Estudos moleculares da interação entre o Mycobacterium leprae e a célula de Schwann: uma abordagem de expressão gênica global

Submitted by Alessandra Portugal ([email protected]) on 2013-10-01T13:18:46Z No. of bitstreams: 1 Anna Beatriz Robottom Ferreira_Tese.pdf: 9570445 bytes, checksum: 839dd23c42d983f3e4b4e6a6658089cd (MD5)Made available in DSpace on 2013-10-01T13:18:46Z (GMT). No. of bitstreams: 1 Anna Beatriz Robottom Ferreira_Tese.pdf: 9570445 bytes, checksum: 839dd23c42d983f3e4b4e6a6658089cd (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilA hanseníase é uma doença infecciosa crônica causada pelo Mycobacterium leprae, onde tanto fatores do hospedeiro quanto ambientais têm papel no desfecho da doença. A hanseníase é uma das causas predominantes de incapacidade no nervo por infecção e os pacientes exibem altas taxas de morbidade, o que tem grande impacto na saúde pública. Apesar disso, os mecanismos de imunopatogênese induzidos pelo M. leprae são pouco conhecidos, especialmente nos tempos iniciais da infecção neural. Nesse estudo foi realizada uma análise global da expressão gênica por microarranjos numa tentativa de esclarecer a interação entre o M. leprae e seu hospedeiro humano. Células de Schwann primárias humanas infectadas pelo M. leprae apresentaram como diferencialmente expressos a classe de genes induzidos por interferon do tipo I. Desses genes, o OASL foi o gene que apresentou a maior alteração de expressão. Esse aumento de expressão foi confirmado por RT-PCR em tempo real em células de Schwann primárias infectadas pelo M. leprae. Foi demonstrado que a proteína OASL migra para o núcleo frente à infecção pelo M. leprae morto em linhagens de células de Schwann e de macrófagos THP1, onde ela provavelmente atua através da repressão epigenética da transcrição. O estudo de polimorfismos nesse gene envolvendo 521 casos e 498 controles indicou que o alelo A do SNP rs3213545 está associado à resistência a hanseníase. Por fim, carreadores do alelo A do SNP rs3213545 apresentaram menor expressão de RNAm de OASL em biópsias de nervo de indivíduos com neuropatia periférica não hansênica, mas não em pacientes com hanseníase, sugerindo que de fato o OASL parece funcionar como um supressor da ativação da resposta imune protetora para a hanseníase. Assim, a produção diminuída de OASL em estágios iniciais da infecção poderia conferir resistência à hanseníase. Conjuntamente, esses dados sugerem que o aumento da expressão do gene OASL favorece, nas etapas iniciais da infecção pelo M. leprae, a sobrevivência intracelular do bacilo no seu hospedeiro.Leprosy is a chronic infectious disease caused by Mycobacterium leprae, in which both host and environmental factors play a role in disease outcome. Leprosy is the most important cause of infectious nerve incapacity and patients exhibit high rates of morbidity, which impacts public health. Nevertheless, the mechanisms of M. leprae-induced imunopathogenesis are not completely understood, especially in the nerve during the early stages of infection. A global gene expression analysis using microarrays was carried out in an attempt to better understand the host-M. leprae interaction. The class of genes induced by type I IFN was identified as differentially expressed. Of these genes, OASL presented the highest change in expression, which was confirmed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in M. leprae infected primary human Schwann cells. We also showed that OASL protein migrates to the nucleus upon infection with dead M. leprae in both a Schwann cell lineage and THP1 macrophages, where it probably functions as an epigenetic repressor of transcription. A case-control study conducted using single nucleotide polymorphisms (SNPs) in this gene involving 521 cases and 498 controls indicated that the A allele of the rs3213545 SNP is associated with leprosy resistance. Furthermore, carriers of this allele expressed less OASL mRNA in nerve biopsies of patients presenting non-leprous peripheral neuropathy but the same was not observed in patients diagnosed with leprosy, suggesting that OASL seems to function by suppressing the activation of the protective immune response. Thus, an increased production of OASL in the initial stages of the infection by M. leprae may favor intracellular survival of the bacilli within its host

