IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1ß production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

C3 Glomerulopathy: Pathogenesis and Treatment.

C3 glomerulopathy (C3G) is a rare set of kidney diseases with 2 patterns: C3 glomerulonephritis (C3GN) and dense deposit disease. Pathogenesis of both diseases is due to complement dysregulation in the alternative pathway. Acquired or genetic alterations of the regulatory proteins of the complement pathway result in C3G. Although the disease is characterized by low C3 levels in serum and C3-dominant staining by immunofluorescence on biopsy, other disease entities such as infection-related glomerulonephritis and masked monoclonal deposits can present similarly. Both the C3GN and dense deposit disease variants of C3G are progressive and recur in transplanted kidneys. Although no direct treatment is available, complement blockers are either available or in the clinical trial phase. This review will survey the pathogenesis of C3GN and current treatment options.

Effects of Lactobacillus rhamnosus ATCC 7469 on Different Parameters Related to Health Status of Rainbow Trout (Oncorhynchus mykiss) and the Protection Against Yersinia ruckeri.

In the current study, we investigated the effect of a probiotic bacterium (Lactobacillus rhamnosus ATCC 7469) microencapsulated with alginate and hi-maize starch and coated with chitosan on improving growth factors, body composition, blood chemistry, and the immune response of rainbow trout (initial weight: 18.41 ± 0.32 g). Four experimental diets were formulated to feed fish for 60 days. They were control diet without any additive (C), diet added with beads without probiotic (E), a probiotic sprayed to the diet (L.r), and encapsulated probiotic supplemented diet (E-L.r). The results indicated that feeding with E-Lr significantly improved weight gain (84.98 g) and feed conversion ratio (0.95) compared to the other groups (P < 0.05). Also, fish fed E-Lr diet had a significantly higher value of whole-body protein (17.51%), total protein in the blood (4.98 g/dL), lysozyme (30.66 U/mL), alternative complement pathway hemolytic activity (134 U/mL), superoxide dismutase (203 U/mg protein), and catalase (528.33 U/mg protein) (P < 0.05) as compared to those fed the control diet. Similarly, a higher relative expression of immune-related genes such as interleukin-1 (Il-1) and tumor necrosis factor-alpha (TNF-1α) were reported in those fed E-L.r and L.r diets respectively. Interestingly, the fish fed dietary E-L.r had a significantly lower value of lipid in the whole body (4.82%) and cholesterol in the blood (160.67%) in comparison with those fed the control diet (P < 0.05). At the end of the experiment, all groups were challenged by Yersinia ruckeri where the survival rate of rainbow trout fed dietary E-L.r (70.36%) was statistically higher than that of the others (P < 0.05). Overall, the results suggested that encapsulated probiotic Lact. rhamnosus ATCC 7469 acted better than unencapsulated probiotic and has a potential to improve growth performance, flesh quality, and the immune response of rainbow trout.

Recurrence and Outcomes of Complement-Related Renal Disease After Pediatric Renal Transplantation.

Complement dysregulation is related to different glomerular pathologies. Patients with complement dysregulation have high recurrence risk after transplant; however, with trough-effective therapeutics, renal transplant can be an option for these patients. Here, we present 2 boys with renal disease related to complement dysregulation and their outcomes after renal transplant. Patient 1 had atypical hemolytic uremic syndrome, which was treated with eculizumab before renal transplant; eculizumab therapy was also continued after transplant as preventive therapy. Eculizumab therapy was stopped at year 2 post-transplant. At year 4 post-transplant, his serum creatinine level was 0.87 mg/dL. Patient 2, who had chronic renal disease related to C3 glomerulopathy, was not responsive to eculizumab before renal transplant. At month 4 posttransplant, C3 glomerulopathy recurrence was demonstrated with biopsy, and serum creatinine level was 1.96 mg/dL at this time. Eculizumab was started as a rescue therapy. At year 4 posttransplant, his serum creatinine level was 2.07 mg/dL. In our 2 patients with complement dysregulation, eculizumab was an effective and preventive therapy after renal transplant. However, more studies are needed to understand the long-term efficacy and safety of eculizumab after renal transplant.

