Tuberculosis (TB) is an important cause of morbidity and mortality worldwide. The high incidence is still found in Taiwan. There is strong evidence that host genes influence individual susceptibility to tuberculosis. Young children, like immunocompromised patients, once infected are at increased risk for TB disease and progression to extrapulmonary disease. Thus far, to identify the genes responsible for the variation in the human susceptibility/resistance to TB has remained elusive. Mannose-binding lectin (MBL) activates the complement system in an antibody-independent manner, enhances complement-mediated phagocytosis, and plays an important role in innate immunity in the regulation of inflammatory cytokine release by monocytes. It is one of the molecules that have been suggested to have a link to human susceptibility or protection against infection. According to some studies (mostly conducted in adult populations) , low levels of MBL associated with variant alleles at the promoter and exon 1 regions of MBL protect against tuberculosis. Other investigators instead claim that protection against the disease is associated with high levels of MBL. In this study we aimed to investigate the relationships between the susceptibility to TB and MBL gene polymorphisms in children with cervical mycobacterial lymphadenitis infected by M. tuberculosis.139 case patients with cervical mycobacterial lymphadenitis and 102 unrelated healthy control subjects were tested by real-time PCR for polymorphisms at the promoter and the exon 1 regions of the MBL gene. Diagnosis of mycobacterial lymphadenitis infected by M. tuberculosis, based on findings of pathological examination of the lymph nodes, was confirmed by acid-fast stain and TB PCR.The frequency of A allele was significantly higher in TB+ patients compared with TB- controls (82.7% vs 72.6%; odds ratio 1.813; p=0.007). The frequency of high-producer MBL2 genotypes (A/A) was higher in TB+ patients than in TB- subjects (70.5% vs 45.1%, odds ratio 2.91, p<0.001), while patients carried the B alleles (A/B and B/B) that have decreased levels of MBL was inversely associated with mycobacterial infectivity (29.5% vs 54.9%; odds ratio 2.910; p<0.001). The frequencies of MBL promoter -550 genotypes also revealed a significant difference between TB+ and TB- groups (p = 0.046), but in contrast, with significantly higher frequency of L/L genotype (of low MBL level) in TB+ patients (34.5% vs 21.6%; odds ratio 1.918; p=0.029). The frequencies of MBL promoter -221 genotypes (X and Y) was similar in TB+ and TB- groups.This study supports the conclusion that MBL can protect or predispose the host to tuberculosis, depending on the host¡¦s haplotype pair.

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

Background.  Sepsis is a syndrome associated with high mortality rates, substantial morbidity and high costs of care. The incidents of sepsis is reported to be high and controversy exists whether gender affect severity or outcome. Little is known about factors determining suscepti­bility for developing the syndrome and severity of the syndrome once developed. Early detection and adequate antibiotic administration are the mainstay of treatment and means to identify patients with particular high risk of adverse outcome are desirable. There are data to suggest that the course of sepsis and outcome from the syndrome may be influenced by inherited differences in the immunological response among humans Aims: Paper I: Assess incidence and outcome for ICU-treated sepsis patients in this region; Paper II: Assess if there are gender differences related to characteristics, aspects of treatment or out­come in sepsis in this region. Paper III: Assess the association of baseline levels of leptin and adiponectin and future sepsis event, and association of these adipokines in the cute phase and sepsis severity and outcome. Paper IV: Assess association of baseline levels of mannose-binding lectin (MBL) and future sepsis event, and MBL levels in the acute phase in relation to sepsis severity and outcome. Results. Paper I:  Overall ICU mortality rate was 25%, while the ICU mortality for patients with septic shock was 58% in this retrospective single university hospital cohort analysis. Cardio­vascular disease and diabetes were the most prevalent comorbidities among patients who died during hospital stay.  Paper II:  No gender-related differences in mortality or length of stay was found in this prospective single center observational study. Differences in aspects of treatment were related to differences in site of infection. Men had more often infections in skin and skin-structures, whereas women more often had abdominal infections. Early organ dysfunction asses­sed as SOFA score at admission was a stronger predictor for hospital mortality for women than for men. The discrepancy was related to the SOFA coagulation-sub score.  Paper III: In this nes­ted case-referent study hyperleptinemia at baseline predicted a first-ever sepsis event, even after adjustment for BMI and other cardiovascular risk factors. Hyperleptinemia in the acute sepsis phase was associated with reduced risk of in-hospital death in men, but associated with increased risk of in-hospital   death in women.  Paper IV: In the same matched cohort as in Paper III high baseline levels of MBL predicted a first ever sepsis event. High MBL levels in the acute phase or an increase from baseline to the acute phase associate with increased in-hospital death in women but not in men. Low MBL levels was not identified as a risk for acute sepsis or in-hospital death. Conclusions. Mortality from severe sepsis is high, equally affecting men and women. There are differences in patient characteristics and inflammatory markers, which associate with in-hospital mortality differentially in men and women. Aspects of gender should be mandatory, and genetic analysis are desired in future sepsis research.

