Ficolin-1 and ficolin-3 polymorphisms and susceptibility to rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease, which compromises the synovial membrane resulting in chronic inflammation. Ficolins are key proteins of the lectin pathway of complement able to recognize pathogen-associated molecular patterns, apoptotic cells, and cellular debris mediating the clearance by phagocytes. High ficolin-1 and ficolin-3 levels have been observed in RA patients, however, the influence of polymorphisms in the FCN1 gene in RA is not completely established, while no study evaluated FCN3 gene polymorphisms in RA to date. We investigated the influence of FCN1 and FCN3 gene polymorphisms in the susceptibility and clinical presentation of RA. A total of 148 patients with RA and up to 160 controls from Southern Brazil were genotyped by sequence-specific PCR (PCR-SSP) for five FCN1 promoter polymorphisms (rs2989727, rs10120023, rs17039495, rs10117466, and rs10858293) and three FCN3 gene variants (rs532781899, rs28362807, and rs4494157). The FCN1 g.-542GG (rs10120023) genotype and g.-542G allele, were associated with increased susceptibility to RA (p = .025, OR = 1.69 [1.07-2.69]; p = .041, OR = 1.47 [1.02-2.12], respectively) and related to decreased FCN1 gene expression in whole blood (p < .00001), according to gene expression databases. In addition, the FCN1 AAGAG haplotype was more prevalent in rheumatoid factor seronegative in comparison to seropositive patients (p = .006, OR = 0.042 [0.002-0.80]). There was no association of FCN3 polymorphisms with the susceptibility or clinical characteristics of RA. Our results indicate that the FCN1 rs10120023 [g.-542G>A] polymorphism in the promoter region might contribute to RA susceptibility, probably by impacting FCN1 gene expression.

Novel findings on the role of ficolins and colectins in the innate response against Leishmania braziliensis.

Leishmania (Viannia) braziliensis is the main agent of mucocutaneous Leishmaniasis, a neglected tropical disease that affects thousands of people in Brazil. It has been shown that complement plays a critical role at early stages of Leishmania infection and that is involved in the invasion of macrophages by the promastigotes. Ficolins and collectins are soluble pattern recognition and triggering molecules of the lectin complement pathway. We investigated here whether lectin pathway activators ficolin-1, ficolin-2, ficolin-3 and CL-11 bind to live L. braziliensis promastigotes in vitro. Promastigote forms in the stationary growth phase were incubated with normal human serum (NHS) or recombinant ficolins 1, 2 and 3, MBL and CL-11, and protein binding was evaluated by confocal microscopy and flow cytometry. Ficolins 1, 2 and 3, MBL and CL-11 were able to bind to the surface of live promastigotes after incubation with either NHS or recombinant proteins. A partial inhibition by N-acetyl-d-glucosamine characterizing the participation of acetylated groups in the deposition of ficolins and CL-11 to glycoconjugates on the surface of L. braziliensis was observed. These evidences highlight a role for the lectin pathway in the innate response to L. braziliensis.

Ficolin-1 and Ficolin-3 Plasma Levels Are Altered in HIV and HIV/HCV Coinfected Patients From Southern Brazil.

The complement system is a key component of the innate immune system, participating in the surveillance against infectious agents. Once activated by one of the three different pathways, complement mediates cell lysis, opsonization, signalizes pathogens for phagocytosis and induces the adaptive immune response. The lectin pathway is constituted by several soluble and membrane bound proteins, called pattern recognition molecules (PRM), including mannose binding lectin (MBL), Ficolins-1, -2, and -3, and Collectin 11. These PRMs act on complement activation as recognition molecules of pathogen-associated molecular patterns (PAMPs) such as N-acetylated, found in glycoproteins of viral envelopes. In this study, Ficolin-1 and Ficolin-3 plasma levels were evaluated in 178 HIV patients (93 HIV; 85 HIV/HCV) and 85 controls from southern Brazil. Demographic and clinical-laboratory findings were obtained during medical interview and from medical records. All parameters were assessed by logistic regression, adjusted for age, ancestry, and sex. Significantly lower levels of Ficolin-1 were observed in HIV/HCV coinfected when compared to HIV patients (p = 0.005, median = 516 vs. 667 ng/ul, respectively) and to controls (p < 0.0001, 1186 ng/ul). Ficolin-1 levels were lower in males than in females among HIV patients (p = 0.03) and controls (p = 0.0003), but no association of Ficolin-1 levels with AIDS was observed. On the other hand, Ficolin-3 levels were significantly lower in controls when compared to HIV (p < 0.0001, medians 18,240 vs. 44,030 ng/ml, respectively) and HIV/HCV coinfected (p < 0.0001, 40,351 ng/ml) patients. There was no correlation between Ficolin-1 and Ficolin-3 levels and age, HIV viral load or opportunistic infections. However, Ficolin-3 showed a positive correlation with T CD4 cell counts in HIV monoinfected patients (p = 0.007). We provide here the first assessment of Ficolin-1 and-3 levels in HIV and HIV/HCV coinfected patients, which indicates a distinct role for these pattern recognition molecules in both viral infections.

