C3 Activation Research Articles

Potent, selective, and subunit-dependent activation of TRPC5 channels by a xanthine derivative.

Background and PurposeThe TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium‐permeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthine‐based TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthine‐based activators of these channels.Experimental ApproachAn analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4–C1, TRPC5–C1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+]i) and by patch‐clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels.Key ResultsAM237 potently activated TRPC5:C5 channels (EC50 15–20 nM in [Ca2+]i assay) and potentiated their activation by sphingosine‐1‐phosphate but suppressed activation evoked by (−)‐englerin A (EA). In patch‐clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237‐induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4–C1, TRPC5–C1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EA‐dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels.Conclusions and ImplicationsThis study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine‐based compounds can activate, potentiate, or inhibit these channels depending on subunit composition.

Community Coalitions for Change and the Policy, Systems, and Environment Model: A Community-Based Participatory Approach to Addressing Obesity in Rural Tennessee.

Four rural counties in Tennessee adopted the policy, systems, and environment (PSE) approach to address the obesity epidemic in their communities. The community-based participatory initiative, Community Coalitions for Change (C3), was embraced by 67,400 community members and 67 organizations. During year 1, coalition members discussed a need to return to long-held traditions of collective community engagement and action to address rural obesity rates. In response, C3 established 25 community gardens and supported 10 existing gardens, resulting in 8,300 community members who received garden produce. Sites began with an average number of 11 physical activity resources, which increased by year 3 to an average of 13 resources as a result of C3 activities. Overall, 61% (248 of 405) of survey respondents participating in direct education programs reported being more physically active as a result of participating in the programs, 59% (117 of 199) reported eating more fruit, and 66% (131 of 199) reported eating more vegetables. Implications for public health include timing and aligning obesity prevention activities with ongoing initiatives that are working toward similar goals.

Neutrophil extracellular traps-associated markers are elevated in patients with systemic lupus erythematosus.

Neutrophil extracellular traps (NETs) are the main source of autoantigens in systemic lupus erythematosus (SLE). The aim of this study was to evaluate the clinical importance of NETs-associated markers in SLE. We compared NETs-associated markers in SLE patients (n = 111) with healthy controls (n = 50). Moreover, in 35 patients with drug-naïve SLE (n = 35), we investigated correlation between NETs-associated markers [DNase I concentration, myeloperoxidase (MPO) activity, anti-MPO antibodies, cell-free DNA (cfDNA), NETolytic activity] with serological parameters [anti-dsDNA antibodies, C3, C4 and B-cell activating factor (BAFF) levels] and disease activity measured by modified SLE Disease Activity Index (M-SLEDAI-2K). In comparison with healthy controls, SLE patients had higher cfDNA, MPO activity, anti-MPO antibodies (p < 0.001), BAFF and DNase I concentration (p < 0.01). Contrary, NETolytic activity was lower in SLE patients (p < 0.05), despite higher concentration of DNase I. MPO activity and cfDNA levels showed correlation with DNase I concentration (p < 0.001, p < 0.01, respectively). BAFF levels correlated with cfDNA, DNase I concentration and MPO activity (p < 0.05). Anti-dsDNA antibodies showed correlation with MPO activity (p < 0.01), cfDNA and BAFF levels (p < 0.001). Anti-dsDNA and C3 levels were independent predictors of M-SLEDAI-2K in multivariate analysis (p < 0.01). We demonstrated that sera of SLE patients have decreased NETolytic activity, leading to increased levels of various NETs-associated markers, which correlate with anti-dsDNA antibodies in drug-naïve SLE. We showed that BAFF participates in a complex relationship between NETosis and anti-dsDNA antibodies production. These findings have important implications for a better understanding of SLE pathogenesis and development of therapy that inhibits NETs persistence and disease progression.

Structural Basis for Properdin Oligomerization and Convertase Stimulation in the Human Complement System.

Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.

