Understanding Mechanisms and Diversity of Leishmania-Mediated CXCL10 Suppression

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2021

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Abstract

Leishmaniasis is a neglected tropical disease with diverse disease outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. With no vaccines available and treatment options limited by significant costs and side effects, understanding the pathophysiology of leishmaniasis is paramount. However, the complex interactions between parasites in the Leishmania genus and their hosts make this task challenging. This dissertation dissects host and pathogen regulation of a chemokine, CXCL10, that can provide protection against leishmaniasis. First, a novel mechanism by which Leishmania parasites suppress CXCL10 by proteolytic cleavage is identified. Second, variation in CXCL10 between Leishmania spp. that cause different forms of disease is characterized. Third, CXCL10 signaling is studied in a murine model by using small molecule agonists to circumvent parasite immune evasion.

The first goal of this project was to identify how Leishmania parasites are able to suppress CXCL10, a chemokine with diverse antimicrobial functions, including T cell recruitment. Infection with L. major in a human monocyte cell line induced robust CXCL10 transcription without increasing extracellular CXCL10 protein concentrations. CXCL10 suppression was found to require the virulence factor and protease, glycoprotein-63 (gp63). Specifically, GP63 cleaves CXCL10 after amino acid A81 at the base of a C-terminal alpha-helix. Cytokine cleavage by GP63 demonstrated specificity, as GP63 cleaved CXCL10 and its homologues, which all bind the CXCR3 receptor, but not distantly related chemokines, such as CXCL8 and CCL22. Further characterization demonstrated that CXCL10 cleavage activity by GP63 was produced by both extracellular promastigotes and intracellular amastigotes. Crucially, CXCL10 cleavage impaired T cell chemotaxis in vitro, indicating that cleaved CXCL10 cannot signal through CXCR3. Consistent with CXCL10 cleavage during infection, we observed GP63-mediated impairment of activated CD8+ T-cells in the draining lymph nodes of C57BL/6JWT mice. Correspondingly, in C57BL/6JWT mice, gp63 deletion resulted in slower lesion development and a smaller maximum lesion size. However, infection in C57BL/6Jcxcr3-/- mice revealed the delay to lesion development required CXCR3 signaling. Ultimately, we propose CXCL10 suppression is a convergent mechanism of immune evasion by intracellular pathogens, as Salmonella enterica and Chlamydia trachomatis also suppress CXCL10. This commonality suggests that counteracting CXCL10 suppression may provide a generalizable therapeutic strategy against intracellular pathogens.

Although cutaneous leishmaniasis is a classic example of type-1 immunity leading to well controlled self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania diversity in both pathogen sensing by the host and immune evasion by the parasite contributes to variation in the polarization and robustness of the immune response. Here, I demonstrate that unlike other Leishmania (L.) spp., L. Viannia (V.) panamensis is unable to suppress CXCL10 due to reduced cleavage activity by GP63. Similarly, other parasites in the Viannia subgenus, which cause the immunopathologic mucocutaneous form of leishmaniasis, have reduced CXCL10 cleavage capacity. To understand how this altered phenotype emerged, we used protein-protein interaction modeling of the primary amino acid sequence and known crystal structures to identify a putative CXCL10 binding site on GP63. The putative CXCL10 binding site, which varies between the Leishmania and Viannia subgenera, was found to be in a region under significant positive selection. Finally, we experimentally confirmed that the predicted binding site and adjacent positively selected amino acids are involved in CXCL10 suppression by demonstrating a specific reduction in CXCL10 cleavage after mutagenesis of the wild-type L. (L.) major gp63 to the L. (V.) panamensis allele at the binding site. These results demonstrate how genetic diversity of parasites contribute to variation in the host immune response to Leishmania spp. Unraveling additional molecular mechanisms whereby parasite genetic diversity contributes to heterogeneity of host immune response will be vital to understanding variation in the pathophysiology leishmaniasis.

Because CXCL10 contributes to the recruitment and activation of immune cell subsets that confer protection against leishmaniasis, it has the potential to serve as a host-directed therapy for leishmaniasis. However, the consequences of stimulating its receptor, CXCR3, during infection remains uncertain, due to reported protective and exacerbating effects of CXCR3 ligands in different infection contexts and the discovery here that Leishmania spp. cleave CXCR3 ligands. In the final chapter, small molecule CXCR3 agonists were leveraged to circumvent parasite chemokine cleavage and interrogate how specific downstream CXCR3 signaling patterns impact disease outcome in C57BL6/J mice. A G-protein-biased agonist (VUF11418) decreased lesion size, but this beneficial impact was due to an off-target effect. Unexpectedly, both a CXCR3 -arrestin-biased agonist (VUF10661) and a CXCR3 antagonist (AMG487) increased the size of the cutaneous lesion, despite having opposite effects on T-cell recruitment in vitro. We next determined whether -arrestin-biased CXCR3 signaling increased lesion size due to excess recruitment of anti-parasitic immune cell types or due to altered polarization and activation of the inflammatory cells recruited. Despite not improving outcomes in C57BL6/J mice, the observed increase in inflammation by CXCR3 stimulation in response to L. major highlights the importance of understanding the host, pathogen, and environmental factors that determine when CXCR3 signaling is protective or pathologic after Leishmania spp. infection.

Together these studies highlight the complexity of host interactions with Leishmania parasites. Future work is needed to elucidate the clinical relevance of the mechanisms and diversity of CXCL10 suppression described here. However, this work demonstrates how parasite diversity can be leveraged to unravel the molecular basis for pathogenesis and inform the design of novel treatment strategies for leishmaniasis.

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Antonia, Alejandro (2021). Understanding Mechanisms and Diversity of Leishmania-Mediated CXCL10 Suppression. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/22946.

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