Collaborative Research: Macrophages: Guardians of amphibian skin antifungal defenses

真菌感染是野生动物日益关注的问题，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，壶菌巴氏杆菌(Bd)感染两栖动物皮肤，导致壶菌病，这是世界范围内两栖动物衰退和灭绝的主要原因。由于它最近出现，寄主范围广，并不断向新的地理区域扩散，因此被认为是一种新的病原体。游动的Bd游动孢子定植于成年两栖动物角化的皮肤和蝌蚪角化的口器。这些入侵的游动孢子发育成骨灰盒状的游动孢子囊，其中新的游动孢子发育并游出，感染皮肤的其他区域或新宿主。与其他进展到远端器官的真菌感染不同，Bd仍然局限在皮肤上，通过干扰皮肤上必要的离子运输而导致死亡，从而导致心脏骤停。由于巨噬细胞系细胞，如朗格汉斯细胞，是皮肤监测和随后的免疫反应不可或缺的细胞，这些细胞无疑对皮肤局部Bd感染的抗真菌防御至关重要。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-1R)，该受体由CSF-1和无关的白细胞介素34(IL-34)细胞因子连接。我们先前的研究表明，经重组(R)CSF-1和rIL-34分化的非洲爪哇蛙巨噬细胞具有明显不同的识别和应答各种病原体的能力。通过这些由NSF资助的研究，我们将研究这些功能上的二分青蛙巨噬细胞亚群如何促进青蛙Bd的易感性和抵抗力。该项目将吸收所有级别的学生参与，私营部门将主办年度讲习班，探讨妇女和少数群体在科学领域面临的挑战和机会，并将通过教学和一个新的合作网站吸引公众对勃起功能障碍日益增长的威胁的关注。朗格汉斯细胞的分化取决于IL-34，而CSF-1在这些过程中的作用仍有待充分确定。与之相关的是，皮肤巨噬细胞的数量越多，青蛙对Bd的易感性越高，而皮肤IL-34-巨噬细胞数量越多的蛙，对这种病原体的抵抗力就越强。因此，指导我们研究的中心假设是，皮肤对Bd的有效免疫反应依赖于龟裂菌入侵的皮肤细胞的“警报反应”，这与IL-34-巨噬细胞一起，导致其他效应器的招募。相反，青蛙对Bd的免疫反应受损是由于真菌、受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，导致驻留在皮肤的巨噬细胞和皮肤上皮细胞未能产生适当的反应。我们的研究目标是：确定两栖巨噬细胞亚群与Bd之间的相互作用；确定Bd对青蛙巨噬细胞与淋巴细胞之间相互作用的影响；以及在Bd感染的背景下，确定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进行了深入的了解，这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一个亟需的视角。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.