Regulation of the immune system during experimental cutaneous leishmaniasis in gene-deficient mice

Kling, Jessica Christine (2013) Regulation of the immune system during experimental cutaneous leishmaniasis in gene-deficient mice. PhD thesis, James Cook University.

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The obligate intracellular protozoan parasite Leishmania major is transmitted by sandfly vectors and causes cutaneous leishmaniasis in infected hosts, which results in the development of lesions at the site of infection. In the mouse model, genetically resistant mouse strains, such as C57BL/6, are able to contain the pathogen and spontaneously heal the lesions, while genetically susceptible mice, such as BALB/c, are unable to confine the parasite and heal the lesions, and therefore finally succumb to the disease. This genetically programmed outcome of leishmaniasis is determined primarily by cytokine expression patterns, which have been attributed to CD4⁺ helper T cell subsets. Large amounts of the Th1-type cytokines IL-12 and IFN-γ are associated with a protective immune response, while expression of the Th2-type cytokines IL-4 and IL-13 coincide with host susceptibility. Two further cytokines, IL-10 and TGF-β can modify the immune response to Leishmania by suppressing the immune system, and thus preventing complete eradication of the parasites.

Immunosuppression during cutaneous leishmaniasis is important for controlling collateral damage that can occur as a result of overzealous inflammation, as well as limiting the immune response once the parasite is eliminated from the host. However, the degree of immunosuppression needs to be finely balanced, since amplified suppression can lead to chronic disease or death due to uncontrolled parasite growth, while insufficient suppression can lead to excessive inflammation causing collateral damage and host injury. A variety of cells from both the innate and adaptive branches of the immune system can mediate immunosuppression. These cells include alternatively activated macrophages (AAMs) and myeloid-derived suppressor cells (MDSCs), which represent the innate branch of the immune system, as well as Tregs, which are the main regulators within the adaptive branch. Since the maintenance of an appropriate immune response relies on the balance of inflammation and immunoregulation, my hypothesis was that during cutaneous leishmaniasis, dysfunctional regulation of the immune response by immunoregulatory cells contributes to pathogenesis and undesirable clinical outcomes.

Two gene-deficient mouse models on the genetically resistant C57BL/6 background were used to analyse regulatory aspects of the immune response to L. major. One of these mouse models is deficient for the pro-inflammatory cytokine tumour necrosis factor (TNF) and succumbs to L. major, while the other model, which is deficient for the chemokine receptor CCR7, develops a chronic infection. The susceptible or partly susceptible disease outcomes of these two mouse models differs from the normally resistant phenotype of the C57BL/6 strain, indicating that there is a lack of induction of immunoregulatory cells resulting in collateral damage or increased immunosuppression during infection leading to parasite dissemination. Therefore, both models are well suited to study the ensuing implications of immunoregulation during cutaneous leishmaniasis.

Firstly, in the absence of TNF, mice develop a Th1 response and exhibit a lack of Th2 cytokine production in response to L. major, comparable to control mice, and a possible cause for the death of B6.TNF⁻/⁻ mice has yet to be elucidated. In B6.TNF⁻/⁻ mice, but not in control mice, I observed a large percentage of AAMs in the infected footpad and in the draining lymph node. At the site of infection, these AAMs are defined as F4/80⁺CD206⁺ monocytes/macrophages that express arginase 1 (Arg1). In the draining lymph node, two monocytic populations are present, as defined by Ly6C expression, and in the absence of TNF, the population with low expression of Ly6C accumulates at the peak of disease. Both the CD11b⁺Ly6Clo and CD11b⁺Ly6Chi monocytic populations in B6.TNF⁻/⁻ mice display markers for alternative activation, such as Arg1 and CD206, while B6.WT monocytic populations have minimal expression of the same markers. These results indicate that TNF has an antagonistic role in the development of AAMs, and describe a potential mechanism for the death of B6.TNF⁻/⁻ mice after L. major infection.

