of innate immune stimulation, studying the relationship between complement activation and cytokine release in an in vitro CS model holds promise to better understand CS and identify new therapies against it. We used peripheral blood mononuclear cells (PBMCs) cultured in the presence of autologous serum to test the impact of complement activation and inhibition on cytokine release, testing the effects of liposomal amphotericin B (AmBisome), zymosan and bacterial lipopolysaccharide (LPS) as immune activators and heat inactivation of serum, EDTA and mini-factor H (mfH) as complement inhibitors. These activators induced significant rises of complement activation markers C3a, C4a, C5a, Ba, Bb, and sC5b-9 at 45 min of incubation, with or without ~5- to ~2,000-fold rises of IL-1α, IL-1β, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13 and TNFα at 6 and 18 h later. Inhibition of complement activation by the mentioned three methods had differential inhibition, or even stimulation of certain cytokines, among which effects a limited suppressive effect of mfH on IL-6 secretion and significant stimulation of IL-10 implies anti-CS and anti-inflammatory impacts. These findings suggest the utility of the model for in vitro studies on CS, and the potential clinical use of mfH against CS.

Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting complement activation and upregulation of miR-499.

The complement system plays a vital role in myocardial ischemia/reperfusion (I/R) injury. microRNA (miR)-499 is involved in the cardioprotection of ischemic postconditioning (IPostC). The present study aimed to study the role of the complement system and miR-499 in IPostC. Rat hearts were subjected to coronary ligation for 30 min, followed by reperfusion for 2 h. IPostC was introduced at the onset of reperfusion with three cycles of reperfusion for 30 sec and coronary artery occlusion for 30 sec. To study the role of miR-499 in IPostC, adeno-associated virus (AAV) vectors of miR-499-5p (AAV-miR-499-5p) and miR-499-5p-sponge (AAV-miR-499-5p-sponge) were transfected via tail vein injection, followed by IPostC protocols. Cardiac injury as well as the status of local and systemic complement activation and inflammation were assessed. IPostC significantly attenuated I/R-induced rat cardiomyocyte apoptosis and the myocardial infarct size. These beneficial effects were accompanied by decreased local and circulating complement component (C)3a and C5a levels, decreased inflammatory marker expression, decreased NF-κB signaling and increased cardiac miR-499 expression. AAV-miR-499-5p prevented local and systemic complement activation and inflammation as well as enhanced the cardioprotection of IPostC, whereas AAV-miR-499-5p-sponge produced the opposite effects. In summary, IPostC protected the rat myocardium against I/R injury, by inhibiting local and systemic complement activation; inflammation; NF-κB signaling; and upregulation of miR-499. As such, miR-499 may have a critical role in IPostC-mediated cardioprotection against I/R injury.