Clinical relevance of membrane attack complex deposition in children with IgA nephropathy and Henoch-Schönlein purpura.

BACKGROUND: IgA nephropathy (IgAN) and Henoch-Schönlein purpura are common glomerular disorders in children sharing the same histopathologic pattern of IgA deposits within the mesangium, even if their physiopathology may be different. Repeated exposure to pathogens induces the production of abnormal IgA1. The immune complex deposition in the renal mesangium in IgAN or potentially in small vessels in Henoch-Schönlein purpura induces complement activation via the alternative and lectin pathways. Recent studies suggest that levels of membrane attack complex (MAC) in the urine might be a useful indicator of renal injury. Because of the emerging availability of therapies that selectively block complement activation, the aim of the present study is to investigate whether MAC immunostaining might be a useful marker of IgA-mediated renal injury. METHODS: We conducted immunohistochemistry analysis of the MAC on renal biopsies from 67 pediatric patients with IgAN and Henoch-Schönlein purpura. We classified their renal biopsies according to the Oxford classification, retrieved symptoms, biological parameters, treatment, and follow-up. RESULTS: We found MAC expression was significantly related to impaired renal function and patients whose clinical course required therapy. MAC deposits tend to be more abundant in patients with decreased glomerular filtration rate (p = 0.02), patients with proteinuria > 0.750 g/day/1.73 m2, and with nephrotic syndrome. No correlation with histological alterations was observed. CONCLUSIONS: We conclude that MAC deposition could be a useful additional indicator of renal injury in patients with IgAN and Henoch-Schönlein purpura, independent of other indicators.

Complement System: a Neglected Pathway in Immunotherapy.

Approved for the treatment of autoimmune diseases, hematological malignancies, and solid cancers, several monoclonal antibodies (mAb) make use of complement in their mechanism of action. Such an assessment is based on comprehensive investigations that used mouse models, in vitro studies, and analyses from patients at initiation (basal level to highlight deficiencies) and after treatment initiation (mAb impact on complement), which have further provided key insights into the importance of the complement activation and/or complement deficiencies in mAb activity. Accordingly, new approaches can now be developed with the final objective of increasing the clinical efficacy of mAb. These improvements include (i) the concurrent administration of fresh frozen plasma during mAb therapy; (ii) mAb modifications such as immunoglobulin G subclass switching, Fc mutation, or IgG hexamerization to improve the fixation and activation of C1q; (iii) optimization of the target recognition to induce a higher complement-dependent cytotoxicity (CDC) and/or complement-dependant cellular cytotoxicity (CDCC); and (iv) the control of soluble and cellular complement inhibitors.

Complement-driven anemia: more than just paroxysmal nocturnal hemoglobinuria.

Atypical hemolytic uremic syndrome (aHUS); hemolysis, elevated liver function tests, and low platelets syndrome; and transplant-associated thrombotic microangiopathy are related conditions, in that many patients harbor germline heterozygous mutations in genes that regulate the alternative pathway of complement (APC). Penetrance is variable because development of clinically significant disease appears to require supervention of a process such as inflammation. Complement activation on the endothelial surfaces leads to endothelial damage, platelet consumption, microthrombi, and a mechanical hemolytic anemia with schistocytes. Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic disease caused by expansion of a stem cell that harbors a somatic mutation in PIGA PIGA mutant blood cells are deficient in the complement regulator proteins CD55 and CD59, making them susceptible to intravascular hemolysis due to a failure to regulate the APC on erythrocytes. Eculizumab is a monoclonal antibody that binds to C5 and inhibits terminal complement by interfering with the cleavage of C5 by the C5 convertases. The drug is approved by the US Food and Drug Administration for the treatment of aHUS and PNH; however, a new generation of complement inhibitors that block C5 and other components of the complement cascade is showing promise in preclinical and clinical trials.