Leishmania (Viannia) braziliensis: Influence of successive in vitro cultivation on the expression of promastigote proteinases

Cysteine proteinases are an important virulence factor in Leishmania parasites. In this study we analyzed the cysteine proteinase expression of infective Leishmania (Viannia) braziliensis promastigotes, examining the expression induced by successive in vitro passages in culture. We observed that this parasite presents a decrease in its virulence over BALB/c macrophages, after successive passages in culture, but still they present proteinase activity, being capable of hydrolyzing the substrate pGlu-Phe-Leu-p Nitroanilide at pH 7.0. This proteinase activity also decreases in the course of the successive passages. Additionally, the decrease in the amount of CPB proteins following successive passages of promastigotes was verified by immunoblotting assays, using an anti-CPB antiserum. Real-time PCR assays were performed to assess the relative cpb expression when compared to a housekeeping gene in promastigote cDNA preparations from the first, fourth and seventh passages. Interestingly, the data indicate a relative increase in cpb gene transcripts as the promastigotes were maintained under in vitro culture: 2.2 times higher for fourth and 2.7 times higher for seventh passages when compared to the first passage. Thus, the information gathered here shows that the expression of cysteine proteinases is modified during in vitro cultivation of L. (V.) braziliensis promastigotes

Parasite Load Induces Progressive Spleen Architecture Breakage and Impairs Cytokine mRNA Expression in <i>Leishmania infantum</i>-Naturally Infected Dogs

<div><p>Canine Visceral Leishmaniasis (CVL) shares many aspects with the human disease and dogs are considered the main urban reservoir of <i>L</i>. <i>infantum</i> in zoonotic VL. Infected dogs develop progressive disease with a large clinical spectrum. A complex balance between the parasite and the genetic/immunological background of the host are decisive for infection evolution and clinical outcome. This study comprised 92 Leishmania infected mongrel dogs of various ages from Mato Grosso, Brazil. Spleen samples were collected for determining parasite load, humoral response, cytokine mRNA expression and histopathology alterations. By real-time PCR for the ssrRNA Leishmania gene, two groups were defined; a low (lowP, n = 46) and a high parasite load groups (highP, n = 42). When comparing these groups, results show variable individual humoral immune response with higher specific IgG production in infected animals but with a notable difference in CVL rapid test optical densities (DPP) between highP and lowP groups. Splenic architecture disruption was characterized by disorganization of white pulp, more evident in animals with high parasitism. All cytokine transcripts in spleen were less expressed in highP than lowP groups with a large heterogeneous variation in response. Individual correlation analysis between cytokine expression and parasite load revealed a negative correlation for both pro-inflammatory cytokines: IFNγ, IL-12, IL-6; and anti-inflammatory cytokines: IL-10 and TGFβ. TNF showed the best negative correlation (r² = 0.231; p<0.001). Herein we describe impairment on mRNA cytokine expression in leishmania infected dogs with high parasite load associated with a structural modification in the splenic lymphoid micro-architecture. We also discuss the possible mechanism responsible for the uncontrolled parasite growth and clinical outcome.</p></div

Quantification of parasite load in spleen samples from dogs naturally infected with <i>Leishmania infantum</i>.

<p>Quantification was carried out using real time PCR with primers specific for a <i>Leishmania</i> sp. DNA sequence of the small subunit ribosomal RNA (ssrRNA). The canine HPRT gene was used in order to normalize initial concentrations of DNA in each sample. (A) Histogram and normal density lines of <i>L</i>. <i>infantum</i> infected animals classified into low (n = 46), red line, or high (n = 42), green line, parasite load by the fitting of an optimum number of mixtures of normal distributions. (B) Minimum and maximum values of parasite per 10⁶ canine cells. Each sample was quantified in duplicate for each target. a,b Statistically significant differences (p < 0.0001, Unpaired T-test).</p

<i>Leishmania infantum</i> naturally infected dogs present increased levels of immunoglobulins.

