MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of approximately 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator

MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro. In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways

Sodium Iodate-Induced Degeneration Results in Local Complement Changes and Inflammatory Processes in Murine Retina

Age-related macular degeneration (AMD), one of the leading causes of blindness worldwide, causes personal suffering and high socioeconomic costs. While there has been progress in the treatments for the neovascular form of AMD, no therapy is yet available for the more common dry form, also known as geographic atrophy. We analysed the retinal tissue in a mouse model of retinal degeneration caused by sodium iodate (NaIO3)-induced retinal pigment epithelium (RPE) atrophy to understand the underlying pathology. RNA sequencing (RNA-seq), qRT-PCR, Western blot, immunohistochemistry of the retinas and multiplex ELISA of the mouse serum were applied to find the pathways involved in the degeneration. NaIO3 caused patchy RPE loss and thinning of the photoreceptor layer. This was accompanied by the increased retinal expression of complement components c1s, c3, c4, cfb and cfh. C1s, C3, CFH and CFB were complement proteins, with enhanced deposition at day 3. C4 was upregulated in retinal degeneration at day 10. Consistently, the transcript levels of proinflammatory ccl-2, -3, -5, il-1β, il-33 and tgf-β were increased in the retinas of NaIO3 mice, but vegf-a mRNA was reduced. Macrophages, microglia and gliotic Müller cells could be a cellular source for local retinal inflammatory changes in the NaIO3 retina. Systemic complement and cytokines/chemokines remained unaltered in this model of NaIO3-dependent retinal degeneration. In conclusion, systemically administered NaIO3 promotes degenerative and inflammatory processes in the retina, which can mimic the hallmarks of geographic atrophy

Ophthalmology

OBJECTIVE: In the current study we aimed to identify metabolites associated with age-related macular degeneration (AMD) by performing the largest metabolome association analysis in AMD to date. In addition, we aimed to determine the effect of AMD-associated genetic variants on metabolite levels, and aimed to investigate associations between the identified metabolites and activity of the complement system, one of the main AMD-associated disease pathways. DESIGN: Case-control assocation analysis of metabolomics data. SUBJECTS: 2,267 AMD cases and 4,266 controls from five European cohorts. METHODS: Metabolomics was performed using a high-throughput H-NMR metabolomics platform, which allows the quantification of 146 metabolite measurements and 79 derivative values. Metabolome-AMD associations were studied using univariate logistic regression analyses. The effect of 52 AMD-associated genetic variants on the identified metabolites was investigated using linear regression. In addition, associations between the identified metabolites and activity of the complement pathway (defined by the C3d/C3 ratio) were investigated using linear regression. MAIN OUTCOME MEASURES: Metabolites associated with AMD RESULTS: We identified 60 metabolites that were significantly associated with AMD, including increased levels of large and extra-large HDL subclasses and decreased levels of VLDL, amino acids and citrate. Out of 52 AMD-associated genetic variants, seven variants were significantly associated with 34 of the identified metabolites. The strongest associations were identified for genetic variants located in or near genes involved in lipid metabolism (ABCA1, CETP, APOE, LIPC) with metabolites belonging to the large and extra-large HDL subclasses. In addition, 57 out of 60 metabolites were significantly associated with complement activation levels, and these associations were independent of AMD status. Increased large and extra-large HDL levels and decreased VLDL and amino acid levels were associated with increased complement activation. CONCLUSIONS: Lipoprotein levels were associated with AMD-associated genetic variants, while decreased essential amino acids may point to nutritional deficiencies in AMD. We observed strong associations between the vast majority of the AMD-associated metabolites and systemic complement activation levels, independent of AMD status. This may indicate biological interactions between the main AMD disease pathways, and suggests that multiple pathways may need to be targeted simultaneously for successful treatment of AMD

Modeling the activation of the alternative complement pathway and its effects on hemolysis in health and disease.

The complement system is a powerful mechanism of innate immunity poised to eliminate foreign cells and pathogens. It is an intricate network of >35 proteins, which, once activated, leads to the tagging of the surface to be eliminated, produces potent chemoattractants to recruit immune cells, and inserts cytotoxic pores into nearby lipid surfaces. Although it can be triggered via different pathways, its net output is largely based on the direct or indirect activation of the alternative pathway. Complement dysregulation or deficiencies may cause severe pathologies, such as paroxysmal nocturnal hemoglobinuria (PNH), where a lack of complement control proteins leads to hemolysis and life-threatening anemia. The complexity of the system poses a challenge for the interpretation of experimental data and the design of effective pharmacological therapies. To address this issue, we developed a mathematical model of the alternative complement pathway building on previous modelling efforts. The model links complement activation to the hemolytic activity of the terminal alternative pathway, providing an accurate description of pathway activity as observed in vitro and in vivo, in health and disease. Through adjustment of the parameters describing experimental conditions, the model was capable of reproducing the results of an array of standard assays used in complement research. To demonstrate its clinical applicability, we compared model predictions with clinical observations of the recovery of hematological biomarkers in PNH patients treated with the complement inhibiting anti-C5 antibody eculizumab. In conclusion, the model can enhance the understanding of complement biology and its role in disease pathogenesis, help identifying promising targets for pharmacological intervention, and predict the outcome of complement-targeting pharmacological interventions