Transcriptome of Porcine PBMCs over Two Generations Reveals Key Genes and Pathways Associated with Variable Antibody Responses post PRRSV Vaccination.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a virus susceptible to antibody dependent enhancement, causing reproductive failures in sows and preweaning mortality of piglets. Modified-live virus (MLV) vaccines are used to control PRRS in swine herds. However, immunized sows and piglets often generate variable antibody levels. This study aimed to detect significant genes and pathways involved in antibody responsiveness of pregnant sows and their offspring post-PRRSV vaccination. RNA sequencing was conducted on peripheral blood-mononuclear cells (PBMCs), which were isolated from pregnant sows and their piglets with high (HA), median (MA), and low (LA) PRRS antibody levels following vaccination. 401 differentially expressed genes (DEGs) were identified in three comparisons (HA versus MA, HA versus LA, and MA versus LA) of sow PBMCs. Two novel pathways (complement and coagulation cascade pathway; and epithelial cell signaling in H. pylori infection pathway) revealed by DEGs in HA versus LA and MA versus LA were involved in chemotactic and proinflammatory responses. TNF-α, CCL4, and NFKBIA genes displayed the same expression trends in subsequent generation post-PRRS-MLV vaccination. Findings of the study suggest that two pathways and TNF-α, CCL4, and NFKBIA could be considered as key pathways and potential candidate genes for PRRSV vaccine responsiveness, respectively.

Kidney Diseases Associated With Alternative Complement Pathway Dysregulation and Potential Treatment Options.

Atypical hemolytic uremic syndrome and C3 glomerulopathy (dense deposit disease and C3 glomerulonephritis) are characterized as inappropriate activation of the alternative complement pathway. Genetic mutations affecting the alternative complement pathway regulating proteins (complement factor H, I, membrane cofactor protein and complement factor H-related proteins) and triggers (such as infection, surgery, pregnancy and autoimmune disease flares) result in the clinical manifestation of these diseases. A decade ago, prognosis of these disease states was quite poor, with most patients developing end-stage renal disease. Furthermore, renal transplantation in these conditions was associated with poor outcomes due to graft loss to recurrent disease. Recent advances in targeted complement inhibitor therapy resulted in significant improvement in disease remission, renal recovery, health-related quality of life and allograft survival.

C3 glomerulopathy and current dilemmas.

C3 glomerulopathy (C3G) is a recently identified disease entity caused by dysregulation of the alternative complement pathway, and dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are its components. Because laboratory detection of complement dysregulation is still uncommon in practice, "dominant C3 deposition by two orders greater than that of immunoglobulins in the glomeruli by immunofluorescence", as stated in the consensus report, defines C3G. However, this morphological definition possibly includes the cases with glomerular diseases of different mechanisms such as post-infectious glomerulonephritis. In addition, the differential diagnosis between DDD and C3GN is often difficult because the distinction between these two diseases is based solely on electron microscopic features. Recent molecular and genetic advances provide information to characterize C3G. Some C3G cases are found with genetic abnormalities in complement regulatory factors, but majority of cases seem to be associated with acquired factors that dysregulate the alternative complement pathway. Because clinical courses and prognoses among glomerular diseases with dominant C3 deposition differ, further understanding the background mechanism, particularly complement dysregulation in C3G, is needed. This may resolve current dilemmas in practice and shed light on novel targeted therapies to remedy the dysregulated alternative complement pathway in C3G.

Small-molecule factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 µM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

Severe active C3 glomerulonephritis triggered by immune complexes and inactivated after eculizumab therapy.

BACKGROUND: Understanding the role of alternative complement pathway dysregulation in membranoproliferative glomerulonephritis (MPGN) has led to a dramatic shift in its classification into two subgroups: immune complex-mediated MPGN and complement-mediated MPGN, consisting of dense deposit disease and C3 glomerulonephritis (C3GN). A limited number of C3GN cases have been published to date with not yet conclusive results since the novel therapeutic approach with eculizumab was introduced. CASE PRESENTATION: We report the clinical follow-up of a 16-year-old patient in whom a diagnosis of C3GN was confirmed by immunofluorescence and electron microscopy in second and third kidney biopsies, while the first biopsy revealed idiopathic immune complex-mediated MPGN type III, Anders and Strife variant, which failed to improve after several attempts at conventional immunosuppression therapy. Although applied late in an already fairly advanced stage of the severe active form of MPGN, the efficacy of eculizumab on C3GN was evidenced clinically and pathohistologically. Its beneficial influence on pathomorphogenesis was demonstrated by a unique follow-up in the last three biopsies, despite the recent observation, confirmed in this study, of eculizumab binding within the kidney tissue. CONCLUSIONS: Clinicians and pathologists should be aware that, in some patients, an underlying genetic or acquired complement alternative pathway abnormality can be masked by an initial immune complex-mediated mechanism, which subsequently triggers an unbalanced excessive continual driving of complement terminal pathway activation and the development of C3GN. In such a patient, supplementary steroids in addition to eculizumab appear necessary to achieve an adequate response.