Binuclear ruthenium(II) complexes of 4,4′-azopyridine bridging ligand as anticancer agents: synthesis, characterization, and in vitro cytotoxicity studies

Four new binuclear Ru(II)-arene complexes with N-ferrocenyl amino acid ligands and 4,4′-azopyridine as the bridging group [(p-cym)(Fc-AA)Ru(μ-azpy)Ru(Fc-AA)(p-cym)]Cl (C1–C4; where p-cym = p-cymene, MeC6H4Pri; Fc-AA = N-ferrocenyl amino acid conjugate ligands (Fc-tyr 1, Fc-phe 2, Fc-leu 3, Fc-trp 4) and azpy = 4,4′-azopyridine) were synthesized and characterized by 1H NMR, UV—visible, FTIR and mass spectroscopy, and CHN analysis. The structure of one of the precursor mononuclear complexes, [(p-cym)Ru(Fc-phe)Cl], was determined by single-crystal X-ray diffraction studies. Binding of the complexes with calf-thymus DNA and bovine serum albumin (BSA) has been evaluated using absorption and emission spectral studies. Ethidium bromide-displacement studies and DNA-viscosity measurements affirmed intercalative interaction mode between complexes and CT-DNA. Fluorescence titration was used to explore interaction of the complexes with BSA. MTT assay was adopted to evaluate the anticancer activity of C1–C4 on HeLa cell line and the corresponding IC50 values were calculated. The IC50 values of the complexes were higher than that for cisplatin but much lower than for NAMIA and RAPTA-C. The lower cytotoxicity of C1–C4 compared to cisplatin is explained based on nucleotide excision repair mechanism.

Synthesis and evaluation of antiviral activities of triterpenic conjugates with 2-aminobutan-1-ol as potent microbicidal agents

The effect of the synthetic modifications of the triterpenic A ring on the level of antiviral activity of triterpenic C3, C28 amides with a residue of racemic, (S), or (R)-enantiomeric 2-aminobutan-1-ol against herpes simplex viruses type I (HSV-I) and type II (HSV-II), as well as against human immunodeficiency virus type I (HIV-1) was investigated. The 2,3-secolupane racemic amide 5a was selected as a potent microbicidal agent with the highest virus inhibitory (against HSV-I and HSV-II) and virucidal (against HSV-1 and HIV-1) actions, the antiviral activity of which was provided by the (S)-enantiomeric conjugate 5b.

Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth.

Clear-cell renal cell carcinoma (ccRCC) possesses an unmet medical need, particularly at the metastatic stage, when surgery is ineffective. Complement is a key factor in tissue inflammation, favoring cancer progression through the production of complement component 5a (C5a). However, the activation pathways that generate C5a in tumors remain obscure. By data mining, we identified ccRCC as a cancer type expressing concomitantly high expression of the components that are part of the classical complement pathway. To understand how the complement cascade is activated in ccRCC and impacts patients' clinical outcome, primary tumors from three patient cohorts (n = 106, 154, and 43), ccRCC cell lines, and tumor models in complement-deficient mice were used. High densities of cells producing classical complement pathway components C1q and C4 and the presence of C4 activation fragment deposits in primary tumors correlated with poor prognosis. The in situ orchestrated production of C1q by tumor-associated macrophages (TAM) and C1r, C1s, C4, and C3 by tumor cells associated with IgG deposits, led to C1 complex assembly, and complement activation. Accordingly, mice deficient in C1q, C4, or C3 displayed decreased tumor growth. However, the ccRCC tumors infiltrated with high densities of C1q-producing TAMs exhibited an immunosuppressed microenvironment, characterized by high expression of immune checkpoints (i.e., PD-1, Lag-3, PD-L1, and PD-L2). Our data have identified the classical complement pathway as a key inflammatory mechanism activated by the cooperation between tumor cells and TAMs, favoring cancer progression, and highlight potential therapeutic targets to restore an efficient immune reaction to cancer.