The second model I used to test my hypothesis was the L. major infection of CCR7-deficient mice. This infection model allowed me to study the expansion of Tregs in response to parasitic infection, and in addition, to address more general questions regarding the role of CCR7, since there is currently a general lack of studies examining L. major-infected B6.CCR7-/- mice. Therefore, both the innate and lymphocytic infiltrates during infection were analysed to provide a foundation for further research. In the absence of CCR7, cells crucial for the clearance of parasites, such as inflammatory iNOS-expressing monocytes, were delayed in their migration to the site of infection. Furthermore, B6.CCR7-/- mice had increased expression of the Th2 cytokines IL-4 and IL-10 within the lymph node, while there were no changes in IFN-γ expression. Additionally, there was an increased percentage of Tregs in the draining lymph node of B6.CCR7⁻/⁻ mice throughout infection. Since CCR7 is required for the migration of antigen-presenting DCs into the lymph node, B6.CCR7⁻/⁻ mice were injected with B6.WT DCs to increase DC migration, promote efficient antigen presentation and support an effective Th1 response. Instead, the addition of B6.WT DCs into B6.CCR7⁻/⁻ mice or B6.CCR7⁻/⁻ DCs into B6.WT mice caused exacerbated disease compared to control host mice. Moreover, the injection of B6.WT DCs further increased the percentage of Tregs in the lymph nodes of B6.CCR7⁻/⁻ mice. Taken together, I show that CCR7 is required for maintaining a pro-inflammatory response to L. major.

Overall, increased immunosuppression during cutaneous leishmaniasis is an underlying cause for fatal or chronic disease outcomes. Specifically, I showed that TNF negatively regulates the development of AAMs during L. major infection, providing a plausible explanation for the death of TNF-deficient mice. Furthermore, I provide evidence that CCR7 modulates immunosuppression and is required to elicit a Th1 response, as an additional function to its known role in cell migration. Therefore, as hypothesised, disturbed cellular regulation from both the innate and adaptive branches of the immune system contribute to undesired outcomes of L. major infection.

Item ID: 40328
Item Type: Thesis (PhD)
Keywords: animal models; cutaneous; host parasite interactions; immune response; immune system; immunosuppression; infectious diseases; leishmania; leishmaniasis; lymph nodes; monocytes; mouse models; parasitic diseases; protozoan; regulatory t cells
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Kling, Jessica, Darby, J., and Körner, H. (2014) CCR7 facilitates the pro-inflammatory function of dendritic cells in experimental leishmaniasis. Parasite Immunology, 36 (4). pp. 177-185.

Fromm, Phillip D., Kling, Jessica, Mack, Matthias, Sedgwick, Jonathon D., and Koerner, Heinrich (2012) Loss of TNF signaling facilitates the development of a novel Ly-6C(low) macrophage population permissive for Leishmania major infection. Journal of Immunology, 188 (12). pp. 6258-6266.

Kling, Jessica, Gollan, Rene, Fromm, Phillip, and Körner, Heinrich (2011) Redundancy of interleukin-6 in the differentiation of T cell and monocyte subsets during cutaneous leishmaniasis. Experimental Parasitology, 129 (3). pp. 270-276.

Kling, Jessica C., and Körner, Heinrich (2013) Different regulatory mechanisms in protozoan parasitic infections. International Journal for Parasitology, 43 (6). pp. 417-425.

Kling, Jessica C., Mack, Matthias, and Körner, Heinrich (2013) The absence of CCR7 results in dysregulated monocyte migration and immunosuppression facilitating chronic cutaneous leishmaniasis. PLoS ONE, 8 (10). pp. 1-16.

Date Deposited: 01 Oct 2015 01:16
FoR Codes: 06 BIOLOGICAL SCIENCES > 0603 Evolutionary Biology > 060307 Host-Parasite Interactions @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 50%
92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920109 Infectious Diseases @ 50%
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