IMMUNE REGULATION AND VACCINE DEVELOPMENT IN LEISHMANIASIS

组织驻留巨噬细胞(TRMS)对组织内稳态/修复至关重要，其发育受组织生态位特异性信号的调控。尽管这些滋养信号对TRMS执行组织特异性功能的重要性已经进行了广泛的研究，但关于TRMS如何在炎症过程中保持自身的动态平衡特性，特别是感染驱动的皮肤炎症，仍然没有得到充分的研究。我们先前的研究表明，嗜酸性粒细胞与TRM的协同作用对于在主要致炎乳杆菌感染的强致炎环境中维持真皮TRMS的M2样属性非常重要；嗜酸性粒细胞向真皮TRMS提供IL-4，而IL-4刺激的真皮TRMS产生CCL24(嗜酸性粒细胞趋化因子)，而CCL24(嗜酸性粒细胞趋化因子)反过来又能招募更多的嗜酸性粒细胞。在我们目前的研究中，我们已经确定了两个独立的真皮TRMS亚群，MHCII+甘露糖受体(MR)低和MHCII-MR Hi群体。单细胞RNA序列显示，真皮TRMS的MHCII-MR Hi亚群是感染皮肤中唯一产生CCL24的亚群，也是激活固有淋巴样细胞2(ILC2)所需的ALARIN胸腺基质淋巴生成素(TSLP)的唯一来源，ILC2产生IL-5以放大嗜酸性粒细胞-TRM相互作用。选择性去除IL-5+ILC2或基因消融TSLP都会损害维持M2样真皮TRMS数量和功能所需的局部TH2回路，从而改善疾病进展。因此，在大型乳杆菌感染真皮强烈的促炎环境中，真皮TRM通过产生CCL24和TSLP来主动维持自身，局部TH2回路的任何破坏都会改变它们的数量和激活状态，促进更强的抗感染能力。 内脏利什曼病(VL)是由杜氏利什曼原虫(L.donovani/L.infantum)复合体内的利什曼原虫引起的一种热带疾病。肝、脾和骨髓中的巨噬细胞是感染的主要靶细胞。Kupffer细胞(KCs)是胚胎来源的、驻留在肝脏的巨噬细胞，其特征是表达Clec4f和Tim4。在动态平衡中，KCs通过自我增殖来维持其数量，但在某些炎症环境中，它们可能会死亡，并被单核细胞来源的细胞(MoKCs)取代。在小鼠VL模型中，KCs对于寄生虫在肝脏中的初始生长和肉芽肿的形成都是重要的，这与该组织最终的保护性反应有关。在VL中，从来没有研究过KC死亡，它们被moKCs取代，以及KCs和moKCs的功能。在感染婴儿乳杆菌的C57BL/6小鼠中，在感染后42天，我们发现了KC凋亡的证据，用共聚焦显微镜裂解的caspase 3染色来检测，用流式细胞仪的脂质过氧化检测来检测铁下垂。KCs中Clec4f和Tim4的表达持续下降，而Ly6c和CD11c等单核细胞标志物表达增强。作为慢性感染期间它们起源于单核细胞的进一步证据，CCR2-/-小鼠的KC数量比WT小鼠低，在涉及同源异生小鼠的实验中，肉芽肿内的KC表达两种同源标记。用Clec4f-Cre-DTR小鼠在感染前选择性去除KC并用moKCs替代，表明moKCs的优势导致较低的寄生虫量，表明moKCs在控制感染方面更有效。因此，KCs的死亡和它们被单核细胞来源的细胞替代似乎是该器官保护性反应的中心。

Tissue-resident macrophages (TRMs) are critical for tissue homeostasis/repair, and their development is governed by tissue niche-specific signals. Although the importance of these nurturing signals for TRMs to perform tissue-specific functions has been extensively studied, it remain poorly addressed how TRMs maintain their homeostatic properties during inflammation, in particular infection-driven inflammation in the skin. We previously showed the importance of eosinophil-TRM cooperative interactions for the maintenance of the M2-like properties of dermal TRMs within the strong pro-inflammatory environment of the L. major infected dermis; eosinophils provide IL-4 to dermal TRMs, and IL-4-stimulated dermal TRMs produce CCL24 (eotaxin) which in turn functions to recruit more eosinophils. In our current studies, we have identified two independent subsets of dermal TRMs, MHCII+ mannose receptor (MR) low and MHCII- MR hi populations. By single cell RNA seq, the MHCII- MR hi subset of dermal TRMs was shown to be the sole producer of CCL24 in the infected skin, and was also found to be the sole source of the alarmin thymic stromal lymphopoietin (TSLP) required to activate innate lymphoid cells 2 (ILC2), which produce IL-5 to amplify eosinophil-TRM interactions. Both selective depletion of IL-5+ ILC2 or genetic ablation of TSLP impaired the localized TH2 circuitries required to maintain the numbers and functionality of M2-like dermal TRMs, and disease progression was ameliorated. Thus, in the setting of the strong pro-inflammatory environment of the L. major infected dermis, dermal TRMs actively maintain themselves by producing CCL24 and TSLP, and any disruption of the localized TH2 circuitries will alter their number and activation states, and promote stronger resistance to infection. Visceral leishmaniasis (VL) is a tropical disease caused by Leishmania species within the L. donovani complex (L. donovani/L. infantum). Macrophages in the liver, spleen, and bone-marrow are the principal target cells for infection. Kupffer cells (KCs) are the embryonic-derived, liver resident macrophages, and are characterized by Clec4f and Tim4 expression. In homeostasis, KCs maintain their numbers via self-proliferation, but in some inflammatory settings they can die and be replaced by monocyte-derived cells (moKCs). In the murine VL model, KCs are important for both the initial growth of the parasite in the liver and for granuloma formation, which is associated with the eventual protective response in this tissue. KC death, their replacement by moKCs, and the functionality of KCs vs moKCs has never been investigated in VL. In C57BL/6 mice infected with L. infantum, at 42 days post-infection we found evidence of KC apoptosis, measured by cleaved caspase 3 staining by confocal microscopy, and ferroptosis, by lipid peroxidation detection by flow cytometry. Consistently, Clec4f and Tim4 expression in KCs was reduced, while monocyte markers such as Ly6C and CD11c were enhanced. As further evidence of their monocytic origin during chronic infection, KC numbers were lower in CCR2-/- mice when compared to WT mice, and in experiments involving congenic parabiotic mice, the KCs present within the granulomas expressed both congenic markers. Selective KC depletion and replacement by moKCs prior to infection using Clec4f-Cre-DTR mice, demonstrated that the predominance of moKCs resulted in lower parasite loads, indicating that moKCs are more effective in controlling infection. Thus, KCs death and their replacement by monocyte-derived cells appears to be central to the protective response in this organ.