Regulating Immune Mediated Pathology In Cutaneous Leishmaniasis: Roles For Il-22 And Skin Microbiota

Cutaneous leishmaniasis is a chronic disease characterized by ulcerating and disfiguring skin lesions. Infection with different species of Leishmania parasites is responsible for the initiation of this disease, yet most of the pathology observed is mediated by an unregulated immune response. The work presented in this thesis investigated the roles of IL-22 and the skin microbiota in regulating immune mediated pathology during cutaneous leishmaniasis. We found that IL-22, a cytokine important in wound repair in the skin, was required to limit pathology when mice were infected with L. major. In order to promote lesion resolution, IL-22 induced keratinocyte migration and decreased IL-1α and IL-1β production, both important stages in tissue repair. Interestingly, this protective role for IL-22 was only observed with a high dose of infection, suggesting a threshold of inflammation is required for IL-22 to limit pathology. We also found that the L. major infection in mice, as well as, L. braziliensis infection in humans caused a dysbiosis in the skin microbiota on lesional skin and nearby skin sites, characterized by a dominance of Staphylococcus spp. or Streptococcus spp. Interestingly, this dysbiotic microbiota was also transmissible to co-housed na�ve skin and exacerbated skin inflammation during L. major infection and during an acute contact hypersensitivity model. These data are the first to demonstrate that a dysbiotic skin microbiota can be transmitted to non-inflamed tissue and demonstrate how a naturally occurring dysbiosis can worsen disease during cutaneous leishmaniasis. Work presented in this thesis demonstrates that both IL-22 and the skin microbiota have distinct roles during cutaneous leishmaniasis. Future studies will be aimed at how these factors can be regulated to aid in the treatment of the disease

Utilization, retention and bio-efficacy studies of PermaNet® in selected villages in Buie and Fentalie districts of Ethiopia

<p>Abstract</p> <p>Background</p> <p>Malaria remains a major public health problem in Ethiopia. Pyrethroid-treated mosquito nets are one of the major tools available for the prevention and control of malaria transmission. PermaNet^®is a long-lasting insecticide-treated net (LLIN) recommended by WHO for malaria control.</p> <p>Objective</p> <p>The objective of the study was to assess utilization and retention of PermaNet^®nets distributed for malaria control in Buie and Fentalie districts and monitor the bio-efficacy of the nets using the WHO cone bioassay test procedures.</p> <p>Methods</p> <p>A cross sectional study was carried out by interviewing household heads or their representative in Buie and Fentalie districts. The two districts were selected based on a priori knowledge of variations on ethnic background and housing construction. Clusters of houses were chosen within each of the study villages for selection of households. 20 households that had received one or more PermaNet^®nets were chosen randomly from the clusters in each village. A total of eight used PermaNet^®nets were collected for the bio-efficacy test. The bio-efficacy of PermaNet^®nets was monitored according to the standard WHO procedures using a susceptible colony of <it>Anopheles arabiensis </it>to deltamethrin.</p> <p>Results</p> <p>A total of 119 household heads were interviewed during the study. The retention rate of nets that were distributed in 2005 and 2006 season was 72%. A total of 62.2% of the interviewees claimed children under five years of age slept under LLIN, while only 50.7% of the nets were observed to be hanged inside houses when used as a proxy indicator of usage of LLIN. For the bio-efficacy test the mean knock-down was 94% and 100%, while the mean mortality rate observed after 24 hr holding period was 72.2% and 67% for Buie and Fentalie districts respectively.</p> <p>Conclusion</p> <p>The study revealed a moderately high retention of PermaNet^®in the study villages and effectiveness of the nets when tested according to the standard WHO procedure.</p

Leishmania mexicana induces limited recruitment and activation of monocytes and monocyte-derived dendritic cells early during infection.

While C57BL/6 mice infected in the ear with L. major mount a vigorous Th1 response and resolve their lesions, the Th1 response in C57BL/6 mice infected with L. mexicana is more limited, resulting in chronic, non-healing lesions. The aim of this study was to determine if the limited immune response following infection with L. mexicana is related to a deficiency in the ability of monocyte-derived dendritic cells (mo-DCs) to prime a sufficient Th1 response. To address this issue we compared the early immune response following L. mexicana infection with that seen in L. major infected mice. Our data show that fewer monocytes are recruited to the lesions of L. mexicana infected mice as compared to mice infected with L. major. Moreover, monocytes that differentiate into mo-DCs in L. mexicana lesions produced less iNOS and migrated less efficiently to the draining lymph node as compared to those from L. major infected mice. Treatment of L. mexicana infected mice with α-IL-10R antibody resulted in increased recruitment of monocytes to the lesion along with greater production of IFN-γ and iNOS. Additionally, injection of DCs into the ear at the time of infection with L. mexicana also led to a more robust Th1 response. Taken together, these data suggest that during L. mexicana infection reduced recruitment, activation and subsequent migration of monocytes and mo-DCs to the draining lymph nodes may result in the insufficient priming of a Th1 response

IL-22 Protects against Tissue Damage during Cutaneous Leishmaniasis.