A leishmaniose visceral (LV) ou calazar é uma doença endêmica, crônica, grave e de alta letalidade se não tratada. Os estudos apontam a proteína Lectina Ligante de Manose (MBL), codificada pelo gene MBL2, como uma peça-chave na imunidade inata, dada a sua função no reconhecimento microbiano, na eliminação, inflamação e morte celular. Neste trabalho realizamos um estudo do tipo caso-controle que teve como objetivo investigar a associação entre variantes no gene MBL2 e a suscetibilidade à LV em indivíduos residentes em áreas endêmicas da Ilha de São Luís-MA. A amostra foi constituída por 322 indivíduos, sendo 161 casos com LV, não aparentados, de ambos os sexos, residentes em áreas endêmicas da doença na Ilha de São Luís e 161 controles saudáveis, não infectados e não aparentados da mesma região. A identificação dos casos de LV se deu por meio do contato constante com os principais hospitais e ambulatórios de referência para a doença na cidade. Também foram feitas buscas de pacientes com LV em ambiente domiciliar, a partir de registros da FUNASA-MA. A análise molecular consistiu na genotipagem de 6 variantes localizadas na região promotora [posições -550 (C>G), -221(G>C), +4(C>T)] e codificadora [códons 52 (C>T), 54 (G>A) e 57 (G>A)] do gene MBL2, através da reação em cadeia da polimerase e sequenciamento automático. A dosagem da proteína MBL no soro foi realizada pelo teste de ELISA. Verificamos que os fenótipos MBL dependem do conjunto de alelos presentes no gene MBL2, sendo nítido o efeito que as variantes defectivas causam nos níveis da proteína. Não encontramos diferença significativa entre casos e controles em relação à distribuição dos genótipos MBL2 e dos níveis séricos de MBL. As frequências alélicas das variantes exônicas na amostra total mostram que o alelo A é o mais comum (74,8%) e que os alelos defectivos (B, C e D) se encontram principalmente em heterozigose (36,6%), o que reforça a ideia de que alelos MBL2 defectivos são mantidos na população por conferirem vantagem seletiva aos heterozigotos. Em relação aos 3 principais polimorfismos existentes na região promotora, verificamos ser a variante -221G (Y) a mais frequente (88%) seguida de +4C (P) (73%) e de -550C (L) (67%). Identificamos oito haplótipos em MBL2 num total de 644 cromossomos avaliados, em 30 combinações diferentes, sendo HYPA e LYQA os mais frequentes e HYPD e HYPB os mais raros. Todos os portadores de combinações de haplótipos homozigotos para alelos defectivos apresentaram níveis séricos de MBL indetectáveis. Os genótipos LYQA/LYQA e HYPA/HYPA apresentaram as maiores concentrações médias de MBL no soro. A combinação entre SNPs no éxon 1 e na região promotora do gene MBL2 resulta em grande variação nas concentrações de MBL em indivíduos saudáveis. Consideramos que o conjunto de dados gerados é uma contribuição valiosa que poderá ser expandida para outros cenários. / Visceral leishmaniasis (VL), also known as kalazar, is an endemic, chronic, severe and highly lethal disease when not treated. Studies have shown that the protein Mannose-biding lectin (MBL), encoded by the gene MBL2, is the major player in innate immune system due its role in microbial recognition, elimination and inflammation as well as in the cell death. In the current work, we conducted a case-control study which aimed to investigate the association between variants in the gene MBL2 and the susceptibility to VL in individuals living in endemic areas of the São Luís - MA. 322 individuals participated in this study. Of these, 161 were VL cases being unrelated individuals of both sexes, and inhabitants from endemic areas of the disease in São Luís. The other 161 individuals were uninfected healthy controls, being unrelated and from the same region. The identification of VL cases occurred by visiting reference hospitals and clinics in the city. VL patients were identified in the household