Sickening or Healing the Heart? The Association of Ficolin-1 and Rheumatic Fever.

Rheumatic fever (RF) and its subsequent progression to rheumatic heart disease (RHD) are chronic inflammatory disorders prevalent in children and adolescents in underdeveloped countries, and a contributing factor for high morbidity and mortality rates worldwide. Their primary cause is oropharynx infection by Streptococcus pyogenes, whose acetylated residues are recognized by ficolin-1. This is the only membrane-bound, as well as soluble activator molecule of the complement lectin pathway (LP). Although LP genetic polymorphisms are associated with RF, FCN1 gene's role remains unknown. To understand this role, we haplotyped five FCN1 promoter polymorphisms by sequence-specific amplification in 193 patients (138 with RHD and 55, RF only) and 193 controls, measuring ficolin-1 serum concentrations in 78 patients and 86 controls, using enzyme-linked immunosorbent assay (ELISA). Patients presented lower ficolin-1 serum levels (p < 0.0001), but did not differ according to cardiac commitment. Control's genotype distribution was in the Hardy-Weinberg equilibrium. Four alleles (rs2989727: c.-1981A, rs10120023: c.-542A, rs10117466: c.-144A, and rs10858293: c.33T), all associated with increased FCN1 gene expression in whole blood or adipose subcutaneous tissue (p = 0.000001), were also associated with increased protection against the disease. They occur within the *3C2 haplotype, associated with an increased protection against RF (OR = 0.41, p < 0.0001) and with higher ficolin-1 levels in patient serum (p = 0.03). In addition, major alleles of these same polymorphisms comprehend the most primitive *1 haplotype, associated with increased susceptibility to RF (OR = 1.76, p < 0.0001). Nevertheless, instead of having a clear-cut protective role, the minor c.-1981A and c.-144A alleles were also associated with additive susceptibility to valvar stenosis and mitral insufficiency (OR = 3.75, p = 0.009 and OR = 3.37, p = 0.027, respectively). All associations were independent of age, sex or ethnicity. Thus, minor FCN1 promoter variants may play a protective role against RF, by encouraging bacteria elimination as well as increasing gene expression and protein levels. On the other hand, they may also predispose the patients to RHD symptoms, by probably contributing to chronic inflammation and tissue injury, thus emphasizing the dual importance of ficolin-1 in both conditions.

Ficolin Gene Polymorphisms in Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Systemic lupus erythemathosus (SLE) and rheumatoid arthritis (RA) are complex autoimmune diseases characterized by an immune balance breakdown and by chronic inflammation. Several findings link SLE and RA development with the complement system and ficolin components have emerged as candidates for disease development. Since genetic association studies with ficolin genes in SLE and RA have not yet been conducted in a Brazilian population, the aim of this study was to determine whether polymorphisms of ficolin-1(FCN1) and ficolin-2 (FCN2) genes are associated with SLE and RA susceptibility as well as disease manifestation. Two SNPs within FCN1 (rs2989727 and 1071583) and three in FCN2 (rs17514136, rs3124954, and rs7851696) were studied in 208 SLE and184 RA patients as well as 264 healthy individuals in a Southeast Brazilian population. For SLE patients, the FCN2 rs17514136 SNP was associated with a more severe disease (SLICC) (p = 0.0067). Furthermore, an association between the occurrence of nephritis and the T/T genotype for FCN2 rs3124954 SNP (p = 0.047, OR = 3.17, 95%CI = 1.34-7.5) was observed. No association was observed between the studied polymorphisms and RA development. Thus, our data support involvement of the FCN2 gene in the SLE phenotype.