A non-coated enzyme-linked immunosorbent assay for screening zika virus envelope protein

To establish a non-coated enzyme-linked immunosorbent assay (ELISA) based on zika virus envelope (E) protein for detecting the expression of E protein in infected cells. Adherent Vero-143 cells infected with zika virus in a 96-well plate were fixed, and the antibodies against zika virus E protein were added at an optimized concentration to establish the non-coated ELISA method for E protein. The antiviral activities of lignans compound C1 was evaluated using this method. The accuracy of this non-coated ELISA was verified by RT-PCR, and the cross reaction with dengue virus was assessed. After optimization, the background absorbance at 450 nm of uninfected cells was reduced to about 0.20. The antiviral activities of lignans compound C1 detected by this method were basically consistent with the results of RT-PCR. No cross reaction with dengue virus was found in this assay. A non- coated ELISA method based on zika virus E protein was established, which can be used for screening antiviral agents against zika virus.

Salidroside Reduces Inflammation and Brain Injury After Permanent Middle Cerebral Artery Occlusion in Rats by Regulating PI3K/PKB/Nrf2/NFκB Signaling Rather than Complement C3 Activity.

Salidroside, an active constituent of Rhodiola rosea, is neuroprotective after transient middle cerebral artery occlusion (tMCAO). However, its effects in other experimental stroke models are less understood. Here, we investigated the effect of daily intraperitoneal injections of salidroside in rats after permanent MCAO (pMCAO). Cerebral infarct volumes at 1day after pMCAO were significantly reduced by treatment with 100mg/kg/day salidroside, but not by 25 or 50mg/kg/day, and this benefit of salidroside increased significantly over at least 7days of treatment, when it was also accompanied by decreased neurological deficit scores. These observations led us to investigate the underlying mechanism of action of salidroside. 100 mg/kg salidroside for 1day increased NeuN, Nrf2, and its downstream mediator HO-1, while it reduced nuclear NFκB p50, IL-6, and TNFα. Brusatol, a Nrf2 inhibitor, blocked the actions of salidroside on Nrf2, NFκB p50, IL-6, and TNFα. Salidroside also increased the ratio of p-PKB/PKB at 1day after pMCAO even in the presence of brusatol. LY294002, a PI3K inhibitor, prevented all these effects of salidroside, including those on NeuN, p-PKB/PKB, Nrf2, HO-1, and pro-inflammatory mediators. In contrast, salidroside had no significant effect on the level of cerebral complement C3 after pMCAO, or on the activity of C3 as measured by the expression of cerebral Egr1. Our findings therefore suggest that salidroside reduces neuroinflammation and neural damage by regulating the PI3K/PKB/Nrf2/NFκB signaling pathway after pMCAO, and that this neuroprotective effect does not involve modulation of complement C3 activity.

Mitigating placental injuries through up-regulating DAF in experimental APS mice: new mechanism of progesterone.

Anti-phospholipid syndrome (APS) is characterized by recurrent pathological pregnancy, arterial or venous thrombosis in the presence of anti-phospholipid antibody (aPL). Complement activation is recognized as an intermediate link leading to placental thrombosis and placental inflammation in APS model mice. Decay accelerating factor (DAF, CD55), MAC-inhibitory protein (MAC-IP, CD59) and membrane co-factor protein (MCP, CD46) are important complement inhibitory proteins (CIPs) highly expressed in normal placenta to curb excessive complement activation and its mediated injuries. Anti-β2 glycoprotein I (anti-β2GPI) antibody is an important aPL. We found that placental DAF and CD46 decreased in β2GPI passively immunized APS model mice, accompanied by C3 deposition, neutrophil infiltration and increased proinflammatory cytokine levels detected in its placenta. Progesterone supplement can up-regulate DAF but not CD46 expression, curb C3 activation and decrease proinflammatory cytokines levels to reduce fetal loss frequency. Progesterone receptor antagonist (mifepristone) or knock-down DAF with specific siRNA, above the protective effects of progesterone, were significantly weakened. Another sex hormone, oestrogen, has no significant effect on placental DAF and C3 contents and fetal loss frequency in the APS mice model. This may be an important mechanism by which progesterone induces maternal-fetal immune tolerance. At the same time, it may provide evidence for the use of progesterone in APS abortion patients.

C3- and CR3-dependent microglial clearance protects photoreceptors in retinitis pigmentosa.