environment through the records of FUNASA-MA. Molecular analysis consisted in genotyping six variants located in the promoter region [positions -550 (C> G), -221 (G> C), +4 (C> T)] and coding region [codons 52 (C> T), 54 (G> A) and 57 (G> A)] of the MBL2 gene by polymerase chain reaction and automated DNA sequencing. The concentrations of MBL protein in the serum was performed by ELISA. We found that MBL phenotypes depend on the number of alleles present in the gene MBL2, being clear the consequence of the defective variants in the protein levels. There was no significant difference between cases and controls regarding the distribution of MBL2 genotypes and MBL serum levels. The allele frequencies of exon variants in the overall sample showed that the A allele is the most common (74.8%) and that the defective alleles (B, C and D) are mainly heterozygous (36.6%). This highlights the idea that defective MBL2 alleles are maintained in the population to confer selective advantage to heterozygotes. Concerning the three main existing polymorphisms in the promoter region, we noticed that the variant-221G (Y) is more frequent (88%) followed by +4 C (P) (73%) and 550C-(L) (67%) variants. We identified eight haplotypes in MBL2 in a total of 644 chromosomes evaluated in 30 different combinations, being the HYPA and LYQA the most frequent haplotypes and HYPD and HYPB the rarest ones. All carriers with combinations of homozygous haplotypes for defective alleles had undetectable serum levels of MBL. Genotypes LYQA / LYQA and HYPA / HYPA had the highest mean concentrations of MBL in the serum. Combination between SNPs in exon 1 and in the promoter region of the gene MBL2 results in a great variation of MBL concentrations in healthy individuals. We consider that the data set that was generated is a valuable contribution that can be expanded to others cenarios.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-101).

Η λεκτίνη ΜΒL συμμετέχει στην φυσική ανοσία, αφ΄ενός σαν ενεργοποιητής του συστήματος του συμπληρώματος και αφ΄ετέρου σαν οψωνίνη που προσδένεται σε συγκεκριμένες υδατανθρακικές δομές των μικροοργανισμών. Οι λεκτίνες τύπου C, είναι ασβέστιο εξαρτώμενες και φέρουν περιοχή δέσμευσης σε υδατάνθρακες (CRD). Προκειμενου να διασαφηνιστεί περαιτέρω η εξελικτική πορεία της λεκτινικής οδού του συμπληρώματος, απομονώθηκαν, κλωνοποιήθηκαν και χαρακτηρίστηκαν δύο ισομορφές της λεκτίνης ΜBL, οι ΜΒL1 και MBL2, στην ιριδίζουσα πέστροφα (Oncorhynchus mykiss). Οι συναγώμενες αμινοξικές αλληλουχίες των MBL1 και ΜΒL2 είναι 185 και 186 αμινοξέα, αντίστοιχα, παρουσιάζουν μεταξύ τους ταυτοσημία της τάξης του 83% ενώ εμφανίζουν το υψηλότερο σκορ ταυτοσημίας, 43% και 41% αντίστοιχα με τους τελεόστεους ιχθύες Αtlantic salmon και zebrafish. Το σκορ ταυτοσημίας των πρωτεϊνών της πέστροφας με τις αντίστοιχες πρωτεΐνες των πτηνών και των θηλαστικών κυμαίνεται μεταξύ 28 και 32%. Επιπλέον,οι περιοχές CRD των πρωτεϊνών αυτών χαρακτηρίζονται από την ύπαρξη του δομικού μοτίβου EPN που σχετίζεται με την εξειδίκευση της δέσμευσης ως προς την μαννόζη. Τα αποτελέσματα έδειξαν επίσης πως τα μόρια MBL1 και ΜBL2, εκφράζονται, σε επίπεδο mRNA, στο ήπαρ και στον σπλήνα της πέστροφας, αντίστοιχα. / Mannose-binding lectin(MBL) participates in the innate immune system as an activator of the complement system and as an opsonin after binding to certain carbohydrate structures on microorganisms. C-type lectins are all Ca2+ -dependent and they share a tightly folded carbohydrate recognition domain (CRD). In order to stydy the evolution of the complement lectin pathway, we report the isolation and characterization of two mannose-binding lectin isoforms, MBL1 and MBL2, in rainbow trout (Oncorhynchus mykiss). The deduced amino acid sequences of trout MBL1 and MBL2 are 185 and 186aa, respectively, presenting 83% identity to each other. These proteins exhibit the highest identity score 43 and 41% with the atlantic salmon and zebrafish counterparts. The identity with the bird and mammalian MBLs ranges from 28 to 32%. The trout MBL molecules contain in the CRD domain the EPN motif of mannose-binding C-type lectins, which is important for mannose specificity. Trout MBL1 and MBL2 are expressed exclusively in liver and spleen, respectively.