Studies of the binding of ficolin-2 and ficolin-3 from the complement lectin pathway to Leptospira biflexa, Pasteurella pneumotropica and Diarrheagenic Escherichia coli.

Ficolins recognize pathogen associated molecular patterns and activate the lectin pathway of complement system. However, our knowledge regarding pathogen recognition of human ficolins is still limited. We therefore set out to explore and investigate the possible interactions of the two main serum ficolins, ficolin-2 and ficolin-3 with different Gram-negative bacteria. We used recombinant ficolin molecules and normal human serum, which were detected with anti-ficolin monoclonal antibodies. In addition we investigated the capacity of these pathogens to activate the lectin pathway of complement system. We show for the first time that human ficolin-2 recognizes the nonpathogenic spirochete Leptospira biflexa serovar Patoc, but not the pathogenic Leptospira interrogans serovar Kennewicki strain Fromm. Additionally, human ficolin-2 and ficolin-3 recognize pathogenic Pasteurella pneumotropica, enteropathogenic Escherichia coli (EPEC) serotype O111ab:H2 and enteroaggregative E. coli (EAEC) serogroup O71 but not four enterohemorrhagic E. coli, three EPEC, three EAEC and two nonpathogenic E. coli strains (DH5α and HB101). The lectin pathway was activated by Pasteurella pneumotropica, EPEC O111ab:H2 and EAEC O71 after incubation with C1q depleted human serum. In conclusion, this study provide novel insight in the binding and complement activating capacity of the lectin pathway initiation molecules ficolin-2 and ficolin-3 towards relevant Gram-negative pathogens of pathophysiological relevance.

Trypanosoma cruzi calreticulin inhibits the complement lectin pathway activation by direct interaction with L-Ficolin.

Trypanosoma cruzi, the agent of Chagas' disease, the sixth neglected tropical disease worldwide, infects 10-12 million people in Latin America. Differently from T. cruzi epimastigotes, trypomastigotes are complement-resistant and infective. CRPs, T-DAF, sialic acid and lipases explain at least part of this resistance. In vitro, T. cruzi calreticulin (TcCRT), a chaperone molecule that translocates from the ER to the parasite surface: (a) Inhibits the human classical complement activation, by interacting with C1, (b) As a consequence, an increase in infectivity is evident and, (c) It inhibits angiogenesis and tumor growth. We report here that TcCRT also binds to the L-Ficolin collagenous portion, thus inhibiting approximately between 35 and 64% of the human complement lectin pathway activation, initiated by L-Ficolin, a property not shared by H-Ficolin. While L-Ficolin binds to 60% of trypomastigotes and to 24% of epimastigotes, 50% of the former and 4% of the latter display TcCRT on their surfaces. Altogether, these data indicate that TcCRT is a parasite inhibitory receptor for Ficolins. The resulting evasive activities, together with the TcCRT capacity to inhibit C1, with a concomitant increase in infectivity, may represent T. cruzi strategies to inhibit important arms of the innate immune response.

Association of L-ficolin levels and FCN2 genotypes with chronic Chagas disease.

BACKGROUND: L-ficolin (encoded by FCN2) binds to acetylated sugar moieties of many pathogens, including Trypanosoma cruzi, promoting their phagocytosis and lysis by the complement system. METHODS: We investigated L-ficolin levels in 160 T. cruzi infected patients with chronic Chagas disease and 71 healthy individuals, and FCN2 polymorphisms (-986 G>A, -602 G>A, and -4 A>G in the promoter and A258S in exon 8) in 243 patients, being 88 indeterminate (asymptomatic), 96 with cardiac, 23 with digestive and 33 with cardiodigestive manifestations (two were unspecified) and 305 controls (135 for A258S). RESULTS: Patients presented lower L-ficolin plasma levels than controls (p<0.0001). Among the different groups of cardiac commitment, individuals with moderate forms had higher L-ficolin levels than the severe forms (P = 0.039). Lower L-ficolin levels were found associated with the 258S variant in the patients (P = 0.034). We found less -4A/G heterozygotes in the cardiac patients, than in the controls (OR = 0.56 [95% CI = 0.33-0.94], P = 0.034). Heterozygote -4A/G genotypes with the 258S variant and 258SS homozygotes were nevertheless more frequent among cardiodigestive patients than in controls (OR = 14.1 [95% CI = 3.5-56.8], P = 0.0001) and in indeterminate patients (OR = 3.2 [95% CI = 1.1-9.4], P = 0.037). We also found an association of the allelic frequency of the 258S variant with cardiodigestive Chagas disease compared to controls (OR = 2.24 [95% CI = 1.1-4.5], P = 0.037). Thus, decreased patient levels of L-ficolin reflect not only protein consumption due to the disease process, but also the higher frequency of the 258S variant in patients with cardiodigestive symptoms. CONCLUSION: The very first study on Brazilian cohort associates both L-ficolin plasma levels and FCN2 variants to Chagas disease and subsequent disease progression. The prognostic value of L-ficolin levels and the FCN2*A258S polymorphism should be further evaluated in other settings.