Complement factor D homolog involved in the alternative complement pathway of rock bream (Oplegnathus fasciatus): Molecular and functional characterization and immune responsive mRNA expression analysis.

The complement system serves conventional role in the innate defense against common invading pathogens. Complement factor D (CfD) is vital to alternative complement pathway activation in cleaving complement factor B. This catalytic reaction forms the alternative C3 convertase that is crucial for complement-mediated pathogenesis. In this study, rock bream (Oplegnathus fasciatus) CfD (OfCfD) was characterized and OfCfD mRNA expression was investigated. OfCfD encodes 277 amino acids (aa) for a 30-kDa polypeptide. A domain analysis of the deduced OfCfD aa sequence showed a single serine protease trypsin superfamily domain, a serine active region, three active sites, and three substrate-binding sites. Pairwise sequence comparisons indicated that OfCfD has the highest identity (84.5%) with Oreochromis niloticus CfD. The phylogenetic tree revealed a common ancestral origin of CfD members, with fish CfD distinct from other vertebrate orthologs. The structural arrangement of the OfCfD gene (2451 bp) contained five exons interrupted by four introns. A spatial transcriptional analysis indicated that OfCfD transcripts constitutively expressed in all of the examined rock bream tissues, and that they were highest in the spleen and liver. In addition, OfCfD transcripts were immunologically upregulated by lipopolysaccharide (LPS) (12 h p.i.), Streptococcus iniae (12 h p.i.), rock bream iridovirus (RBIV) (6-12 h p.i.), and poly I:C (6 h p.i.) in spleen tissue. OfCfD is a trypsin protease and its recombinant protein showed strong protease activity similar to that of trypsin, indicating its catalytic function in the alternative pathway. Together, our findings suggest that OfCfD might be involved in immune responses in rock bream.

A Targeted Inhibitor of the Alternative Complement Pathway Accelerates Recovery From Smoke-Induced Ocular Injury.

PURPOSE: Morphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP. METHODS: Mice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR. RESULTS: The CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM. CONCLUSIONS: Taken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD.

Coagulation induced by C3aR-dependent NETosis drives protumorigenic neutrophils during small intestinal tumorigenesis.

Excessive activation of blood coagulation and neutrophil accumulation have been described in several human cancers. However, whether hypercoagulation and neutrophilia are linked and involved in cancer development is currently unknown. Here we show that spontaneous intestinal tumorigenesis correlates with the accumulation of low-density neutrophils with a pro-tumorigenic N2 phenotype and unprompted neutrophil extracellular traps (NET) formation. We find that increased circulating lipopolysaccharide induces upregulation of complement C3a receptor on neutrophils and activation of the complement cascade. This leads to NETosis, induction of coagulation and N2 polarization, which prompts tumorigenesis, showing a novel link between coagulation, neutrophilia and complement activation. Finally, in a cohort of patients with small but not large intestinal cancer, we find a correlation between neutrophilia and hypercoagulation. This study provides a mechanistic explanation for the tumour-promoting effects of hypercoagulation, which could be used as a new biomarker or as a therapeutic target.

Identification of peptidic inhibitors of the alternative complement pathway based on Staphylococcus aureus SCIN proteins.