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Mannose binding lectin (MBL) belongs to the collectin family of soluble pattern recognition molecules that elicit diverse biologic activities. Via multiple carbohydrate-recognition domains (CRD), MBL binds to mannose and N-acetyl-glucosamine oligosaccharides present on the surface of bacteria, fungi and yeast. Following pathogen recognition, MBL activates the complement system via MBL associated serine proteases in a manner independent of antibody and C1 complex. Deficiency in function and level of MBL is found in 25% of otherwise apparently healthy individuals, representing the most prevalent innate immune deficiency. MBL deficiency is a risk factor for the development of infections in humans and mice. The role of MBL as a modulator of infection is complex. MBL deficiency may influence proinflammatory cytokine production, expression of leukocyte adhesion molecules, or vascular damage, during the course of infection. Given that dendritic cells (DC) are antigen presenting cells (APC) with potent capacity to respond to microbial stimulation, I hypothesized that MBL deficiency may be reflected in DC functions associated with microbial stimulation. Initially, I investigated the association of MBL with human immune cells and demonstrated that in both MBL-Sufficient (MBL-S) and MBL-Deficient (MBL-D) individuals, MBL was particularly associated with monocytes. RT-PCR analysis demonstrated MBL was not transcribed by monocytes or other immune cells investigated (T, B, and NK cells, CD11c+DC, immature monocyte derived DC [MoDC], LPS matured MoDC, and granulocytes), suggesting MBL association with the cell surface may be via an adapter or co-receptor. Magnetically separated monocytes but not MoDC bound exogenous purified human plasma MBL (hpMBL). Addition of hpMBL (5 -15 µg/mL) did not induce MoDC activation, and MBL added together with lipopolysaccharide (LPS) did not induce MoDC activation above the level induced by LPS only. In the second part of this study, I used the particulate MBL ligand zymosan (Zy) as a pathogenic stimulus in a whole blood model to gain a greater understanding of the consequences of MBL deficiency. I compared surface phenotype, inflammatory cytokine production and antigen presenting capacity of blood myeloid (M)DC of MBL-D and MBL-S individuals following stimulation with Zy and MBL opsonised Zy (MBL-Zy). Blood MDC in MBL-D individuals, unlike their counterpart in MBL-S individuals, displayed unique functional characteristics, including higher production of proinflammatory cytokines IL-6 and TNF-, but poor capacity for allo-T cell effector cell induction. It appeared that stimulation with MBL-Zy reduced elevated production of IL-6 but not TNF- by blood MDC in MBL-D individuals. In the third part, expression microarray analysis was utilised to provide broad information on the genes and potential signalling pathways involved in the MDC responses in MBL-D and MBL-S individuals following stimulation with Zy and MBL-Zy. MBL-S individuals demonstrated greater capacity to induce T cell and NK cell signalling pathways than MBL-D individuals. Further, MBL acted as a regulator of important inflammatory molecules, namely T-cell receptor zeta (CD247), IFN-γ and perforin 1. The data presented in this study provides novel information on blood MDC function in MBL-S and MBL-D individuals in response to pathogen stimulation, and provided insight into mechanisms involved in the increased frequency of infection observed in MBL-D individuals.