Susceptibility to leprosy is associated with M-ficolin polymorphisms.

PURPOSE: Mycobacterium leprae exploits complement activation and opsonophagocytosis to infect phagocytes. M-ficolin is encoded by the FCN1 gene and initiates the lectin pathway on monocyte surfaces. We investigated FCN1 promoter polymorphisms that could be responsible for the high interindividual variability of M-ficolin levels and for modulating leprosy susceptibility. METHODS: We genotyped rs2989727 (-1981 G > A), rs28909068 (-791 G > A), rs10120023 (-542 G > A), rs17039495 (-399 G > A), rs28909976 (-271IndelT), rs10117466 (-144C > A) and rs10858293 (+33 T > G) in 400 controls and 315 leprosy patients from Southern Brazil, and in 296 Danish healthy individuals with known M-ficolin levels. RESULTS: Ten haplotypes were identified with sequence-specific PCR and/or haplotype-specific sequencing. We found evidence for a protective codominant additive effect of FCN1*-542A-144C with leprosy in Euro-Brazilians (P=0.003, PBf =0.021, OR=0.243 [CI95% =0.083-0.71]), which was independent of age, ethnic group and gender effects (P=0.029). There was a trend for a positive association of the -399A variant in Afro-Brazilians (P=0.022, PBf =0.154, OR=4.151 [CI95% =1.115-15.454], as well as for a negative association of the FCN1*3A haplotype with lepromatous leprosy, compared with less severe forms of the disease (P=0.016, PBf =0.112, OR=0.324 [CI95% =0.123-0.858]). Danish individuals with this haplotype presented M-ficolin levels higher than the population average of circa 1,000 ng/ml, and -542A-144C, which is able to modify the recognition of transcription factors in silico, occurred in individuals with levels under the 25 percentil (P=0.031). CONCLUSIONS: Our data provide the first evidence that FCN1 polymorphisms are associated with leprosy. M-ficolin may represent a novel key to understand the immunopathogenesis of M. leprae infection.

Mannan-binding lectin and ficolin deposition in skin lesions of pemphigus.

Pemphigus is characterized by circulating autoantibodies directed against desmossomal antigens that, once bound to target antigens, induce disruption in the cell-cell adhesion of the epidermis and mucosal epithelium, leading to blister formation. Evidence has indicated a role for complement in the physiopathology of pemphigus, with complement deposition in intercellular spaces of skin and mucous membrane lesions. Mannan-binding lectin (MBL) and Ficolin-2 are recognition proteins of innate immunity, which by binding to specific molecular patterns on pathogens surfaces trigger the activation of complement, leading to phagocytosis and lyses of target cells and inflammation. In this study we report for the first time the deposition of MBL and ficolins in pemphigus lesions. Eight biopsies of skin lesions of pemphigus vulgaris were studied for in situ deposition of IgG and the complement components MBL, Ficolin 1, Ficolin-2, C1q, C3 and membrane attack complex C5b-9. All biopsies presented deposition of IgG and C3 in the intercellular spaces (ICS) of epidermis. MBL deposition was found in the ICS and basal membrane zone (BMZ) of all specimens, whereas C5b-9 showed deposition only in the ICS, with irregular distribution. Ficolin-2 were positive in 50% (4/8) of biopsies showing deposition in the BMZ. On the other hand, ficolin-1 and C1q were negative in all specimens. Our study suggest that MBL and to a lesser extend Ficolin-2 may bind to altered intercellular structures in the skin and lead to the activation of complement in situ, contributing to tissue damage in pemphigus.

