CTLA-4阻断改善了肺副球虫病小鼠模型的抗真菌治疗结果

摘要(中文译文)

副球孢子菌病（PCM）是一种在美洲流行的系统性真菌感染，在巴西发病率最高。目前的治疗依赖于两性霉素B（AmB）、伊曲康唑和氟康唑等抗真菌药物。然而，长期治疗常因药物毒性、慢性炎症和纤维化而导致治疗中断和/或后遗症，这些问题可严重损害器官功能和生活质量。因此，免疫调节疗法应运而生，包括使用针对检查点分子CTLA-4的单克隆抗体。此前，我们已证明CTLA-4阻断可通过增强保护性免疫反应来降低真菌负荷、减轻肺部病变严重程度并提高生存率。因此，本研究旨在评估抗CTLA-4与两性霉素B的联合治疗。C57BL/6小鼠经气管内接种1×10⁶个巴西副球孢子菌酵母细胞，感染6周后接受抗CTLA-4和/或AmB治疗。治疗两周后，通过菌落形成单位（CFU）计数、组织病理学和生存分析评估疾病进展。通过酶联免疫吸附试验（ELISA）和流式细胞术评估免疫反应。抗CTLA-4联合AmB治疗实现了更好的疾病控制，表现为肺、肝和脾中真菌负荷降低，肺组织损伤减轻，与单药治疗相比生存率提高。此外，肺部树突状细胞和中性粒细胞浸润减少、活化标志物CD80和CD86表达降低以及CD4⁺T细胞数量减少，提示抗原刺激减弱使疾病得到控制。这些发现表明，CTLA-4介导的免疫调节与两性霉素B的抗真菌活性协同作用以控制小鼠PCM，支持联合治疗优于单药治疗。

Abstract

Paracoccidioidomycosis (PCM) is a systemic fungal infection endemic in the Americas, with the highest incidence in Brazil. Current treatment relies on antifungal agents such as amphotericin B (AmB), itraconazole, and fluconazole. However, long-term therapy frequently leads to treatment discontinuation and/or sequelae due to drug toxicity, chronic inflammation, and fibrosis, which can severely impair organ function and quality of life. Accordingly, immunomodulatory therapies have emerged, including the use of monoclonal antibodies against the checkpoint molecule CTLA-4. Previously, we demonstrated that CTLA-4 blockade reduced fungal burden, decreased lung lesion severity, and increased survival by enhancing protective immune responses. Therefore, this study aimed to evaluate a combined therapy of anti-CTLA-4 and amphotericin B. C57BL/6 mice were inoculated intratracheally with 1 × 10⁶ Paracoccidioides brasiliensis yeast cells and, after 6 weeks of infection, treated with anti-CTLA-4 and/or AmB. Two weeks after treatment, disease progression was evaluated by CFU counts, histopathology, and survival analysis. Immune responses were assessed by ELISA and flow cytometry. The combined anti-CTLA-4 + AmB therapy resulted in improved disease control, evidenced by reduced fungal burden in the lungs, liver, and spleen, decreased pulmonary tissue damage, and increased survival compared to monotherapies. Additionally, reduced infiltration of dendritic cells and neutrophils, lower expression of activation markers CD80 and CD86, and fewer CD4⁺ T cells in the lungs suggest controlled disease due to diminished antigenic stimulation. These findings demonstrate that CTLA-4-mediated immune modulation and the antifungal activity of amphotericin B act complementarity to control murine PCM, supporting combined therapy as superior to monotherapy.

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