Complement activation has been implicated as contributing to neurodegeneration in retinal and brain pathologies, but its role in retinitis pigmentosa (RP), an inherited and largely incurable photoreceptor degenerative disease, is unclear. We found that multiple complement components were markedly up-regulated in retinas with human RP and the rd10 mouse model, coinciding spatiotemporally with photoreceptor degeneration, with increased C3 expression and activation localizing to activated retinal microglia. Genetic ablation of C3 accelerated structural and functional photoreceptor degeneration and altered retinal inflammatory gene expression. These phenotypes were recapitulated by genetic deletion of CR3, a microglia-expressed receptor for the C3 activation product iC3b, implicating C3-CR3 signaling as a regulator of microglia-photoreceptor interactions. Deficiency of C3 or CR3 decreased microglial phagocytosis of apoptotic photoreceptors and increased microglial neurotoxicity to photoreceptors, demonstrating a novel adaptive role for complement-mediated microglial clearance of apoptotic photoreceptors in RP. These homeostatic neuroinflammatory mechanisms are relevant to the design and interpretation of immunomodulatory therapeutic approaches to retinal degenerative disease.

Identification of functionally important amino acid sequences in cobra venom factor using human C3/Cobra venom factor hybrid proteins

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF,Bb is an enzyme that cleaves C3 and C5. However, CVF,Bb exhibits significantly different functional properties from C3b,Bb. The CVF,Bb convertase is physico-chemically very stable, and completely resistant to an activation by Factors H and I. These two properties, in contrast to C3b,Bb, allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the functional differences between CVF and C3, we have created several hybrid proteins of CVF and human C3. Here we report that replacing the C-terminal 168 amino acid residues of human C3 with the corresponding residues from CVF results in a hybrid protein (HC3-1496) which is essentially a human C3 derivative exhibiting the functional properties of CVF. This result demonstrates that the important structures for the CVF-specific functions reside within the C-terminal 168 amino acid residues of CVF. We further demonstrate that reverting the 46 C-terminal CVF residues of HC3-1496 to human C3 sequence results in a hybrid protein (HC3-1496/1617) that exhibits a physico-chemically unstable convertase with only residual complement depleting activity. This result demonstrates that most, but not all, structural requirements for CVF activity reside within the 46 C-terminal amino acid residues. We also investigated the potential role of position 1633, which is an acidic residue in human C3 (glutamic acid) but a basic amino acid residue (histidine) in CVF. However, the charge at position 1633 appears to be of no functional relevance. Exchanging the neutral amino acids present in CVF at positions 1499 and 1501 with the two charged amino acids at these positions in human C3 (aspartic acid and lysine) resulted in a hybrid protein that exhibited significantly slower convertase formation although both binding to Factor B and C3 cleavage was not affected, demonstrating that the charged amino acid residues at these two positions interfere with the formation of the convertase. In conclusion, our work demonstrates that hybrid proteins of human C3 and CVF present valuable tools to identify functionally important amino acid residues in CVF.

Effects of acute hypoxic stress on biochemical parameters, immune regulation and metabolic capacity of the blood in genetically improved farmed tilapia (GIFT,Oreochromis niloticus)

Dissolved oxygen (DO) in the culture water is an important environmental factor in fish farming. This study tested whether genetically improved farmed tilapia (GIFT, Oreochromis niloticus) challenged with a hypoxic episode would show inhibited growth, increased metabolic stress and a reduced immune response. GIFT subjected to 72 hr of semi-lethal hypoxia (determined by linear regression as DO = 0.64 mg/L) showed changes in blood biochemistry, energy metabolism and related immune responses. In the early stages of stress, GIFT showed increased anaerobic respiration and increased lactic acid accumulation. Compared with a control group, white and red blood cell counts, hematocrit and blood hemoglobin concentration in GIFT exposed to 4 hr of semi-lethal hypoxic stress were not significantly changed. However, the levels of glutamic-pyruvic transaminase, glucose, cholesterol and cortisol were significantly higher under hypoxic stress at 2 and 4 hr. In the later stages of stress, GIFT displayed increased oxygen utilization and increased aerobic respiration associated with increased red blood cell count, hematocrit and hemoglobin concentration. However, the white blood cell count, serum lysozyme and complement C3 activities began to decline, associated with increased mortality. Reverting to a normal DO environment (5.0 mg/L) for an additional 72 hr, GIFT showed strong recovery, with respiratory metabolic enzymes, immune indicators and associated energy metabolites restored to near normal levels. Our observations contribute to a better understanding of the mechanisms of energy and immune regulation in fish, and will help reduce the damage caused by hypoxic stress during culture.

Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b

Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomography structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4 °C. The IgM-C1-C4b complexes revealed C4b product release as the temperature-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helices, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab2 and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.

Inefficient and abortive classical complement pathway activation by the calcium inositol hexakisphosphate component of the Echinococcus granulosus laminated layer

Persistent extracellular tissue-dwelling pathogens face the challenge of antibody-dependent activation of the classical complement pathway (CCP). A prime example of this situation is the larva of the cestode Echinococcus granulosus sensu lato, causing cystic echinococcosis. This tissue-dwelling, bladder-like larva is bounded by a cellular layer protected by the outermost acellular “laminated layer” (LL), to which host antibodies bind. The LL is made up of a mucin meshwork and interspersed nano-deposits of calcium inositol hexakisphosphate (calcium InsP6). We previously reported that calcium InsP6 bound C1q, apparently initiating CCP activation. The present work dissects CCP activation on the LL. Most of the C1 binding activity in the LL corresponded to calcium InsP6, and this binding was enhanced by partial proteolysis of the mucin meshwork. The remaining C1 binding activity was attributable to host antibodies, which included CCP-activating IgG isotypes. Calcium InsP6 made only a weak contribution to early CCP activation on the LL, suggesting inefficient C1 complex activation as reported for other polyanions. CCP activation on calcium InsP6 gave rise to a dominant population of C3b deposited onto calcium InsP6 itself that appeared to be quickly inactivated. Apparently as a result of inefficient initiation plus C3b inactivation, calcium InsP6 made no net contribution to C5 activation. We propose that the LL protects the underlying parasite cells from CCP activation through the combined effects of inefficient permeation of C1 through the mucins and C1 retention on calcium InsP6. This mechanism does not result in C5 activation, which is known to drive parasite-damaging inflammation.

Structural and photosynthetic re-acclimation to low light in C4 maize leaves that developed under high light.

C4 plants have higher photosynthetic capacity than C3 plants, but this advantage comes at an energetic cost that is problematic under low light. In the crop canopy, lower leaves first develop under high light but later experience low light because of mutual shading. To explore the re-acclimation of C4 leaves to low light, we investigated the structural and physiological changes of the leaves of maize plants grown in shaded pots. Plants were first grown under high light, and then some of them were shaded (20 % of sunlight) for 3 weeks. Four types of leaves were examined: new leaves that developed under low light during shading (L), new leaves that developed under high light (H), mature leaves that developed under high light before shading and were then subjected to low light (H-L) and mature leaves that always experienced high light (H-H). The leaf mass per area, nitrogen and chlorophyll contents per unit leaf area, chlorophyll a/b ratio and activities of C3 and C4 photosynthetic enzymes were lower in H-L than in H-H leaves and in L than in H leaves. Unlike L leaves, H-L leaves maintained the thickness and framework of the Kranz anatomy of H leaves, but chloroplast contents in H-L leaves were reduced. This reduction of chloroplast contents was achieved mainly by reducing the size of chloroplasts. Although grana of mesophyll chloroplasts were more developed in L leaves than in H leaves, there were no differences between H-L and H-H leaves. The light curves of photosynthesis in H-L and L leaves were very similar and showed traits of shade leaves. Mature maize leaves that developed under high light re-acclimate to low-light environments by adjusting their biochemical traits and chloroplast contents to resemble shade leaves but maintain the anatomical framework of sun leaves.