The complement system plays a central role in a number of human inflammatory diseases, and there is a significant need for development of complement-directed therapies. The discovery of an arsenal of anti-complement proteins secreted by the pathogen Staphylococcus aureus brought with it the potential for harnessing the powerful inhibitory properties of these molecules. One such family of inhibitors, the SCINs, interact with a functional "hot-spot" on the surface of C3b. SCINs not only stabilize an inactive form of the alternative pathway (AP) C3 convertase (C3bBb), but also overlap the C3b binding site of complement factors B and H. Here we determined that a conserved Arg residue in SCINs is critical for function of full-length SCIN proteins. Despite this, we also found SCIN-specific differences in the contributions of other residues found at the C3b contact site, which suggested that a more diverse repertoire of residues might be able to recognize this region of C3b. To investigate this possibility, we conducted a phage display screen aimed at identifying SCIN-competitive 12-mer peptides. In total, seven unique sequences were identified and all exhibited direct C3b binding. A subset of these specifically inhibited the AP in assays of complement function. The mechanism of AP inhibition by these peptides was probed through surface plasmon resonance approaches, which revealed that six of the seven peptides disrupted C3bBb formation by interfering with factor B/C3b binding. To our knowledge this study has identified the first small molecules that retain inhibitory properties of larger staphylococcal immune evasion proteins.

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum.

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

Smoke exposure causes endoplasmic reticulum stress and lipid accumulation in retinal pigment epithelium through oxidative stress and complement activation.

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD.

Cholesterol crystals induce complement-dependent inflammasome activation and cytokine release.

Inflammation is associated with development of atherosclerosis, and cholesterol crystals (CC) have long been recognized as a hallmark of atherosclerotic lesions. CC appear early in the atheroma development and trigger inflammation by NLRP3 inflammasome activation. In this study we hypothesized whether CC employ the complement system to activate inflammasome/caspase-1, leading to release of mature IL-1ß, and whether complement activation regulates CC-induced cytokine production. In this study we describe that CC activated both the classical and alternative complement pathways, and C1q was found to be crucial for the activation. CC employed C5a in the release of a number of cytokines in whole blood, including IL-1ß and TNF. CC induced minimal amounts of cytokines in C5-deficient whole blood, until reconstituted with C5. Furthermore, C5a and TNF in combination acted as a potent primer for CC-induced IL-1ß release by increasing IL-1ß transcripts. CC-induced complement activation resulted in upregulation of complement receptor 3 (CD11b/CD18), leading to phagocytosis of CC. Also, CC mounted a complement-dependent production of reactive oxygen species and active caspase-1. We conclude that CC employ the complement system to induce cytokines and activate the inflammasome/caspase-1 by regulating several cellular responses in human monocytes. In light of this, complement inhibition might be an interesting therapeutic approach for treatment of atherosclerosis.

Excessive activation of the alternative complement pathway in autosomal dominant polycystic kidney disease.

OBJECTIVES: The complement system is involved in many immune complex-mediated kidney diseases, yet its role in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD) has not been examined in detail. METHODS AND RESULTS: Screening of the glycoproteome of urine samples from ADPKD patients revealed that levels of complement factor B (CFB), serpin peptidase inhibitor, complement component 1 inhibitor (SERPING1) and complement component 9 (C9) increased, whereas complement component 1, r subcomponent-like (C1RL), CD55 and CD59 levels decreased with disease progression. Immunostaining and Western blot analysis confirmed the enhanced expression of CFB and C9 in cystic kidneys from ADPKD patients. Immunostaining also showed that the expressions of CFB and C9 in renal biopsy tissues from patients with other types of chronic kidney disease were lower than in tissues from ADPKD patients. The effect of the complement inhibitor rosmarinic acid (RMA) was evaluated in Pkd1(-/-) mice and Han:SPRD Cy/+ rats. Compared with vehicle-treated Pkd1(-/-) animals, RMA-treated mice had significantly lower serum creatinine (-50%) and blood urea nitrogen (-78%) levels, two kidneys/body weight ratio (-60%) and renal cystic index (-60%). Similar results were found in Cy/+ rats. Lower numbers of Ki67-positive nuclei and inflammatory cells and reduced fibrosis were observed in both animal models upon treatment with RMA. CONCLUSIONS: These results suggest that excessive activation of the alternative complement pathway is associated with ADPKD progression, probably mediated by cyst-lining epithelial cell proliferation, tubulointerstitial inflammatory cell infiltration and fibrosis. Targeting the complement system might represent a new therapeutic strategy for ADPKD.