Adding MASP1 to the lectin pathway-Leprosy association puzzle: Hints from gene polymorphisms and protein levels.

BACKGROUND: Deposition of complement factors on Mycobacterium leprae may enhance phagocytosis. Such deposition may occur through the lectin pathway of complement. Three proteins of the lectin pathway are produced from the gene MASP1: Mannan-binding lectin-associated serine protease 1 (MASP-1) and MASP-3 and mannan-binding lectin-associated protein of 44 kDa (MAp44). Despite their obvious importance, the roles played by these proteins have never been investigated in leprosy disease. METHODOLOGY: We haplotyped five MASP1 polymorphisms by multiplex sequence-specific PCR (intronic rs7609662*G>A and rs13064994*C>T, exon 12 3'-untranslated rs72549262*C>G, rs1109452*C>T and rs850314*G>A) and measured MASP-1, MASP-3 and MAp44 serum levels in 196 leprosy patients (60%, lepromatous) and 193 controls. PRINCIPAL FINDINGS: Lower MASP-3 and MAp44 levels were observed in patients, compared with controls (P = 0.0002 and P<0.0001, respectively) and in lepromatous, compared with non-lepromatous patients (P = 0.008 and P = 0.002, respectively). Higher MASP-3 levels were present in controls carrying variants/haplotypes associated with leprosy resistance (rs13064994*T, rs1109452_rs850314*CG within GT_CCG and rs850314*A: OR = 0.5-0.6, Pcorr = 0.01-0.04). Controls with rs1109452*T, included in susceptibility haplotypes (GT_GTG/GT_CTG: OR = 2.0, Pcorr = 0.03), had higher MASP-1 and lower MASP-3 levels (P≤0.009). Those with GC_CCG, presented increasing susceptibility (OR = 1.7, Pcorr = 0.006) and higher MAp44 levels (P = 0.015). MASP-3 expression decreased in patients, compared with controls carrying rs1109452_rs850314*CA or CG (P≤0.02), which may rely on exon 12 CpG methylation and/or miR-2861/miR-3181 mRNA binding. CONCLUSION: Polymorphisms regulating MASP-3/MAp44 availability in serum modulate leprosy susceptibility, underlining the importance of lectin pathway regulation against pathogens that exploit phagocytosis to parasitize host macrophages.

Hepatitis B Virus Infection Among Leprosy Patients: A Case for Polymorphisms Compromising Activation of the Lectin Pathway and Complement Receptors.

Thousands of leprosy patients not only suffer from physical deformities, but also either have or have had hepatitis B virus (HBV) coinfection. Polymorphisms of the complement system modulate susceptibility to leprosy, but genetic susceptibility to past or present HBV infection is unknown. We used sequencing and multiplex sequence-specific PCR to genotype 72 polymorphisms of seven genes (MBL2, FCN1, FCN2, FCN3, MASP1, MASP2, C3) encoding components of the lectin pathway, and two genes encoding complement receptors (CR1, VSIG4) in 190 patients, of which 74 were positive for HBsAg and/or anti-HBc (HBV+, 93.2% with a resolved infection) and 116 lepromatous patients, and 408 HBV-blood donors. In addition, we tested for levels of proteins of the lectin pathway. We found no difference between serum concentrations of mannan-binding lectin (MBL), MBL-associated serine proteins (MASP-1, MASP-2, MASP-3, MAp44), ficolin-3 (FCN-3), soluble complement receptor 1 (sCR1) and MBL mediated C4 activation, measured by ELISA or TRIFMA in up to 167 HBV+ and HBV- patients. Haplotypes lowering protein levels or encoding dysfunctional proteins increased susceptibility to HBV infection: MBL2*LYQC (OR = 3.4, p = 0.02), MASP1*AC_CC (OR = 4.0, p = 0.015) and MASP2*1C2-l (OR = 5.4, p = 0.03). Conversely, FCN1*3C2 haplotype, associated with higher gene expression, was protective (OR = 0.56, P = 0.033). Other haplotypes associated with HBV susceptibility were: MASP2*2B1-i (OR = 19.25, P = 0.003), CR1*3A (OR = 2.65, P = 0.011) and VSIG4*TGGRCG (OR = 12.55, P = 0.014). Some polymorphisms in ficolin genes associated with lower protein levels increased susceptibility to leprosy/HBV infection: FCN*1 (OR = 1.66, P = 0.029), FCN2*GGGCAC (OR = 6.73, P = 0.008), and FCN3*del_del_C (OR = 12.54, P = 0.037), and to lepromatous disease/HBV infection: FCN2*TA (OR = 2.5, P = 0.009), whereas FCN2*MAG was associated with increased FCN-2 expression and resistance against coinfection (OR = 0.29, P = 0.026). These associations were independent of demographic factors and did not increase susceptibility to leprosy per se, except MASP2*1C2-l. Associations for FCN2, FCN3, MASP1, MASP2, and VSIG4 variants were also independent of each other. In conclusion, polymorphisms compromising activation of the lectin pathway of complement increase susceptibility to HBV infection, with ficolin polymorphisms playing a major role in modulating the susceptibility among leprosy patients.