<p><i>Leishmania</i>-specific total IgG (A and C) and total IgM (B) levels in sera from noninfected dogs (control, n = 15) and from naturally infected dogs with either low (lowP, n = 46) or high (highP, n = 42) parasite load in spleen. Optical density of serum samples were determined by ELISA (A and B) using crude protein derived from <i>L</i>. <i>infantum</i> promastigotes IOC/L3128 (MCAN/BR/2009/BOB-FÍGADO). Reflectance values were determined by the rapid immunocromatographic test (Dual Path Platform, DPP, BioManguinhos). Horizontal bars indicate mean values. The dashed line indicates the cut-off, 0.549 for IgG and 0.645 for IgM. (***) p < 0.001; (**) p < 0.0002 indicate statistically significant differences, Mann Whitney test.</p

Splenic mRNA cytokines are down regulated in <i>Leishmania infantum</i> infected dogs with higher parasite loads.

<p>Ex-vivo analyses of relative mRNA levels for indicated genes in the splenic compartments of mongrel dogs infected with <i>L</i>. <i>infantum</i> and classified as low (lowP) or high (highP) parasite load. Gene expression levels of each tested cytokine were normalized using HPRT and RP32 expression. Error bars indicate the standard error of mean for each group. Significant differences are indicated by, p < 0.05 and **, p < 0.01 (nonparametric T-test with 1,000 permutations).</p

Most animals with high parasite load present clinical signs.

<p>Clinical signs in <i>Leishmania infantum</i> infected animals classified into low (n = 46) or high (n = 42) parasite DNA load measured by real-time PCR for the <i>Leishmania</i> ssrRNA gene in spleen.</p

Morphological changes in the spleen of dogs naturally infected by <i>Leishmania infantum</i>.

<p>(A) Organized splenic white pulp compartments with well-formed follicle; (B) Moderately disorganized splenic white pulp when the white pulp was evident, however, its regions were poorly individualized or indistinct; (C) Disorganized splenic white pulp without compartment distinction; (D, E and F) Intracelullar amastigotes (arrows). HE, Scale bar 20 μm (A, B and C); 10 μm (D, E and F). WP—white pulp; RP—red pulp; BCA—B cell area; PALS—periarterial lymphoid sheat; CA—central artery.</p

Gene Expression Profiling Specifies Chemokine, Mitochondrial and Lipid Metabolism Signatures in Leprosy

<div><p>Herein, we performed microarray experiments in Schwann cells infected with live <i>M. leprae</i> and identified novel differentially expressed genes (DEG) in <i>M. leprae</i> infected cells. Also, we selected candidate genes associated or implicated with leprosy in genetic studies and biological experiments. Forty-seven genes were selected for validation in two independent types of samples by multiplex qPCR. First, an <i>in vitro</i> model using THP-1 cells was infected with live <i>Mycobacterium leprae</i> and <i>M. bovis</i> bacillus Calmette-Guérin (BCG). In a second situation, mRNA obtained from nerve biopsies from patients with leprosy or other peripheral neuropathies was tested. We detected DEGs that discriminate <i>M. bovis</i> BCG from <i>M. leprae</i> infection. Specific signatures of susceptible responses after <i>M. leprae</i> infection when compared to BCG lead to repression of genes, including <i>CCL2</i>, <i>CCL3</i>, <i>IL8</i> and <i>SOD2</i>. The same 47-gene set was screened in nerve biopsies, which corroborated the down-regulation of <i>CCL2</i> and <i>CCL3</i> in leprosy, but also evidenced the down-regulation of genes involved in mitochondrial metabolism, and the up-regulation of genes involved in lipid metabolism and ubiquitination. Finally, a gene expression signature from DEG was identified in patients confirmed of having leprosy. A classification tree was able to ascertain 80% of the cases as leprosy or non-leprous peripheral neuropathy based on the expression of only <i>LDLR</i> and <i>CCL4</i>. A general immune and mitochondrial hypo-responsive state occurs in response to <i>M. leprae</i> infection. Also, the most important genes and pathways have been highlighted providing new tools for early diagnosis and treatment of leprosy.</p></div