A leishmaniose visceral (LV) ou calazar é uma doença endêmica, crônica, grave e de alta letalidade se não tratada. Os estudos apontam a proteína Lectina Ligante de Manose (MBL), codificada pelo gene MBL2, como uma peça-chave na imunidade inata, dada a sua função no reconhecimento microbiano, na eliminação, inflamação e morte celular. Neste trabalho realizamos um estudo do tipo caso-controle que teve como objetivo investigar a associação entre variantes no gene MBL2 e a suscetibilidade à LV em indivíduos residentes em áreas endêmicas da Ilha de São Luís-MA. A amostra foi constituída por 322 indivíduos, sendo 161 casos com LV, não aparentados, de ambos os sexos, residentes em áreas endêmicas da doença na Ilha de São Luís e 161 controles saudáveis, não infectados e não aparentados da mesma região. A identificação dos casos de LV se deu por meio do contato constante com os principais hospitais e ambulatórios de referência para a doença na cidade. Também foram feitas buscas de pacientes com LV em ambiente domiciliar, a partir de registros da FUNASA-MA. A análise molecular consistiu na genotipagem de 6 variantes localizadas na região promotora [posições -550 (C>G), -221(G>C), +4(C>T)] e codificadora [códons 52 (C>T), 54 (G>A) e 57 (G>A)] do gene MBL2, através da reação em cadeia da polimerase e sequenciamento automático. A dosagem da proteína MBL no soro foi realizada pelo teste de ELISA. Verificamos que os fenótipos MBL dependem do conjunto de alelos presentes no gene MBL2, sendo nítido o efeito que as variantes defectivas causam nos níveis da proteína. Não encontramos diferença significativa entre casos e controles em relação à distribuição dos genótipos MBL2 e dos níveis séricos de MBL. As frequências alélicas das variantes exônicas na amostra total mostram que o alelo A é o mais comum (74,8%) e que os alelos defectivos (B, C e D) se encontram principalmente em heterozigose (36,6%), o que reforça a ideia de que alelos MBL2 defectivos são mantidos na população por conferirem vantagem seletiva aos heterozigotos. Em relação aos 3 principais polimorfismos existentes na região promotora, verificamos ser a variante -221G (Y) a mais frequente (88%) seguida de +4C (P) (73%) e de -550C (L) (67%). Identificamos oito haplótipos em MBL2 num total de 644 cromossomos avaliados, em 30 combinações diferentes, sendo HYPA e LYQA os mais frequentes e HYPD e HYPB os mais raros. Todos os portadores de combinações de haplótipos homozigotos para alelos defectivos apresentaram níveis séricos de MBL indetectáveis. Os genótipos LYQA/LYQA e HYPA/HYPA apresentaram as maiores concentrações médias de MBL no soro. A combinação entre SNPs no éxon 1 e na região promotora do gene MBL2 resulta em grande variação nas concentrações de MBL em indivíduos saudáveis. Consideramos que o conjunto de dados gerados é uma contribuição valiosa que poderá ser expandida para outros cenários. / Visceral leishmaniasis (VL), also known as kalazar, is an endemic, chronic, severe and highly lethal disease when not treated. Studies have shown that the protein Mannose-biding lectin (MBL), encoded by the gene MBL2, is the major player in innate immune system due its role in microbial recognition, elimination and inflammation as well as in the cell death. In the current work, we conducted a case-control study which aimed to investigate the association between variants in the gene MBL2 and the susceptibility to VL in individuals living in endemic areas of the São Luís - MA. 322 individuals participated in this study. Of these, 161 were VL cases being unrelated individuals of both sexes, and inhabitants from endemic areas of the disease in São Luís. The other 161 individuals were uninfected healthy controls, being unrelated and from the same region. The identification of VL cases occurred by visiting reference hospitals and clinics in the city. VL patients were identified in the household environment through the records of FUNASA-MA. Molecular analysis consisted in genotyping six variants located in the promoter region [positions -550 (C> G), -221 (G> C), +4 (C> T)] and coding region [codons 52 (C> T), 54 (G> A) and 57 (G> A)] of the MBL2 gene by polymerase chain reaction and automated DNA sequencing. The concentrations of MBL protein in the serum was performed by ELISA. We found that MBL phenotypes depend on the number of alleles present in the gene MBL2, being clear the consequence of the defective variants in the protein levels. There was no significant difference between cases and controls regarding the distribution of MBL2 genotypes and MBL serum levels. The allele frequencies of exon variants in the overall sample showed that the A allele is the most common (74.8%) and that the defective alleles (B, C and D) are mainly heterozygous (36.6%). This highlights the idea that defective MBL2 alleles are maintained in the population to confer selective advantage to heterozygotes. Concerning the three main existing polymorphisms in the promoter region, we noticed that the variant-221G (Y) is more frequent (88%) followed by +4 C (P) (73%) and 550C-(L) (67%) variants. We identified eight haplotypes in MBL2 in a total of 644 chromosomes evaluated in 30 different combinations, being the HYPA and LYQA the most frequent haplotypes and HYPD and HYPB the rarest ones. All carriers with combinations of homozygous haplotypes for defective alleles had undetectable serum levels of MBL. Genotypes LYQA / LYQA and HYPA / HYPA had the highest mean concentrations of MBL in the serum. Combination between SNPs in exon 1 and in the promoter region of the gene MBL2 results in a great variation of MBL concentrations in healthy individuals. We consider that the data set that was generated is a valuable contribution that can be expanded to others cenarios.

