Intestinal dwelling parasites live in close association with the complex microbiota that inhabit our intestinal tracts. The intestinal helminth, Trichuris muris, depends on these bacteria for egg hatching and successful establishment of infection within the epithelium of the caecum and colon. Infection causes significant alterations to the host intestinal microbiota, including a decrease in bacterial diversity and shifts in proportions of certain bacterial groups. This is accompanied by a decrease in Foxp3+ regulatory T cells and changes to the metabolic potential of the host microbiota, consequently impacting host health. However, the factor(s) driving these changes and the existence and role of its own intestinal microbiota is unknown. Infection of C57BL/6 and immunodeficient SCID mice with a high dose (~ 200 embryonated eggs) and a low dose (~ 20 embryonated eggs) of T. muris was used to determine the impact of worm burden and the adaptive immune system on the host intestinal microbiota, in comparison to naïve controls. Microbiota analysis was performed by 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) and Illumina sequencing. This revealed that infection-induced microbiota changes were dose dependent and high level infection caused an increase in the Bacteroidaceae and Enterobacteriaceae families, independently of the host adaptive immune system. Development of a surface sterilisation protocol enabled the internal T. muris microbiota to be analysed by 16S rRNA gene DGGE and fluorescence in situ hybridisation (FISH). The resulting data indicated that T. muris requires its own diverse intestinal microbiota that is derived from, but distinct to, that of its host. A core microbiota is selected and maintained by the parasite regardless of the surrounding host microbiota. The parasite microbiota is important for its fitness, shown in vitro using an antibiotic motility assay and in vivo using germ free (GF) mice. Furthermore, infection with T. muris causes a significant reduction in caecal butyrate concentrations and consequently a decrease in the expression of butyrate transporters in caecal tissue. Interestingly, the T. muris microbiota is able to produce the short-chain fatty acid (SCFA) butyrate, which the parasite is unable to make itself yet secretes into its local environment. Together these strategies promote the long term survival of T. muris within the intestinal niche, adding a new level of complexity to the interaction between the pathogen, the host and their respective microbiotas that underpins successful chronic nematode infection.

Made available in DSpace on 2016-12-23T13:56:03Z (GMT). No. of bitstreams: 1 HELMINtiASE INTESTINAL.pdf: 1507224 bytes, checksum: 6132aca0f1aadf166d4e2b773c982a74 (MD5) Previous issue date: 2009-04-27 / Mycobacterium tuberculosis (MTB) é um exemplo clássico de patógeno para o qual a resposta protetora depende da imunidade celular do tipo Th1, que é caracterizada pela presença de linfócitos T CD4+ produtores de IFN-g. Essa citocina ativa mecanismos microbicidas no macrófago infectado, levando à eliminação do bacilo. Evidências sugerem que a progressão para a tuberculose esteja relacionada à presença de mecanismos imunossupressores mediados por citocinas e por células T reguladoras. Acredita-se que a presença de helmintíase intestinal possa prejudicar o desenvolvimento de uma resposta adaptativa capaz de conter ou eliminar o MTB, tornando assim o indivíduo susceptível ao adoecimento. Para aquilatar a influência da infecção por helmintos intestinais na resposta celular durante a tuberculose pulmonar, neste trabalho, foram avaliados os perfis quantitativo e fenotípico de populações celulares de sangue periférico e o padrão de citocinas em culturas de sangue total estimuladas com antígenos de MTB, em pacientes portadores de tuberculose pulmonar apresentando ou não helmintíase intestinal no momento do diagnóstico e durante a terapia antituberculose. Para isso, foram arrolados 53 pacientes com diagnóstico recente de tuberculose pulmonar. Desses, 26% eram portadores de pelo menos uma espécie de helminto intestinal. Pacientes com tuberculose pulmonar apresentaram uma redução significativa nos números de linfócitos T CD8+, células NK e NKT. Os indivíduos com helmintíase intestinal associada à tuberculose apresentaram uma maior freqüência de células T reguladoras, com ambos os fenótipos CD4+CD25HIGH e CD4+CD25HIGHFoxp3+. Além disso, os resultados sugerem que a presença de infecção por helmintos intestinais tenha induzido um estado de hipoergia em pacientes portadores de tuberculose pulmonar, uma vez que esses pacientes apresentaram concentrações menores das citocinas IL-2, TNF-a, IL-4, IL-5 e IL-10 nos sobrenadantes de culturas em relação às concentrações encontradas no grupo TB e no grupo controle. Portanto, os resultados desse trabalho sugerem que a presença de infecção por helmintos intestinais tenha um impacto negativo na resposta imunitária à tuberculose em pacientes portadores de tuberculose pulmonar. / The protection against Mycobacterium tuberculosis (MTB) depends on a cellmediated Th1 type-immune response. This response is characterized by IFN-g production by CD4+ T cells, which activates macrophages to enhance microbicidal mechanisms leading to the bacillus eradication. Factors related to tuberculosis resistance or susceptibility are not completely understood. There are evidences suggesting that the progress to active disease is related to immune downregulation caused by suppressors cytokines and regulatory T cells. It is believed that the association with helminth infection can disturb the protective immune response that should contain or eliminate MTB. Here, we investigated the role of intestinal helminth infection on M. tuberculosis specific immune response during active pulmonar tuberculosis in patients with associated tuberculosis and intestinal helminth infection at the time of diagnosis and during tuberculosis therapy. Quantitative and phenotypic analyses of peripheral blood cells populations were performed and the MTBstimulated whole blood culture cytokines production was evaluated. Fifty-three patients with newly diagnosed tuberculosis were enrolled for this study. Twenty-six percent of these patients were infected with at least one intestinal helminth (TB + HELM patients). Patients with pulmonary tuberculosis presented a significant reduction in the numbers of TCD8+, NK and NKT cells. Patients with both intestinal helminth infection and tuberculosis presented higher frequency of regulatory T cells, of both phenotype CD4+CD25HIGH and CD4+CD25HIGHFoxp3+, as compared to TB group, to HELM group, and to control group. In addition, the results suggest a hipoergy status in TB + HELM patients because the production of the cytokines IL-2, TNF-a, IL-4, IL-5 and IL-10 decreased in whole blood culture of these patients as compared to both TB patients and healthy controls. The data from this study indicated that the associated intestinal helminth infection has a negative impact on immunity to tuberculosis in patients with tuberculosis.