Complement receptor 1 (CR1, CD35) association with susceptibility to leprosy.

BACKGROUND: Pathophysiological mechanisms are still incompletely understood for leprosy, an urgent public health issue in Brazil. Complement receptor 1 (CR1) binds complement fragments C3b/C4b deposited on mycobacteria, mediating its entrance in macrophages. We investigated CR1 polymorphisms, gene expression and soluble CR1 levels in a case-control study with Brazilian leprosy patients, aiming to understand the role of this receptor in differential susceptibility to the disease. METHODOLOGY: Nine polymorphisms were haplotyped by multiplex PCR-SSP in 213 leprosy patients (47% multibacillary) and 297 controls. mRNA levels were measured by qPCR and sCR1 by ELISA, in up to 80 samples. PRINCIPAL FINDINGS: Individuals with the most common recombinant haplotype harboring rs3849266*T in intron 21 and rs3737002*T in exon 26 (encoding p.1408Met of the York Yka+ antigen), presented twice higher susceptibility to leprosy (OR = 2.43, p = 0.017). Paucibacillary patients with these variants presented lower sCR1 levels, thus reducing the anti-inflammatory response (p = 0.040 and p = 0.046, respectively). Furthermore, the most ancient haplotype increased susceptibility to the multibacillary clinical form (OR = 3.04, p = 0.01) and presented the intronic rs12034383*G allele, which was associated with higher gene expression (p = 0.043), probably increasing internalization of the parasite. Furthermore, there was an inverse correlation between the levels of sCR1 and mannose-binding lectin (initiator molecule of the lectin pathway of complement, recognized by CR1) (R = -0.52, p = 0.007). CONCLUSIONS: The results lead us to suggest a regulatory role for CR1 polymorphisms on mRNA and sCR1 levels, with haplotype-specific effects increasing susceptibility to leprosy, probably by enhancing parasite phagocytosis and inflammation.

Evaluation of rheumatoid factor and anti-citrullinated peptide antibodies in relation to rheumatological manifestations in patients with leprosy from Southern Brazil.

BACKGROUND: Leprosy patients may present several osteoarticular complaints, which require further evaluation of inflammatory diseases, such as rheumatoid arthritis (RA). Therefore, an adequate clinical assessment in addition to testing for rheumatoid factors (RF) and anticyclic citrullinated peptide antibodies (anti-CCP), can be useful in order to establish the correct diagnosis. METHOD: In this study, the relation of RF and anti-CCP with rheumatological manifestations was evaluated in 97 leprosy patients from Southern Brazil. The results were compared to RA patients and healthy controls from the same geographical area and ethnic background. RESULTS: Neuropathy was observed in 71.1% and arthritis in 35.1% of the leprosy patients. A high frequency of RF positivity was observed among the leprosy patients (41.2%, 40/97), with RF immunoglobulin A (IgA) significantly associated with arthritis (OR = 7.9, 95% CI = 1.5-40.6 P = 0.008). Anti-CCP was observed in 9.3% (9/97) of the patients, with anti-CCP2 being the most frequent subtype. Only 4.1% (4/97) of the patients were RF and anti-CCP concomitantly positive. RF IgM showed a significant association with leprosy when compared to healthy controls (P < 0.0001) whereas for anti-CCP2 no significant results were observed (P = 0.0585). However, both biomarkers showed a strong association with RA when compared to leprosy in patients from the same geographical area and ethnic background (anti-CCP2 OR = 38.6; 95% CI = 16.49-90.26; P < 0.0001 and RF IgM OR = 4.51; 95% CI = 2.62-7.77; P < 0.0001). CONCLUSION: Due to the similarity of some rheumatological manifestations in leprosy with other inflammatory diseases, such as RA, clinical and laboratorial evaluation of affected patients must be carefully assessed in order to achieve proper diagnosis and treatment.