La maladie de Crohn (MC) est une maladie inflammatoire chronique de l’intestin qui peut être expliquée par une dysbiose et une dérégulation de la réponse immunitaire. La mannose-binding lectin (MBL), récepteur lectinique et les Toll-like récepteurs jouent un rôle crucial dans la défense contre les pathogènes et dans le développement de la réponse inflammatoire. Ils peuvent reconnaitre de nombreux micro-organismes dont Candida albicans. Cette levure commensale est un immunogène des anticorps anti-Saccharomyces cerevisiae (ASCA), anticorps utilisés dans le diagnostic de la MC. Une association entre un déficit en MBL et des taux élevés d’ASCA a été montrée chez les patients atteints de MC. Ce déficit en MBL est fréquemment associé à un phénotype sévère de la maladie. Le récepteur TLR2, associé à TLR1 ou à TLR6, est également impliqué dans la reconnaissance de C. albicans et le maintien de la barrière intestinale. L’objectif de ce projet a été d’étudier le rôle de la MBL et des récepteurs TLR2/TLR1/TLR6 dans l’homéostasie intestinale et l’élimination de C. albicans du tube digestif. Pour cela, nous avons exploré, les effets de la MBL et des TLR sur la colonisation intestinale à C. albicans et l’inflammation intestinale. Par ailleurs, nous avons déterminé, chez l’homme, l’influence des polymorphismes du gène MBL2 sur la modulation de l’activité et des taux de MBL au cours de la MC.A l’aide d’un modèle murin, nous avons mis en évidence l’expression de MBL-A et de MBL-C par les cellules épithéliales intestinales et leur implication dans l’homéostasie intestinale. Un déficit en MBL favorise la colonisation par C. albicans et une dissémination de la levure, en présence d’une colite chimio-induite. Les récepteurs TLR1 et TLR2 participent également dans la défense contre la colonisation par C. albicans, sans pour autant que leurs déficits n’entrainent de dissémination de la levure. A l’inverse, le récepteur TLR6 favorise la colonisation par C. albicans. La MBL et les récepteurs TLR1/TLR2/TLR6 régulent aussi l’expression des cytokines pro-inflammatoires impliquées dans les réponses Th1 et Th17. Ces résultats expérimentaux ont incité à envisager la poursuite de notre travail en approfondissant l’étude clinique sur des variations quantitatives et qualitatives de la MBL chez les patients atteints de MC et leur lien éventuel avec la persistance de l’inflammation intestinale. L’étude sur la cohorte de patients MC a montré que le gène MBL2 était associé à un déficit quantitatif en MBL et un déficit qualitatif du complexe MBL-MASP chez les sujets sains et les patients. Ce polymorphisme est également associé à des taux élevés d’ASCA chez les patients MC (p<0.05) de la maladie et il est fréquemment associé aux formes sévères de la maladie. De plus, le variant rs2066844 du gène NOD2, variant associé à la MC, est significativement corrélé à une diminution de l’activité fonctionnelle du complexe MBL-MASP, sans diminution quantitative des taux de MBL.Ces données montrent, pour la première fois, la production intestinale de MBL, qui est modulée par la colonisation par C. albicans. Elles confirment le rôle de la MBL et des récepteurs TLR dans l’homéostasie intestinale et dans la défense contre C. albicans. De plus, l’étude clinique sur la cohorte de patients MC révèle un défaut d’activité fonctionnelle du complexe MBL-MASP chez les patients MC ayant des polymorphismes des gènes MBL2 et NOD2, pouvant mener à un phénotype sévère de la maladie. Ces études expérimentales et cliniques apportent des nouvelles données sur le lien entre les récepteurs de l’immunité innée et la maladie de Crohn ainsi que la colonisation/infection fongique et ouvrent des nouvelles perspectives sur le lien entre la persistance de la colonisation fongique et