Dermis resident macrophages orchestrate localized ILC2 eosinophil circuitries to promote non-healing cutaneous leishmaniasis.

Tissue-resident macrophages are critical for tissue homeostasis and repair. We previously showed that dermis-resident macrophages produce CCL24 which mediates their interaction with IL-4+ eosinophils, required to maintain their M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we show that thymic stromal lymphopoietin (TSLP) and IL-5+ type 2 innate lymphoid cells are also required to maintain dermis-resident macrophages and promote infection. Single cell RNA sequencing reveals the dermis-resident macrophages as the sole source of TSLP and CCL24. Generation of Ccl24-cre mice permits specific labeling of dermis-resident macrophages and interstitial macrophages from other organs. Selective ablation of TSLP in dermis-resident macrophages reduces the numbers of IL-5+ type 2 innate lymphoid cells, eosinophils and dermis-resident macrophages, and ameliorates infection. Our findings demonstrate that dermis-resident macrophages are self-maintained as a replicative niche for L. major by orchestrating localized type 2 circuitries with type 2 innate lymphoid cells and eosinophils.

HOP1 and HAP2 are conserved components of the meiosis-related machinery required for successful mating in Leishmania.

Whole genome analysis of Leishmania hybrids generated experimentally in sand flies supports a meiotic mechanism of genetic exchange, with Mendelian segregation of the nuclear genome. Here, we perform functional analyses through the generation of double drug-resistant hybrids in vitro and in vivo (during sand fly infections) to assess the importance of conserved meiosis-related genes in recombination and plasmogamy. We report that HOP1 and a HAP2-paralog (HAP2-2) are essential components of the Leishmania meiosis machinery and cell-to-cell fusion mechanism, respectively, since deletion of either gene in one or both parents significantly reduces or completely abrogates mating competence. These findings significantly advance our understanding of sexual reproduction in Leishmania, with likely relevance to other trypanosomatids, by formally demonstrating the involvement of a meiotic protein homolog and a distinct fusogen that mediates non-canonical, bilateral fusion in the hybridizing cells.

Overexpression of Leishmania major protein arginine methyltransferase 6 reduces parasite infectivity in vivo.

Arginine methylation is catalysed by Protein Arginine Methyltransferases (PRMTs) and can affect how a target protein functions and how it interacts with other macromolecules, which in turn impacts on cell metabolism and gene expression control. Leishmania parasites express five different PRMTs, and although the presence of each individual PRMT is not essential per se, the imbalanced activity of these PRMTs can impact the virulence of Leishmania parasites in vitro and in vivo. Here we created a Leishmania major cell line overexpressing PRMT6 and show that similar to what was observed for the T. brucei homologous enzyme, L. major PRMT6 probably has a narrow substrate range. However, its overexpression notably impairs the infection in mice, with a mild reduction in the number of viable parasites in the lymph nodes. Our results indicate that arginine methylation by LmjPRMT6 plays a significant role in the adaptation of the parasite to the environment found in the mammalian host.

Dermis resident macrophages orchestrate localized ILC2-eosinophil circuitries to maintain their M2-like properties and promote non-healing cutaneous leishmaniasis.

Tissue-resident macrophages (TRMs) are critical for tissue homeostasis/repair. We previously showed that dermal TRMs produce CCL24 (eotaxin2) which mediates their interaction with IL-4 producing eosinophils, required to maintain their number and M2-like properties in the TH1 environment of the Leishmania major infected skin. Here, we unveil another layer of TRM self-maintenance involving their production of TSLP, an alarmin typically characterized as epithelial cell-derived. Both TSLP signaling and IL-5+ innate lymphoid cell 2 (ILC2s) were shown to maintain the number of dermal TRMs and promote infection. Single cell RNA sequencing identified the dermal TRMs as the sole source of TSLP and CCL24. Development of Ccl24-cre mice permitted specific labeling of dermal TRMs, as well as interstitial TRMs from other organs. Genetic ablation of TSLP from dermal TRMs reduced the number of dermal TRMs, and disease was ameliorated. Thus, by orchestrating localized type 2 circuitries with ILC2s and eosinophils, dermal TRMs are self-maintained as a replicative niche for L. major.

Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq.

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low-efficiency crosses between two Leishmania tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains and extends to other species, including Leishmania donovani, Leishmania infantum, and Leishmania braziliensis, a capacity to generate intra- and interspecific hybrids. Whole-genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

Interferon-γ-Producing CD4+ T Cells Drive Monocyte Activation in the Bone Marrow During Experimental Leishmania donovani Infection.

