Collaborative Research: Macrophages: Guardians of amphibian skin antifungal defenses

真菌感染是一个日益严重的野生动物问题，因为它们对生物多样性和生态系统健康造成破坏性影响。例如，壶菌真菌，树枝状瓶壶菌（Batrachochytrium dendrobatium，Bd）感染两栖动物的皮肤，导致壶菌病，这是世界范围内两栖动物减少和灭绝的主要原因。由于Bd最近出现、宿主范围广泛以及持续传播到新的地理区域，因此被认为是一种新兴病原体。游动芽孢杆菌游动孢子寄生在成年两栖动物的角质化皮肤和蝌蚪的角质化口器上。这些侵入的游动孢子发育成壶状游动孢子囊，其中新的游动孢子发育并游出来感染皮肤的其他区域或新的宿主。与其他真菌感染进展到远端器官不同，Bd仍然局限于皮肤，通过干扰穿过皮肤的基本离子运输而导致死亡，从而导致心脏骤停。由于巨噬细胞谱系细胞，如朗格汉斯细胞，是皮肤监视和随后的免疫反应不可或缺的，这些细胞无疑是重要的抗真菌防御皮肤局部的Bd感染。反过来，所有脊椎动物巨噬细胞的分化和功能依赖于集落刺激因子-1受体（CSF-1 R），其由CSF-1和无关的白细胞介素-34（IL-34）细胞因子连接。我们之前表明，通过重组（r）CSF-1和rIL-34分化的非洲爪蟾蛙巨噬细胞具有显着不同的识别和响应各种病原体的能力。通过这些NSF资助的研究，我们将研究这些功能二分的青蛙巨噬细胞亚群如何有助于青蛙Bd的易感性和抵抗力。该项目将吸收各级学生参加，PI将举办年度研讨会，探讨妇女和少数民族在科学领域的挑战和机遇，并通过教学和新的合作网站吸引公众注意力到Bd日益增长的威胁。郎格罕细胞的分化取决于IL-34，而CSF-1在这些过程中的作用仍有待充分界定。相应地，富集X.非洲蛙皮肤CSF-1-巨噬细胞导致更大的Bd易感性，而具有更多皮肤IL-34-巨噬细胞的青蛙表现出对该病原体的显著更大的抗性。因此，指导我们研究的中心假设是皮肤中对Bd的有效免疫应答取决于来自壶菌侵入的皮肤细胞的“警报应答”，其与IL-34-巨噬细胞结合，导致其他效应物的募集。相反，受损的青蛙对Bd的免疫应答源于皮肤驻留的巨噬细胞和皮肤上皮细胞由于真菌、感染的角质形成细胞和/或驻留的CSF-1-巨噬细胞产生免疫抑制因子而未能产生适当的应答。我们的研究的目的是：定义两栖类巨噬细胞亚群和Bd之间的相互作用;定义Bd对青蛙巨噬细胞和淋巴细胞之间的相互作用的影响;并定义在Bd感染的背景下青蛙皮肤内的细胞相互作用。我们的研究对青蛙巨噬细胞和Bd之间的免疫相互作用有了深入的了解，将为青蛙对这种致命真菌的免疫反应的有效性和局限性提供急需的视角。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

Fungal infections are a growing wildlife concern because of their devastating effects on biodiversity and ecosystem health. For example, the chytrid fungus, Batrachochytrium dendrobatidis (Bd) infects amphibian skin resulting in the disease chytridiomycosis, which is a leading cause of worldwide amphibian declines and extinctions. Due to its recent appearance, broad host range, and continuing spread into new geographic areas, Bd is regarded as an emerging pathogen. Swimming Bd zoospores colonize the keratinized skin of adult amphibians and the keratinized mouthparts of tadpoles. These invading zoospores develop into urn-shaped zoosporangia in which new zoospores develop and swim out to infect other areas of the skin or new hosts. Unlike other fungal infections that progress to distal organs, Bd remains confined to the skin, causing death by interfering with the essential ion transport across the skin, thereby leading to cardiac arrest. As macrophage-lineage cells, such as Langerhans cells, are integral to skin surveillance and the ensuing immune responses, these cells are undoubtedly important to the antifungal defenses against the skin-localized Bd infections. In turn, the differentiation and functionality of all vertebrate macrophages depend on the colony-stimulating factor-1 receptor (CSF-1R), which is ligated by CSF-1 and the unrelated interleukin-34 (IL-34) cytokines. We previously showed that the Xenopus laevis frog macrophages differentiated by recombinant (r)CSF-1 and rIL-34 possess markedly distinct capacities to recognize and respond to various pathogens. Through these NSF-funded studies, we will examine how these functionally dichotomous frog macrophage subsets contribute to frog Bd susceptibility and resistance. This project will incorporate students at all levels, the PIs will host annual workshops that explore the challenges and opportunities for women and minorities in sciences and public attention will be drawn to the growing threat of Bd through pedagogy and a new collaborative website.The differentiation of Langerhans cells depends on IL-34 while the roles of CSF-1 in these processes remain to be fully defined. Pertinently, enriching X. laevis skin CSF-1-macrophages results in greater Bd susceptibility whereas frogs with more skin IL-34-macrophages exhibit significantly greater resistance against this pathogen. As such, the central hypothesis guiding our studies is that an effective immune response to Bd in the skin depends on an “alarm response” from chytrid-invaded skin cells, which in conjunction with IL-34-macrophages, results in recruitment of other effectors. Conversely, an impaired frog immune response to Bd stems from failure of the skin-resident macrophages and skin epithelial cells to mount appropriate responses due to the production of immunosuppressive factors by the fungus, the infected keratinocytes, and/or resident CSF-1-macrophages. The objectives of our studies are: to define the interactions between amphibian macrophage subsets and Bd; to define the effects of Bd on the interactions between frog macrophages and lymphocytes; and to define the cellular interactions within the frog skin in the context of Bd infections. The in-depth understanding of immune interactions between frog macrophages and Bd granted by our studies, will provide a much-needed perspective into the effectiveness and limitations of the frog immune responses to this deadly fungus.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.