Marcia Holsbach Beltrame

MBL-associated serine proteases (MASPs) and infectious diseases.

Abstract The lectin pathway of the complement system has a pivotal role in the defense against infectious organisms. After binding of mannan-binding lectin (MBL), ficolins or collectin 11 to carbohydrates or acetylated residues on pathogen surfaces, dimers of MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2) activate a proteolytic cascade, which culminates in the formation of the membrane attack complex and pathogen lysis. Alternative splicing of the pre-mRNA encoding MASP-1 results in two other products, MASP-3 and MAp44, which regulate activation of the cascade. A similar mechanism allows the gene encoding MASP-2 to produce the truncated MAp19 protein. Polymorphisms in MASP1 and MASP2 genes are associated with protein serum levels and functional activity. Since the first report of a MASP deficiency in 2003, deficiencies in lectin pathway proteins have been associated with recurrent infections and several polymorphisms were associated with the susceptibility or protection to infectious diseases. In this review, we summarize the findings on the role of MASP polymorphisms and serum levels in bacterial, viral and protozoan infectious diseases.

The Lectin Pathway of Complement and Rheumatic Heart Disease

The innate immune system is the first line of host defense against infection and is comprised of humoral and cellular mechanisms that recognize potential pathogens within minutes or hours of entry. The effector components of innate immunity include epithelial barriers, phagocytes, and natural killer cells, as well as cytokines and the complement system. Complement plays an important role in the immediate response against microorganisms, including Streptococcus sp. The lectin pathway is one of three pathways by which the complement system can be activated. This pathway is initiated by the binding of mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and Ficolin-3) to microbial surface oligosaccharides and acetylated residues, respectively. Upon binding to target molecules, MBL, CL-K1, and ficolins form complexes with MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which cleave C4 and C2 forming the C3 convertase (C4b2a). Subsequent activation of complement cascade leads to opsonization, phagocytosis, and lysis of target microorganisms through the formation of the membrane-attack complex. In addition, activation of complement may induce several inflammatory effects, such as expression of adhesion molecules, chemotaxis and activation of leukocytes, release of reactive oxygen species, and secretion of cytokines and chemokines. In this chapter, we review the general aspects of the structure, function, and genetic polymorphism of lectin-pathway components and discuss most recent understanding on the role of the lectin pathway in the predisposition and clinical progression of Rheumatic Fever.

Polymorphisms in the 2q33 and 3q21 chromosome regions including T-cell coreceptor and ligand genes may influence susceptibility to pemphigus foliaceus

Signaling through antigen presenting cells is required to activate T lymphocytes. The antigen-specific signal is given by interaction of the T-cell receptor (TCR) with the major histocompatibility complex (MHC)/peptide complex. Also essential for activation is the interaction of the coreceptor CD28 with ligands of the B7 family (CD80 and CD86) on antigen presenting cells. Coreceptor CTLA-4 is a negative regulator and binds the same B7 isoforms, contributing to immunologic homeostasis and peripheral tolerance. CD28 and CTLA4 are homologous and closely linked genes in 2q33, as are CD80 and CD86 in the 3q21 chromosomal region. Pemphigus foliaceus (PF) is a multifactorial autoimmune blistering disease characterized by keratinocyte detachment and by autoantibodies against desmoglein 1, a desmosomal protein. To investigate the involvement of CD28, CTLA4, CD80, and CD86 genes in PF pathogenesis, 18 polymorphisms in 2q33 and 3q21 chromosomal regions were analyzed in 269 patients and 395 controls subdivided according to predominant ancestry in Euro-Brazilian and Afro-Brazilian individuals. Associations were found with CD86 1057G>A, CTLA4-1722T>C, CTLA4-318C>T, CTLA4(AT)n, CD28(CAA)n, and D2S72(CA)n. There is no evidence of gene-gene interactions. We conclude that polymorphisms in the 2q33 and 3q21 chromosomal regions may influence PF disease susceptibility, most likely affecting CTLA4 and possibly inducible T-cell costimulator, (ICOS) expression, and also CD86 function.

Association of MASP2 polymorphisms and protein levels with rheumatic fever and rheumatic heart disease.

MASP-2 is a key protein of the lectin pathway of complement system. Several MASP2 polymorphisms were associated with MASP-2 serum levels or functional activity. Here we investigated a possible association between MASP2 polymorphisms and MASP-2 serum levels with the susceptibility to rheumatic fever (RF) and rheumatic heart disease (RHD). We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 145 patients with history of RF from south Brazil (103 with RHD and 42 without cardiac lesion [RFo]) and 342 healthy controls. MASP-2 levels were determined by ELISA. The low MASP-2 producing p.377A and p.439H variants were negatively associated with RF (P=0.02, OR=0.36) and RHD (P=0.01, OR=0.25). In contrast, haplotypes that share the intron 9 - exon 12 g.1961795C, p.371D, p.377V and p.439R polymorphisms increased the susceptibility to RHD (P=0.02, OR=4.9). MASP-2 levels were associated with MASP2 haplotypes and were lower in patients (P<0.0001), which may reflect protein consumption due to complement activation. MASP2 gene polymorphisms and protein levels seem to play an important role in the development of RF and establishment of RHD.

