IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

National Institute Of Allergy And Infectious Diseases

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Abstract

Tissue-resident macrophages (TRMs) maintain tissue homeostasis, but they can also provide a replicative niche for intracellular pathogens such as Leishmania. We have identified a population of M2-like dermal macrophages that are present under steady state conditions and that are preferentially infected by a strain of Leishmania major isolated from a patient with chronic cutaneous lesions to promote non- healing cutaneous disease in conventionally resistant C57Bl/6 mice. The dermal macrophages are not replaced by blood precursors during infection, but are embryonic derived and locally maintained by IL-4 and IL-10 and retain M2 functionality despite the high levels of IFNg produced in the site. How dermal TRMs proliferate and maintain their M2 properties even in the strong TH1 environment of the L. major infected dermis is not clear. We show that in infected mice lacking IL-4/IL-13 from eosinophils, dermal TRMs shifted to a pro-inflammatory state, their numbers declined, and disease was attenuated. Intravital microscopy revealed a rapid infiltration of eosinophils followed by their tight interaction with dermal TRMs. IL-4-stimulated dermal TRMs, in concert with IL-10, produced a large amount of CCL24 (Eotaxin-2) which functioned to amplify eosinophil influx and their interaction with dermal TRMs. An intraperitoneal helminth infection model also demonstrated a requirement for eosinophil-derived IL-4 to maintain tissue macrophages through a CCL24-mediated amplification loop. CCL24 secretion was confined to resident macrophages in other tissues, implicating eosinophil-TRM cooperative interactions in diverse inflammatory settings. The IL-4/CCL24 mediated cooperative interactions between dermal TRM and eosinophils that we described in L. major infection will be conditioned by a network of additional cells, cytokines and chemokines that are present in the inflammatory site. We have initiated single cell RNA seq analyses of the cells recovered from L. major infected ears of C57Bl/6 mice at different times post-infection. Our initial analysis has allowed us to identify the dermal TRMs within a discreet cluster of myeloid cells that are positive for both MR and CCL24, validating our prior flow cytometric findings. We also identified a discreet cluster of cells that were positive for IL-5 and other ILC2 markers, and we could confirm by flow cytometry that ILC2s were the major source of intralesional IL-5. It is well known that IL-5 promotes eosinophil maturation and activation. We observed that IL-5 deficient mice present a similar phenotype as the mice conditionally deficient in IL-4 production by eosinophils with regard to reduced numbers of both dermal TRMs and lesional parasites. Several recent discoveries have highlighted the crucial role of epithelial alarmins, principally IL-25, IL-33 and thymic stromal lymphopoietin (TSLP), which act solely or in concert to drive type 2 responses by ILC2 in various tissues. Our single cell RNA seq analysis failed to detect a signal for IL-25 in any cell cluster, while a weak signal for IL-33 was detected in a fibroblast cluster, and a strong signal for TSLP was detected in a subset of the dermal TRMs, and not epithelial cells as expected. These initial findings suggest that dermal TRMs actively maintain their M2 properties by orchestrating localized interactions with both ILC2s and eosinophils. Visceral leishmaniasis (VL) is a potentially fatal neglected infectious disease caused by infections with the protozoan parasite Leishmania donovani. Infected people may remain asymptomatic or progress, at variable rates, to develop VL disease affecting the liver, spleen and blood. The reasons why clinical outcomes vary so much is not well understood. We decided to investigate whether a hosts gut microbiota might be a relevant factor. We treated mice and hamsters with antibiotics to disrupt their gut microbiota and then infected them with L. donovani. No effect was observed in mice, however, treated hamsters had slower onset and progression of VL, less severe enlargement of the liver and spleen, and had a reduced mortality rate. This was accompanied by alterations in the levels of key immune response factors. We also found novel aspects of symptomatic VL in hamsters relating to the gut and its microbiota. Specifically, the gut tissues themselves were parasitized by L. donovani and liver tissues became co-infected with both L. donovani and Gram negative bacteria originating from the gut. Overall, our findings support the inclusion of anti-bacterial therapy as part of VL treatment strategies. In the mouse model of VL, parasites establish infection in the liver and spleen. Kupffer cells (KCs) are the liver resident macrophages that help maintain tissue homeostasis and regulate the immune response in this organ. Although KCs have an embryonic origin, in some inflammatory settings they may be replaced by monocyte derived cells. In the L. donovani VL model, KCs have been shown to constitute the core of granulomas and to play an important role in the protective response. We aimed to further understand the role of KCs during L. infantum infection, focusing on the dynamics of granuloma formation, the extent of KCs replacement by monocyte-derived cells, and the activation programs of monocyte- and KC- populations during infection. By live imaging, KCs were observed as the first infected cells in the liver after L. infantum i.v. administration. In the first hours post-infection (p.i.), clusters of KCs were detected, followed by neutrophils infiltration that interacted with infected KCs. At 19 days p.i., mature granulomas were readily detected, with eosinophils observed around a fused KC core. To investigate KCs contribution to parasite establishment in the liver, they were depleted by clodronate-loaded liposomes, resulting in a drastic reduction in parasite burden. Single cell mRNA sequencing of CD45+ liver cells in naive, 19 and 42 days p.i mice identified KCs in 4 different clusters, 3 of which also contained monocytes and monocyte-derived macrophages (mo-macs). By flow cytometry analysis, Arg1 expression was downregulated and iNOS and PD-L1 were upregulated on both KCs and mo-macs. On the other hand, TNF and Axl were upregulated mainly in KCs. Taken together, these data show that KCs rapidly initiate the process of granuloma formation after L. infantum infection, and consist of a heterogeneous population expressing both pro- and anti-inflammatory markers, some of which are also expressed in mo-macs.