IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

组织驻留巨噬细胞(TRM)维持组织内平衡，但它们也可以为利什曼原虫等细胞内病原体提供复制的利基环境。我们已经确定了一组M2样真皮巨噬细胞，它们存在于稳定状态下，并优先感染从一名慢性皮肤损害患者中分离出来的一株主要利什曼原虫，以促进常规耐药C57BL/6小鼠的不可愈合皮肤病。在感染期间，真皮巨噬细胞不被血液前体细胞取代，而是胚胎来源的，由IL-4和IL-10局部维持，并保留M2功能，尽管该部位产生高水平的IFNG。目前尚不清楚真皮TRMS如何在感染L1的强TH1环境中增殖并保持其M2特性。我们发现，在缺乏来自嗜酸性粒细胞的IL-4/IL-13的感染小鼠中，真皮TRM转变为促炎状态，其数量减少，疾病减轻。活体显微镜显示，嗜酸性粒细胞迅速渗入，随后它们与真皮TRMS紧密结合。IL-4刺激的真皮TRMS与IL-10协同产生大量的CCL24(嗜酸性粒细胞趋化因子-2)，其功能是放大嗜酸性粒细胞的流入及其与真皮TRMS的相互作用。一种腹膜蠕虫感染模型也证明了嗜酸性粒细胞来源的IL-4需要通过CCL24介导的扩增环来维持组织巨噬细胞。CCL24的分泌仅限于其他组织中的驻留巨噬细胞，暗示嗜酸性粒细胞-TRM在不同的炎症环境中协同作用。 有大量的实验证据表明，通过受感染的沙蝇叮咬自然传播的利什曼原虫感染在根本上不同于针头接种引发的炎症和免疫反应。我们使用流式细胞术和活体显微镜(IVM)揭示了感染后最初几个小时和几天内，沙蝇传播部位相对于携带大沙蝇的皮肤中吞噬细胞亚群的异质性。通过流式细胞仪分析，真皮停留巨噬细胞(TRMS)在1小时和24小时平均是主要的感染细胞类型。在共聚焦活体显微镜下，主要乳杆菌和中性粒细胞的共存取决于寄生的寄生虫与血管损伤部位的接近程度，这是由中性粒细胞的高度局部化聚集所定义的。可以看到一些真皮TRM通过从寄生的中性粒细胞转移或吞噬寄生的中性粒细胞而获得感染，为特洛伊木马模型提供了直接证据。中性粒细胞被真皮TRMS吞噬的作用，以及Tyro3/Ax1/MerTK受体酪氨酸激酶家族参与这些相互作用和维持真皮TRMS的抗炎计划的作用，得到了在中性粒细胞耗竭和Ax1/MerTK-/-小鼠中观察到的效果的支持。AXL-/-MerTK-/-小鼠的寄生虫负担也有所减轻，但通过沙蝇叮咬传播的L.MERTK-/-小鼠的病理更加严重。我们所描述的沙蝇传播地点相对于寄生虫剂量的异质性以及早期的细胞相互作用可能有助于在小鼠模型和人类中观察到与主要乳杆菌的自然传播相关的广泛的感染结果。 杜氏利什曼原虫引起内脏利什曼病(VL)，如果不治疗，这种疾病通常是致命的。在疾病进展速度和对治疗的反应方面，个体之间存在很大差异。在人类和动物模型中出现异质性疾病的原因还知之甚少。宿主微生物区系已被确定为皮肤形式的利什曼病的一个因素，但这还没有在VL中进行研究。我们通过长期在小鼠和仓鼠的饮用水中使用广谱抗生素来诱导肠道生态失调。在营养不良的小鼠中，疾病表现没有显著差异。相比之下，感染了杜诺瓦尼乳杆菌的反生性仓鼠体重下降的开始和进展都有所延迟。抗生素治疗的仓鼠肝脾肿大也明显减轻，并伴随着明显的细胞因子基因表达谱。这种保护作用不能用寄生虫负荷或血液学特征的差异来解释。我们进一步发现有证据表明，肠道-肝轴是仓鼠致死性VL进展的关键方面，包括肠道寄生虫、细菌移位到肝脏、软斑和铁隔离，这些都没有发生在无进展的小鼠VL中。从VL感染的肝脏中培养出不同的细菌属，其中在ABX处理的仓鼠中特别缺乏Roentibacter，这表明这种病原体可能在促进疾病进展中发挥作用。这些结果为抗生素预防人类VL患者继发细菌感染作为辅助治疗提供了实验支持。

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. 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 the inflammatory and immune reactions 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 intravital microscopy, the co-localization of L. major and neutrophils varied depending on the proximity of deposited parasites to the 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. The heterogeneity of sand fly transmission sites with respect to the dose of parasites and the early cellular interactions that we describe likely contribute to the wide range of infection outcomes that are associated with natural transmission of L. major observed in mouse models and humans. Leishmania donovani causes visceral leishmaniasis (VL), which is typically fatal without treatment. There is substantial variation between individuals in rates of disease progression and response to treatment. The reasons for heterogeneous disease presentation in humans and animal models are poorly understood. Host microbiota has become established as a factor in cutaneous forms of leishmaniasis but this has not been studied in VL. We induced intestinal dysbiosis in mice and hamsters by long-term treatment with broad-spectrum antibiotics in their drinking water. There were no significant differences in disease presentation in dysbiotic mice. In contrast, dysbiotic hamsters infected with L. donovani had delayed onset and progression of weight loss. Antibiotic-treated hamsters also had significantly less severe hepatosplenomegaly, which was accompanied by a distinct cytokine gene expression profile. The protective effect was not explained by differences in parasite loads or hematological profiles. We further found evidence that the gut-liver axis is a key aspect of fatal VL progression in hamsters, including intestinal parasitism, bacterial translocation to the liver, malakoplakia and iron sequestration, none of which occurred in non-progressing murine VL. Diverse bacterial genera were cultured from VL affected livers, of which Rodentibacter was specifically absent from ABX-treated hamsters, indicating this pathobiont may play a role in promoting disease progression. The results provide experimental support for antibiotic prophylaxis against secondary bacterial infections as an adjunct therapy in human VL patients.