Differential ability to resist to complement lysis and invade host cells mediated by MBL in R4 and 860 strains of Trypanosoma cruzi

To produce an infection Trypanosoma cruzi must evade lysis by the complement system. During early stages of infection, the lectin pathway plays an important role in host defense and can be activated by binding of mannan‐binding lectin (MBL) to carbohydrates on the surface of pathogens. We hypothesized that MBL has a dual role during parasite‐host cell interaction as lectin complement pathway activator and as binding molecule to invade the host cell. We used two polarized strains of T. cruzi, R4 (susceptible) and 860 (resistant) strains, to investigate the role of MBL in complement‐mediated lysis. Interestingly R4, but not 860 metacyclic strain, markedly increases the invasion of host cells, suggesting that MBL drives the invasion process while the parasite deactivates the Lectin complement pathway.

Association of a new FCN3 haplotype with high ficolin-3 levels in leprosy

Leprosy is a chronic inflammatory disease caused by Mycobacterium leprae that mainly affects the skin and peripheral nervous system, leading to a high disability rate and social stigma. Previous studies have shown a contribution of genes encoding products of the lectin pathway of complement in the modulation of the susceptibility to leprosy; however, the ficolin-3/FCN3 gene impact on leprosy is currently unknown. The aim of the present study was to investigate if FCN3 polymorphisms (rs532781899: g.1637delC, rs28362807: g.3524_3532insTATTTGGCC and rs4494157: g.4473C>A) and ficolin-3 serum levels play a role in the susceptibility to leprosy. We genotyped up to 190 leprosy patients (being 114 (60%) lepromatous), and up to 245 controls with sequence-specific PCR. We also measured protein levels using ELISA in 61 leprosy and 73 controls. FCN3 polymorphisms were not associated with disease, but ficolin-3 levels were higher in patients with FCN3 *2B1 (CinsA) haplotype (p = 0.032). Median concentration of ficolin-3 was higher in leprosy per se (26034 ng/mL, p = 0.005) and lepromatous patients (28295 ng/mL, p = 0.016) than controls (18231 ng/mL). In addition, high ficolin-3 levels (>33362 ng/mL) were more common in leprosy per se (34.4%) and in lepromatous patients (35.5%) than controls (19.2%; p = 0.045 and p = 0.047, respectively). Our results lead us to suggest that polymorphisms in the FCN3 gene cooperate to increase ficolin-3 concentration and that it might contribute to leprosy susceptibility by favoring M. leprae infection.

Polymorphisms of the promoter and exon 3 of the receptor for advanced glycation end products (RAGE) in Euro- and Afro-Brazilians

The receptor for advanced glycation end products (RAGE or AGER), a member of the immunoglobulin superfamily, is involved in pathologies such as atherosclerosis and diabetes. Over 50 SNPs were reported for RAGE, among which were the promoter region polymorphisms −429T>C (rs1800625), −374T>A (rs1800624) and a 63‐bp deletion (−407 to −345 bp), all related to increased RAGE expression. Additionally, in the exon 3, a putative site of binding ligands, the missense variation G82S (rs2070600) was associated with skin disorders in patients with diabetes. We have determined allele, genotype and haplotype frequencies of RAGE polymorphisms −429T>C, −374T>A, 63‐bp deletion and G82S in Euro‐Brazilians (n = 108) and Afro‐Brazilians (n = 91), characterized according to the predominant ancestry of the individuals. The allele frequencies for Euro‐ and Afro‐Brazilians were as follows: −429C, 12.5% vs. 12.1% (P = 0.90); −374A, 31.5% vs. 26.2% (P = 0.25); 63del, 0.0% vs. 3.8% (P = 0.004); and 82S, 1.9% vs. 0.6% (P = 0.24). Absolute linkage disequilibrium was found between the promoter polymorphisms −429T>C and −374T>A plus the 63‐bp deletion (D′=1.000; P < 0.0001). The haplotype frequencies differed (P = 0.003) between Euro‐ and Afro‐Brazilians. Our results showed that the frequencies of the 63‐bp deletion were higher in Afro‐Brazilians, while the other analysed polymorphisms were similarly distributed in the studied populations. The −374T>A plus 63‐bp deletion polymorphism captures more than 80% of the haplotypic variation in the studied population.

A dual role for Mannan-binding lectin-associated serine protease 2 (MASP-2) in HIV infection.