Cutaneous leishmaniasis is a disease characterized by ulcerating skin lesions, the resolution of which requires an effective, but regulated, immune response that limits parasite growth without causing permanent tissue damage. While mechanisms that control the parasites have been well studied, the factors regulating immunopathologic responses are less well understood. IL-22, a member of the IL-10 family of cytokines, can contribute to wound healing, but in other instances promotes pathology. Here we investigated the role of IL-22 during leishmania infection, and found that IL-22 limits leishmania-induced pathology when a certain threshold of damage is induced by a high dose of parasites. Il22-/- mice developed more severe disease than wild-type mice, with significantly more pathology at the site of infection, and in some cases permanent loss of tissue. The increased inflammation was not due to an increased parasite burden, but rather was associated with the loss of a wound healing phenotype in keratinocytes. Taken together, these studies demonstrate that during cutaneous leishmaniasis, IL-22 can play a previously unappreciated role in controlling leishmania-induced immunopathology

Fewer transferred monocytes migrate to the dLN during <i>L. mexicana</i> infection compared to <i>L. major</i>.

<p>Monocytes enriched from CD45.1 C57BL/6 mice were injected into the ear of CD45.2 C57BL/6 mice that were infected for two weeks with either <i>L. major</i> or <i>L. mexicana</i>. Eighteen hours following injection of the monocytes, ears and dLNs were harvested and processed. (<b>A</b>) Absolute number of transferred monocytes (CD11b⁺ CD45.1⁺) in the ear. Cells are previously gated on total, live cells that are singlets. (<b>B</b>) Mean fluorescence intensity (MFI) of Ly6C on transferred cells recovered from the ears of infected mice. Percentage (<b>C</b>) or absolute number (<b>D</b>) of CD45.1⁺ cells in the dLN. These cells are previously gated on live, singlets that are CD11b^hi cells. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 3 mice per group. The results are representative of two experiments. * significantly different (p<0.05) compared to <i>L. major</i> infected mice.</p

Mo-DCs produce significantly less iNOS during infection with <i>L. mexicana</i> compared to <i>L. major</i>.

<p>C57BL/6 mice were infected and ears were processed as above. In addition to staining for surface markers, intracellular staining for iNOS was performed. Percentage (<b>A</b>) or absolute number (<b>B</b>) of iNOS-producing cells in the ear of naïve or infected mice on day 14. Cells are previously gated on live, singlets that are CD11b^hi CD11c⁺. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for the bar graph) of 3 mice per group. The results are representative of two experiments. * significantly lower (p<0.05) compared to <i>L. major</i> infected mice.</p

Production of IL-10 during <i>L. mexicana</i> infection contributes to less recruitment of monocytes.

<p>Ears from naïve, <i>L. mexicana</i> infected or <i>L. mexicana</i> infected and α-IL-10R treated C57BL/6 mice were processed as above. (<b>A</b>) Histograms of monocytes on day 7 and 14 or mo-DCs on day 14 in the ear of naïve (grey shaded histogram), <i>L. mexicana</i> infected mice (black line, top) or <i>L. mexicana</i> infected and α-IL-10R treated mice (black line, bottom). Absolute numbers of monocytes and mo-DCs are also shown. Monocytes are previously gated on live, singlets that are CD11b^hi CD11c⁻. Mo-DCs are previously gated on live, singlets that are CD11b^hi CD11c⁺. (<b>B</b>) Levels of IFN-γ (ng/mL) from the supernatants of single cell suspensions from the dLN of each group that were stimulated for 72 hours with <i>L. mexicana</i> freeze-thaw antigen. (<b>C</b>) Percentage and absolute number of iNOS-producing cells in the ear of naïve, <i>L. mexicana</i> infected mice or <i>L. mexicana</i> infected and α-IL-10R treated mice on day 14. Cells are previously gated on live, singlets that are CD11b^hi CD11c⁺. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 3–5 mice per group. The results are representative of two experiments. * significantly higher (p<0.05) compared to <i>L. mexicana</i> infected mice in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g006" target="_blank">Fig. 6A and 6C</a> (FACS plots) or p<0.05 between indicated groups in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g006" target="_blank">Fig. 6A, 6B and 6C</a> (bar graphs).</p

Fewer CD11b^hi Ly6C⁺ cells in the ear following <i>L. mexicana</i> infection compared to <i>L. major</i>.

<p>Ears from naïve, as well as <i>L. major</i> or <i>L. mexicana</i> infected C57BL/6 mice were processed to single cell suspensions. (<b>A</b>) Percentage of CD11b^hi cells present in the ear of naïve or infected mice on day 3 and 14. Cells are previously gated on total, single live cells. Histograms of monocytes (<b>B</b>) or mo-DCs (<b>C</b>) in the ear of naïve (grey shaded histogram) or infected mice (black line) on day 3 and 14 and absolute number of monocytes (<b>B</b>) or mo-DCs (<b>C</b>) from naïve, day 3 and day 14 infected mice. Monocytes are pre-gated on CD11b^hi CD11c⁻ cells and mo-DCs are previously gated on CD11b^hi CD11c⁺ cells. The results expressed are the mean percentage (± SD for FACS plots) or the mean number of cells (± SE for bar graphs) of 3 mice per group. The results are representative of two experiments. * significantly lower (p<0.05) compared to <i>L. major</i> infected mice in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g001" target="_blank">Fig. 1A</a> or p<0.05 between indicated groups in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001858#pntd-0001858-g001" target="_blank">Fig. 1B</a> and C.</p