Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cellular Biology
Mary E. Wilson
Leishmania are obligate intracellular protozoan parasites that are inoculated into human skin while a sand fly vector takes a blood meal with the resulting disease coined leishmaniasis. The twenty plus species of Leishmania known to cause human disease are found throughout tropical and subtropical regions of the world. Leishmaniasis affects at least eighty-eight countries with three hundred and fifty million people at risk for infection, resulting in an estimated seventy thousand deaths annually. Different species of Leishmania have developed distinct methods for host defense evasion, leading to a wide spectrum of pathologies within humans.
Prior studies of macrophage infections with Leishmania have shown global changes in macrophage mRNA expression. We hypothesized miRNAs are important modifiers of mRNA changes during Leishmania infection. Analysis of miRNA expression patterns revealed that changes were detected primarily during macrophage infection with the low virulent logarithmically growing promastigotes. Profiling studies of mRNA and miRNA changes upon infection with promastigotes in logarithmic growth revealed a decrease in miR-200b and increase in miR-744 levels whereas infections with the highly virulent metacyclic promastigotes revealed a decrease in miR-708 levels. Furthermore, microarray studies revealed differences in macrophage mRNA levels between macrophages infected with the low virulent promastigotes verses the highly virulent promastigotes. Correlative studies between miRNA and mRNA changes suggested some of Leishmania induced changes in mRNA levels may be modified by miRNAs.
The importance of Toll-like receptors (TLR) in detection of microbial products has been well-documented. Leishmania infection is known to initiate signaling through TLRs 2, 3, 4 and 9, of which TLRs 2, 4 and 9 signal through the adaptor molecule MyD88. We found that miR-200b, a microRNA decreased by infection of macrophages with the low virulent Leishmania promastigotes, regulates signaling through the TLR4 pathway by targeting and repressing MyD88 transcript levels. Furthermore, we have shown that MyD88 repression results in the decreased expression of the downstream effector molecules IL-6, CXCL9 and TNFΑ upon challenge with a TLR4 ligand. The suppression of miR-200b during Leishmania infection could serve to up-regulate inflammatory responses induced through TLR4 and other MyD88 dependent TLRs. This may be responsible, in part, for the decreased virulence of logarithmically growing compared to metacyclic promastigotes. Furthermore, low levels of inflammation may promote parasite survival by promoting the influx of inflammatory phagocytic cells to the site of infection in which the highly virulent parasites can survive.
Microarray studies revealed a remarkable increase in expression of metallothionein (MT) transcripts in macrophages infected with low virulent promastigotes but not in macrophages infected with the highly virulent promastigotes. To explore a possible mechanistic role for metallothioneins in leishmaniasis, we used knock-out mice for MT-1 genes. Bone-marrow derived macrophages from MT-1 knock-out mice (MT-KO) generated higher levels of reactive oxygen species upon incubation with Leishmania promastigotes. Consistently, the initial ROS-induced killing of promastigotes, which occurs during the first hours of infection, was greater twenty-four hours after infection of MT-1 KO bone-marrow macrophages than in our wild type controls. Overall, data presented in this thesis documents changes to macrophage mRNA and miRNA expression patterns upon infection with Leishmania promastigotes that correspond to the overall parasite survival in the host macrophage.
Adenosine, Innate immunity, Leishmania infantum chagasi, Metallothioneins, microRNAs, TLRs
xiv, 154 pages
Includes bibliographical references (pages 136-154).
Copyright 2013 Erik Wendlandt