Involvement of lectin pathway activation in the complement killing of Giardia intestinalis.

Giardia intestinalis (syn. G. lamblia, G. duodenalis) is a flagellated unicellular eukaryotic microorganism that commonly causes diarrheal disease throughout the world. In humans, the clinical effects of Giardia infection range from the asymptomatic carrier state to a severe malabsorption syndrome possibly due to different virulence of the Giardia strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection. The question about how G. intestinalis is controlled by the organism remains unanswered. Here, we investigated the role of the complement system and in particular, the lectin pathway during Giardia infections. We present the first evidence that G. intestinalis activate the complement lectin pathway and in doing so participate in eradication of the parasite. We detected rapid binding of mannan-binding lectin, H-ficolin and L-ficolin to the surface of G. intestinalis trophozoites and normal human serum depleted of these molecules failed to kill the parasites. Our finding provides insight into the role of lectin pathway in the control of G. intestinalis and about the nature of surface components of parasite.

Inefficient complement system clearance of Trypanosoma cruzi metacyclic trypomastigotes enables resistant strains to invade eukaryotic cells.

The complement system is the main arm of the vertebrate innate immune system against pathogen infection. For the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, subverting the complement system and invading the host cells is crucial to succeed in infection. However, little attention has focused on whether the complement system can effectively control T. cruzi infection. To address this question, we decided to analyse: 1) which complement pathways are activated by T. cruzi using strains isolated from different hosts, 2) the capacity of these strains to resist the complement-mediated killing at nearly physiological conditions, and 3) whether the complement system could limit or control T. cruzi invasion of eukaryotic cells. The complement activating molecules C1q, C3, mannan-binding lectin and ficolins bound to all strains analysed; however, C3b and C4b deposition assays revealed that T. cruzi activates mainly the lectin and alternative complement pathways in non-immune human serum. Strikingly, we detected that metacyclic trypomastigotes of some T. cruzi strains were highly susceptible to complement-mediated killing in non-immune serum, while other strains were resistant. Furthermore, the rate of parasite invasion in eukaryotic cells was decreased by non-immune serum. Altogether, these results establish that the complement system recognizes T. cruzi metacyclic trypomastigotes, resulting in killing of susceptible strains. The complement system, therefore, acts as a physiological barrier which resistant strains have to evade for successful host infection.

Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi.

The complement system is the first line of defence against pathogen infection and can be activated by the classic, alternative and lectin pathways. Trypanosoma cruzi, the causative agent of Chagas disease, has to evade complement system killing and invade the host cells to progress in infection. T. cruzi infectious stages resist complement-mediated killing by expressing surface receptors, which dissociate or prevent C3 convertase formation. Here, we present the first evidence that T. cruzi activates the complement lectin pathway. We detected rapid binding of mannan-binding lectin, H-ficolin, and L-ficolin to the surface of T. cruzi, and found that serum depleted of these molecules failed to kill parasites. Furthermore, lectin pathway activation by T. cruzi required the MBL-associated serine protease 2 (MASP2) activity resulting in C2 factor cleavage. In addition, we demonstrate that the infectious stage of T. cruzi inhibits the lectin pathway activation and complement killing expressing the complement C2 receptor inhibitor trispanning (CRIT) protein. Transgenic parasites overexpressing CRIT were highly resistant to complement-mediated killing. CRIT-derived peptides inhibited both C2 binding to the surface of T. cruzi and parasite killing. Biochemical studies revealed that the CRIT extracellular domain 1 inhibits MASP2 cleavage of C2 factor and thereby impairs C3 convertase formation. Our findings establish that the complement lectin pathway recognizes T. cruzi and provide molecular insights into how the infectious stage inhibits this activation to resist complement system killing.

Natural inhibitors of snake venom hemorrhagic metalloproteinases.

Metalloproteinases play an important role in the poisoning process by snake venoms. They evoke systemic injury, by degrading or activating host blood factors, and local damage by acting on endothelial cell surface proteins. Plasma and/or muscle of venomous and non-venomous snakes as well as of some special mammals possess metalloproteinase inhibitors that behave as soluble acceptors available for a rapid inhibition of the deleterious action of these enzymes. The purpose of this review is to describe the state of the art on natural immunity against snake venom metalloproteinases and to overview this field by discussing the available structural and biological properties of the inhibitors protein/gene families.