The differential carbon-fixing and nitrogen-assimilating enzyme activities of Oscillatorian marine cyanobacterium Phormidium valderianum BDU 20041

We have identified an efficient filamentous marine cyanobacterium, Phormidium valderianum BDU 20041, which was adaptive to different concentrations of CO2. In our study, P. valderianum BDU 20041 was grown under ambient (0.04) and elevated (2, 3, 4, 5, 10, and 15%) CO2 concentration. Its optimal growth occurred at 3% CO2 supply. P. valderianum displayed similar carbon fixation activities through Rubisco, phosphoenolpyruvate carboxylase (PEPC), carbonic anhydrase, and malate dehydrogenase. The activity of Rubisco, the primary carbon-fixing enzyme, was found to be approximately 80% higher in P. valderianum grown under 4% CO2 than under ambient air. This was substantiated by Western blot. The nitrogen-assimilating enzymes shared a similar trend at higher CO2 concentrations. Our study is one of the few reports that examined the activities of CO2-fixing and nitrogen-assimilating enzymes together. Due to the active PEPC, this cyanobacterium was found to have active C3 and C4 enzymes that promoted higher CO2 fixation and cellular growth. Thus, it may be an efficient organism for biotechnological exploitation.

Complement C3 activation regulates the production of tRNA-derived fragments Gly-tRFs and promotes alcohol-induced liver injury and steatosis

Complement is known to play a role in alcoholic fatty liver disease (AFLD), but the underlying mechanisms are poorly understood, thereby constraining the development of a rational approach for therapeutic intervention in the complement system. C3 deficiency has been shown to impart protective effects against ethanol-induced hepatic steatosis and inflammation. Here we demonstrate a protection effect in wild-type mice by treatment with CR2-Crry, a specific inhibitor of C3 activation. The expression of glycine transfer (t) RNA-derived fragments (Gly-tRFs) is upregulated in ethanol-fed mice and inhibition of Gly-tRFs in vivo decreases chronic ethanol feeding-induced hepatosteatosis without affecting inflammation. The expression of Gly-tRF was downregulated in C3-deficient or CR2-Crry-treated mice, but not in C5-deficient mice; Gly-tRF expression was restored by the C3 activation products C3a or Asp (C3a-des-Arg) via the regulation of CYP2E1. Transcriptome profiling of hepatic tissues showed that Gly-tRF inhibitors upregulate the expression of sirtuin1 (Sirt1) and subsequently affect downstream lipogenesis and β-oxidation pathways. Mechanistically, Gly-tRF interacts with AGO3 to downregulate Sirt1 expression via sequence complementarity in the 3′ UTR. Notably, the expression levels of C3d, CYP2E1 and Gly-tRF are upregulated, whereas Sirt1 is decreased in AFLD patients compared to healthy controls. Collectively, our findings suggest that C3 activation products contribute to hepatosteatosis by regulating the expression of Gly-tRF. Complement inhibition at the C3 activation step and treatment with Gly-tRF inhibitors may be potential and precise therapeutic approaches for AFLD.

Investigational RNAi Therapeutic Targeting C5 Is Efficacious in Pre-clinical Models of Myasthenia Gravis

Complement-mediated damage to the neuromuscular junction (NMJ) is a key mechanism of pathology in myasthenia gravis (MG), and therapeutics inhibiting complement have shown evidence of efficacy in the treatment of MG. In this study, we describe the development of a subcutaneously administered N-acetylgalactosamine (GalNAc)-conjugated small interfering RNA (siRNA) targeting the C5 component of complement that silences C5 expression in the liver (ALN-CC5). Treatment of wild-type rodents with ALN-CC5 resulted in robust and durable suppression of liver C5 expression. Dose-dependent serum C5 suppression was observed in non-human primates, with a lowering of serum C5 of up to 97.5% and the concomitant inhibition of serum complement activity. C5 silencing was efficacious in ameliorating disease symptoms in two standard rat models of MG, demonstrating the key role of circulating C5 in pathology at the NMJ. Improvement in disease activity scores and NMJ pathology was observed at intermediate levels of complement activity inhibition, suggesting that complete ablation of complement activity may not be required for efficacy in MG. The pre-clinical studies of ALN-CC5 and efficacy of C5 silencing in rat models of MG support further clinical development of ALN-CC5 as a potential therapeutic for the treatment of MG and other complement-mediated disorders.

Haemolytic activity of complement component C3 of Labeo rohita

Haemolytic activity of complement component C3 of Labeo rohita