Tissue-resident macrophages (TRMs) maintain tissue homeostasis, but they can also provide a replicative niche for intracellular pathogens such as Leishmania. How dermal TRMs proliferate and maintain their M2 properties even in the strong TH1 environment of the L. major infected dermis is not clear. Here, we show that, in infected mice lacking IL-4/13 from eosinophils, dermal TRMs shifted to a proinflammatory 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, 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.

There is substantial experimental evidence to indicate that Leishmania infections that are transmitted naturally by the bites of infected sand flies differ in fundamental ways from those initiated by needle inocula. We have used flow cytometry and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some of the dermal TRMs could be visualized acquiring their infections via transfer from or efferocytosis of parasitized neutrophils, providing direct evidence for the “Trojan Horse” model. The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl-/-Mertk-/- mice. The Axl-/-Mertk-/- mice also displayed reduced parasite burdens but more severe pathology following L. major infection transmitted by sand fly bite.

2There is substantial experimental evidence to indicate that Leishmania infections that are 3 transmitted naturally by the bites of infected sand flies differ in fundamental ways from the 4 inflammatory and immune reactions initiated by needle inocula. We have used flow cytometry 5 and intravital microscopy (IVM) to reveal the heterogeneity of sand fly transmission sites with 6 respect to the subsets of phagocytes in the skin that harbor L. major within the first hours and days 7 after infection. By flow cytometry analysis, dermis resident macrophages (TRMs) were on 8 average the predominant infected cell type at 1 hr and 24 hr. By confocal IVM, the co-localization 9 of L. major and neutrophils varied depending on the proximity of deposited parasites to the 10 presumed site of vascular damage, defined by the highly localized swarming of neutrophils. Some 11 of the dermal TRMs could be visualized acquiring their infections via transfer from or 12 efferocytosis of parasitized neutrophils, providing direct evidence for the "Trojan Horse" model. 13 The role of neutrophil engulfment by dermal TRMs and the involvement of the Tyro3/Axl/Mertk 14 family of receptor tyrosine kinases in these interactions and in sustaining the anti-inflammatory 15 program of dermal TRMs was supported by the effects observed in neutrophil depleted and in Axl -/-16 Mertk -/mice. The Axl -/-Mertk -/mice also displayed reduced parasite burdens but more severe 17 pathology following L. major infection transmitted by sand fly bite. 18 19 Summary 20 Sand flies transmit Leishmania major which causes cutaneous leishmaniasis in humans and 21 in non-human hosts. Our analyses of sand fly transmission sites of L. major in the mouse skin 22 revealed that dermis resident macrophages (TRM) were the predominant phagocytes to take up the 23 parasite within the first 24 hr post-bite. The early involvement of neutrophils varied depending 3 24 on the proximity of deposited parasites to the site of tissue damage around which the neutrophils 25 coalesced. By intra-vital microscopy, some of the dermal TRMs could be visualized acquiring 26 their infections by direct transfer from or phagocytosis of parasitized neutrophils. The involvement 27 of the Tyro3/Axl/Mertk family of receptor tyrosine kinases in these cellular interactions and in 28 sustaining the anti-inflammatory functions of dermal TRMs was supported by the reduced parasite 29 burdens but more severe pathology observed in Axl -/-Mertk -/mice. The heterogeneity of sand fly 30 transmission sites with respect to the dose of parasites and the early cellular interactions described 31 here likely contribute to the wide range of infection outcomes that are associated with natural 32 transmission of L. major observed in mouse models and possibly humans. 33 34 35 36 37 Kinetoplastid parasites of the genus Leishmania are phagosomal pathogens transmitted by 38 phlebotomine sand flies that produce a spectrum of diseases in their human hosts, ranging from 39 localized cutaneous lesions, to tissue destructive mucosal i...