le déficit quantitatif/qualitatif de la MBL chez les patients MC. / Crohn’s disease (CD) is an inflammatory bowel disease characterized by a dysregulation of the inflammatory response caused by dysbiosis and immune system disorders. Mannose Binding Lectin (MBL) and Toll like receptors (TLR) are involved in recognition of microorganisms and inflammatory response. They can recognize different patterns on the surface of the pathogens including Candida albicans. This pathogenic yeast is an immunogen for anti-Saccharomyces cerevisiae antibody (ASCA), diagnostic marker of CD. An association between MBL-deficiency and ASCA is observed in CD, and this MBL deficiency is frequently associated with a severe clinical phenotype of CD. In addition to MBL, TLR2, which forms heterodimers with either TLR1 or TLR6, have a major role in the innate immune defense against C. albicans and promotes intestinal homeostasis. In this project, we studied the role of MBL, TLR1, TLR2, and TLR6 in intestinal homeostasis and elimination of C. albicans in the intestinal tract. In the first part this study; we assessed the effect of either MBL or TLR deficiencies on C. albicans colonization and intestinal inflammation in a murine model. In the second part of this study, we assessed the role of MBL polymorphisms in the modulation of MBL level and activity in CD patients.In murin model, we showed that MBL is locally produced by the epithelial cells in response to C. albicans sensing and to intestinal inflammation and this lectin is required for the intestinal homeostasis. MBL-deficient mice had a higher level of colonization than wild-type mice. DSS-induced colitis promoted a high C. albicans colonization and dissemination to the kidneys and lungs of MBL-deficient mice. MBL-deficient mice exhibited reduced expression of Il-1β and IL-6 and elevated expression of IL-17, IL-23, dectin-1 and TLR-4. In terms of mice deficient in TLRs, DSS treatment and C. albicans oral challenge induced greater weight loss, worse clinical signs of inflammation, higher histopathologic scores, and increased mortality rates in TLR1-/- and TLR2-/- mice when compared to TLR6-/- and wild-type mice. Cytokine expression (TNF, IL-1β, IL-10, and IL-17A) was significantly increased in TLR1-/- and TLR2-/- mice, while they were decreased in TLR6-/- mice. In addition to the experimental studies, we observed in the clinical cohort of CD patients that MBL2 variant rs5030737 (codon 52) was associated with a low level of MBL that leads to impaired MBL-MASP functional activities in both CD patients and healthy subjects. Furthermore, this variant was also associated with a higher level of ASCA in CD patients (p<0.05). Increased ASCA levels were found in CD patients with stricture formation and penetrating disease complications, 42% and 21% respectively. Besides, we observed that NOD2 variant rs2066844, associated with susceptibility to CD, is significantly correlated with the impairment of the functional activity of MBL-MASP complex.Overall, this study emphasizes the role of MBL and TLR in intestinal homeostasis and host defense against C. albicans and shows for the first time that MBL could be produced locally in the intestinal epithelial cells in response to C. albicans sensing. In terms of the clinical study, we observed that CD patients with a severe clinical phenotype have an impairment in MBL-MASP functional activity, and that this defect is associated with MBL2 and NOD2 polymorphisms These experimental studies contribute to understand the link between innate immunity receptors, Crohn’s disease and fungal colonization/infection. Finally, this study leads to new objectives which are to study the link between intestinal microbiota and MBL variation in Crohn’s disease patients.