Santos Mariana Souza, da Silva Rodrigo A, de Souza Lucas Nunes, Victorino Nicolli R, Pereira Rayane C B, Xavier José Guilherme, Araújo Ronalda da Silva, Pérez Elizabeth C, Alvares-Saraiva Anuska M, Lallo Maria Anete
Programa de Patologia Ambiental e Experimental, Universidade Paulista (UNIP), São Paulo, Brasil.
CETESB-Companhia Ambiental do Estado de São Paulo, São Paulo, Brasil.
PLoS Negl Trop Dis. 2025 Sep 8;19(9):e0012130. doi: 10.1371/journal.pntd.0012130. eCollection 2025 Sep.
Microsporidia causes opportunistic infections in immunosuppressed individuals. Mammals shed these spores of fungi in feces, urine, or respiratory secretions, which could contaminate water and food, thereby reaching the human body and causing infection. The oral route is the most common route of infection, although experiments have demonstrated that intraperitoneal and intravenous routes may also spread infection. Respiratory tract infection, although considered to be possible, has not been reported to date. The present study, therefore, aimed to demonstrate infection with the microsporidia of Encephalitozoon cuniculi via the oropharyngeal route as a model for opportunistic pneumonia. The objectives were the study of opportunistic pneumonia in general while also confirming transmission via the respiratory route to expand the understanding of the epidemiology of zoonoses. C57BL mice, both male and female, up to 12 weeks of age, and free of specific pathogens (SPF) were inoculated (Infected group) or not infected (Uninfected group) with 1 × 107 spores of E. cuniculi via the oropharyngeal route. The animals immunosuppressed with cyclophosphamide (Cy) were infected (Cy-Infected group) or not infected (Cy-Uninfected group), and then assessed for the influence of immunosuppression on infection. In both groups, animals inoculated with 0.9% saline solution via the oropharyngeal route served as controls (Sham and Cy-Sham groups, respectively). After 14 days of infection, the lungs of all animals were retrieved for histopathological analysis, phenotyping of lung inflammatory cells using flow cytometry, measurement of fungal load using qPCR, and measurement of the serum levels of Th1, Th2, and Th17 cytokines. The results revealed that the infected animals developed interstitial pneumonia characterized by perialveolar inflammatory infiltrative lesions with a predominance of lymphocytes and plasma cells. However, the fungal load and the extent of the inflammatory infiltrate were relatively lower in the Cy-Infected group, with a predominance of the CD8+ T lymphocyte population in the lungs compared to Infected group. In the Infected group not treated with Cy, an increase in the population of alveolar macrophages (F4/80+CD11b-SiglecF+) was noted, along with higher fungal load and inflammatory infiltrate in the lungs, indicating a further pronounced Encephalitozoon pneumonia compared to the immunosuppressed animals. The infected groups presented Th1 cytokine profiles, with the Cy-Infected group exhibiting increased Th17 levels. Collectively, these results demonstrated that oropharyngeal infection promoted pneumonia caused by E. cuniculi in mice treated or not with cyclophosphamide, with greater severity occurring in the non-immunosuppressed mice, thereby establishing this model as a suitable one for interstitial pneumonia via aspiration.
微孢子虫可在免疫抑制个体中引起机会性感染。哺乳动物通过粪便、尿液或呼吸道分泌物排出这些真菌孢子，这些孢子可能污染水和食物，进而进入人体并引发感染。尽管实验表明腹腔内和静脉内途径也可能传播感染，但经口途径是最常见的感染途径。呼吸道感染虽被认为有可能，但迄今为止尚未见报道。因此，本研究旨在通过口咽途径感染兔脑炎微孢子虫，以此作为机会性肺炎的模型。目标是总体研究机会性肺炎，同时确认经呼吸道途径的传播，以加深对人畜共患病流行病学的理解。将12周龄以内、无特定病原体（SPF）的C57BL雌雄小鼠经口咽途径接种1×10⁷个兔脑炎微孢子虫孢子（感染组）或不接种（未感染组）。用环磷酰胺（Cy）免疫抑制的动物进行感染（Cy感染组）或不感染（Cy未感染组），然后评估免疫抑制对感染的影响。在两组中，经口咽途径接种0.9%盐水溶液的动物作为对照（分别为假手术组和Cy假手术组）。感染14天后，取出所有动物的肺进行组织病理学分析、用流式细胞术对肺炎症细胞进行表型分析、用qPCR测量真菌载量以及测量血清中Th1、Th2和Th17细胞因子水平。结果显示，感染动物发生间质性肺炎，其特征为肺泡周围炎性浸润性病变，以淋巴细胞和浆细胞为主。然而，Cy感染组的真菌载量和炎性浸润程度相对较低，与感染组相比，肺中CD8⁺T淋巴细胞群体占优势。在未用Cy治疗的感染组中，肺泡巨噬细胞（F4/80⁺CD11b⁻SiglecF⁺）群体增加，同时肺中的真菌载量和炎性浸润更高，这表明与免疫抑制动物相比，兔脑炎微孢子虫肺炎更为明显。感染组呈现Th1细胞因子谱，Cy感染组Th17水平升高。总体而言，这些结果表明，无论是否用环磷酰胺治疗，口咽感染均可促进小鼠发生由兔脑炎微孢子虫引起的肺炎，在未免疫抑制的小鼠中病情更严重，从而确立该模型为适合吸入性间质性肺炎的模型。