Leprosy association with low MASP-2 levels generated by MASP2 haplotypes and polymorphisms flanking MAp19 exon 5.

BACKGROUND: The gene MASP2 (mannan-binding lectin (MBL)-associated serine protease 2) encodes two proteins, MASP-2 and MAp19 (MBL-associated protein of 19 kDa), bound in plasma to MBL and ficolins. The binding of MBL/MASP-2 and ficolin/MASP-2 complexes to microorganisms activates the lectin pathway of complement and may increase the ingestion of intracellular pathogens such as Mycobacterium leprae. METHODS: We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 219 Brazilian leprosy patients (131 lepromatous, 29 borderline, 21 tuberculoid, 14 undetermined, 24 unspecified), 405 healthy Brazilians and 291 Danish blood donors with previously determined MASP-2 and MAp19 levels. We also evaluated MASP-2 levels in further 46 leprosy patients and 69 Brazilian controls. RESULTS: Two polymorphisms flanking exon 5 of MASP2 were associated with a dominant effect on high MASP-2 levels and an additive effect on low MAp19 levels. Patients presented lower MASP-2 levels (P = 0.0012) than controls. The frequency of the p.126L variant, associated with low MASP-2 levels (below 200 ng/mL), was higher in the patients (P = 0.0002, OR = 4.92), as was the frequency of genotypes with p.126L (P = 0.00006, OR = 5.96). The *1C2-l [AG] haplotype, which harbors p.126L and the deficiency-causing p.439H variant, has a dominant effect on the susceptibility to the disease (P = 0.007, OR = 4.15). Genotypes composed of the *2B1-i and/or *2B2A-i haplotypes, both associated with intermediate MASP-2 levels (200-600 ng/mL), were found to be protective against the disease (P = 0.0014, OR = 0.6). Low MASP-2 levels (P = 0.022), as well as corresponding genotypes with *1C2-l and/or *2A2-l but without *1B1-h or *1B2-h, were more frequent in the lepromatous than in other patients (P = 0.008, OR = 8.8). CONCLUSIONS: In contrast with MBL, low MASP-2 levels increase the susceptibility to leprosy in general and to lepromatous leprosy in particular. MASP2 genotypes and MASP-2 levels might thus be of prognostic value for leprosy progression.

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.

The association between mannan-binding lectin gene polymorphism and clinical leprosy: new insight into an old paradigm.

BACKGROUND: Mannan-binding lectin (MBL), a soluble protein of innate immunity, is known to play a role in pathogen recognition and clearance. For more than a decade, it has been proposed that MBL deficiency may be protective against intracellular pathogens, such as Mycobacterium leprae. METHODS: The polymorphisms at the promoter and exon 1 regions of the MBL2 gene were assessed by polymerase chain reaction and sequencing performed on 264 patients with leprosy and 214 matched healthy control subjects from southern Brazil. RESULTS. The distribution of MBL2-gene polymorphisms in patients was significantly different from that in controls, with a decreased frequency of haplotypes/genotypes associated with low expression of circulating MBL in lepromatous patients when compared with tuberculoid patients (odds ratio [OR] for haplotypes, 0.56 [95% confidence interval {CI}, 0.33-0.93] [P=.020]; OR for genotypes, 0.31 [95% CI, 0.13-0.71] [P=.004]). The LYPA haplotype was associated with susceptibility to leprosy per se (OR, 2.25 [95% CI, 1.31-3.88] [P=.003]) and to progression to the lepromatous (OR, 2.2 [95% CI, 1.21-4.05] [P=.008]) and borderline (OR, 2.98 [95% CI, 1.29-6.87] [P=.008]) forms of the disease. CONCLUSIONS: These results suggest that MBL2-gene polymorphisms play a role in susceptibility to leprosy per se and in the clinical progression of the disease.