BACKGROUND Mannan-binding lectin (MBL) - associated serine protease 2 (MASP-2) co-activates the lectin pathway of complement in response to several viral infections. The quality of this response partly depends on MASP2 gene polymorphisms, which modulate MASP-2 function and serum levels. In this study we investigated a possible role of MASP2 polymorphisms, MASP-2 serum levels and MBL-mediated complement activation in the susceptibility to HIV/AIDS and HBV/HCV coinfection. METHODS A total of 178 HIV patients, 89 (50%) coinfected with HBV/HCV, 51.7% female, average age 40 (12-73) years, and 385 controls were evaluated. MASP-2 levels and MBL-driven complement activation were evaluated by enzyme-linked immunosorbent assay and 11 MASP2 polymorphisms from the promoter to the last exon were haplotyped using multiplex sequence-specific PCR. RESULTS Genotype distribution was in Hardy-Weinberg equilibrium and differed between HIV+ patients and controls (P=0.030), irrespective of HBV or HCV coinfection. The p.126L variant, which was associated with MASP-2 levels <200ng/mL (OR=5.0 [95%CI=1.3-19.2] P=0.019), increased the susceptibility to HIV infection (OR=5.67 [95%CI=1.75-18.33], P=0.004) and to HIV+HBV+ status (OR=6.44 [95%CI=1.69-24.53, P=0.006). A similar association occurred with the ancient haplotype harboring this variant, AGCDV (OR=2.35 [95%CI=1.31-4.23], P=0.004). On the other hand, p.126L in addition to other variants associated with low MASP-2 levels-p.120G, p.377A and p.439H, presented a protective effect against AIDS (OR=0.25 [95%CI=0.08-0.80], P=0.020), independently of age, sex, hepatic function and viral load. MASP-2 serum levels were lower in HIV+ and HIV+HBV+ patients than in controls (P=0.0004). Among patients, MASP-2 levels were higher in patients with opportunistic diseases (P=0.001) and AIDS (P=0.004). MASP-2 levels correlated positively with MBL/MASP2-mediated C4 deposition (r=0.29, P=0.0002) and negatively with CD4+ cell counts (r=-0.21, P=0.018), being related to decreased CD4+ cell counts (OR=5.8 [95%CI=1.23-27.5, P=0.026). CONCLUSIONS Genetically determined MASP-2 levels seem to have a two-edge effect in HIV and probably HCV/HBV coinfection, whereas low levels increase the susceptibility to infection, but on the other side protects against AIDS.

CD80 and CD86 polymorphisms in populations of various ancestries: 5 new CD80 promoter alleles

CD80 and CD86 are closely linked genes on chromosome 3 that code for glycoproteins of the immunoglobulin superfamily, expressed on the surface of antigen-presenting cells. These costimulatory molecules play essential roles for stimulation and inhibition of T cells through binding to CD28 and CTLA-4 receptors. In this study, CD80 promoter and CD86 exon 8 polymorphisms were analyzed to investigate the genetic diversity and microevolution of the 2 genes. We genotyped 1,124 individuals, including Brazilians of predominantly European, mixed African and European, and Japanese ancestry, 5 Amerindian populations, and an African sample. All variants were observed in Africans, which suggests their origin in Africa before the human migrations out of that continent. Five new CD80 promoter alleles were identified and confirmed by cloning and sequencing, and promoter 2 is most likely the ancestral allele. Nucleotide -79 is monomorphic in 4 Amerindian populations, where the presence of the -79 G allele is probably the result of gene flow from non-Amerindians.

Seroprevalence of HCV markers among HIV infected patients from Curitiba and metropolitan region

OBJECTIVE to determine the prevalence and epidemiological factors associated with hepatitis (HCV) coinfection in human immunodeficiency virus (HIV) patients from Curitiba and the metropolitan region. METHODS a study with 303 HIV+ patients, mean age 41.2 years (18-73); 50.5% men, followed at the Hospital de Clínicas, Universidade Federal do Paraná, between April 2008 and March 2009. Clinical and epidemiological data were obtained through questionnaires and retrospective analysis of medical records. Anti-HCV antibodies were detected by chemiluminescence immunoassay. RESULTS a total of 12.9% of HIV+ patients were positive for anti-HCV antibodies, 64.1% were men and 35.9% women, with mean age of 44.5 years (24-66). The frequency of HCV among men was 16.7% and among women 9.1% (p=0.06). HCV prevalence was associated to HIV infection when compared to the general population (p<10-6, OR=100.4; 95CI=13.7-734.9). The parenteral route of transmission was the most frequent among coinfected patients (46.1%), and the sexual transmission among HIV+/HCV- (71.8%) (p=0.02, OR=0.2; 95CI=0.1-0.7). The frequency of intravenous drug users was higher among the coinfected patients (61.5%) compared to the non coinfected (12.6%) (p<10-6, OR=11.1; 95CI=4.5-27.7). CONCLUSION the prevalence of coinfection with HCV in HIV+ patients is 12.9%, 88 times higher than in the general population in Curitiba. The most frequent route of transmission in the coinfected patients is parenteral, but the sexual route is also representative (34.6%).