Ly6Chi inflammatory monocytes develop in the bone marrow and migrate to the site of infection during inflammation. Upon recruitment, Ly6Chi monocytes can differentiate into dendritic cells or macrophages. According to the tissue environment they can also acquire different functions. Several studies have described pre-activation of Ly6Chi monocytes in the bone marrow during parasitic infection, but whether this process occurs during experimental visceral leishmaniasis and, if so, the mechanisms contributing to their activation are yet to be established. In wild type C57BL/6 (B6) mice infected with Leishmania donovani, the number of bone marrow Ly6Chi monocytes increased over time. Ly6Chi monocytes displayed a highly activated phenotype from 28 days to 5 months post infection (p.i), with >90% expressing MHCII and >20% expressing iNOS. In comparison, in B6.Rag2-/- mice <10% of bone marrow monocytes were MHCII+ at day 28 p.i., an activation deficiency that was reversed by adoptive transfer of CD4+ T cells. Depletion of CD4+ T cells in B6 mice and the use of mixed bone marrow chimeras further indicated that monocyte activation was driven by IFNγ produced by CD4+ T cells. In B6.Il10-/- mice, L. donovani infection induced a faster but transient activation of bone marrow monocytes, which correlated with the magnitude of CD4+ T cell production of IFNγ and resolution of the infection. Under all of the above conditions, monocyte activation was associated with greater control of parasite load in the bone marrow. Through reinfection studies in B6.Il10-/- mice and drug (AmBisome®) treatment of B6 mice, we also show the dependence of monocyte activation on parasite load. In summary, these data demonstrate that during L. donovani infection, Ly6Chi monocytes are primed in the bone marrow in a process driven by CD4+ T cells and whereby IFNγ promotes and IL-10 limits monocyte activation and that the presence of parasites/parasite antigen plays a crucial role in maintaining bone marrow monocyte activation.

In Vitro Generation of Leishmania Hybrids.

Protozoan parasites in the genus Leishmania produce a broad spectrum of diseases in their human hosts. The strain and species-specific genes controlling these diverse clinical outcomes have remained poorly tractable using reverse genetics approaches. A cryptic sexual cycle involving a meiotic-like process has been described in Leishmania but is so far confined to parasites growing in the sand fly vector. Here, we describe the reproducible in vitro generation of hybrid clones using axenic culture forms of Leishmania tropica promastigotes. Analysis of SNPs marker inheritance and whole-genome sequencing data indicate that the progeny clones are full genomic hybrids. The demonstration that mating-competent forms arise in culture should facilitate experimental study of the mating biology of Leishmania and the generation of large numbers of recombinant parasites for positional cloning of important genes.

Leishmania braziliensis prostaglandin F2α synthase impacts host infection.

BACKGROUND: Prostaglandins (PG) are lipid mediators derived from arachidonic acid metabolism. They are involved in cellular processes such as inflammation and tissue homeostasis. PG production is not restricted to multicellular organisms. Trypanosomatids also synthesize several metabolites of arachidonic acid. Nevertheless, their biological role in these early-branching parasites and their role in host-parasite interaction are not well elucidated. Prostaglandin F2α synthase (PGF2S) has been observed in the Leishmania braziliensis secreted proteome and in L. donovani extracellular vesicles. Furthermore, we previously reported a positive correlation between L. braziliensis PGF2S (LbrPGF2S) expression and pathogenicity in mice. METHODS: LbrPGF2S gene expression and PGF2α synthesis in promastigotes were detected and quantified by western blotting and EIA assay kit, respectively. To investigate LbrPGF2S localization in amastigotes during bone marrow-derived macrophage infection, parasites expressing mCherry-LbrPGF2S were generated and followed by time-lapse imaging for 48 h post-infection. PGF2S homolog sequences from Leishmania and humans were analyzed in silico using ClustalW on Geneious v6 and EMBOSS Needle. RESULTS: Leishmania braziliensis promastigotes synthesize prostaglandin F2α in the presence of arachidonic acid, with peak production in the stationary growth phase under heat stress. LbrPGF2S is a cytoplasmic protein enriched in the secretory site of the parasite cell body, the flagellar pocket. It is an enzyme constitutively expressed throughout promastigote development, but overexpression of LbrPGF2S leads to an increase of infectivity in vitro. The data suggest that LbrPGF2S may be released from intracellular amastigotes into the cytoplasm of bone marrow-derived macrophages over a 48-hour infection period, using time-lapse microscopy and mCherry-PGF2S (mChPGF2S)-expressing parasites. CONCLUSIONS: LbrPGF2S, a parasite-derived protein, is targeted to the host cell cytoplasm. The putative transfer of this enzyme, involved in pro-inflammatory lipid mediator synthesis, to the host cell suggests a potential role in host-parasite interaction and may partially explain the increased pathogenicity associated with overexpression of LbrPGF2S in L. braziliensis. Our data provide valuable insights to help understand the importance of parasite-derived lipid mediators in pathogenesis.