半乳糖凝集素-3對先天免疫細胞抗真菌功能之調控

Abstract

半乳糖凝集素-3廣泛地分佈在細胞內外甚至是膜上，它具有一個可辨識醣類的碳端及一個胜肽尾端。當許多文獻探討了它在自體免疫疾病、癌細胞轉移、細菌和寄生蟲感染、以及纖維化疾病中的角色，它在真菌感染中的重要性卻鮮少有人著墨。真菌感染是一種隱型殺手──據臨床統計指出，即便接受了抗真菌藥物的治療，真菌造成的全身性感染患者仍有高達一半的死亡率，顯示如何控制真菌感染及提供該感染有效治療的方法是全球的重要議題。而我的研究顯示了內生性的半乳糖凝集素-3會負調控先天免疫細胞抗組織胞漿菌及白色念珠菌的感染。 鮮少有人在組織胞漿菌感染時引發症狀，但若是免疫低落或免疫不全的病人在感染後卻極易發展成全身性感染，進而造成死亡。當肺部組織胞漿症的清除需仰賴第一型和第十七型免疫反應，我的研究指出半乳糖凝集素-3會負調控樹突細胞生成IL-17A相關的細胞激素之能力，進而造成CD4 T細胞和嗜中性顆粒球生成IL-17A的能力下降。IL-17A的存在可協助IFN-γ活化巨噬細胞、促使巨噬細胞內組織胞漿菌之生長被抑制。相較於一般小鼠，半乳糖凝集素-3基因剔除的小鼠感染後體內真菌量也因有較高量IL-17A生成而下降。以靜脈注射方式將樹突細胞送入感染的小鼠體內顯示了半乳糖凝集素-3對宿主IL-17A免疫反應和其體內真菌清除之調控乃是經由抑制樹突細胞生成細胞激素之能力造成。 白色念珠菌分佈在人類的皮膚和黏膜表面，是一種伺機性的雙性型真菌。在台灣醫療照護機構內全身性感染案例中，念珠菌感染佔了第一位。白色念珠菌之感染仰賴嗜中性顆粒球進行清除。我們發現未刺激前人類和小鼠的嗜中性顆粒球的半乳糖凝集素-3僅表現在胞內，而白色念珠菌刺激才誘使其在細胞表面被偵測到。當細胞透過CR3辨識調理過的白色念珠菌後，會透過活化磷酸激酶Syk促使細胞生成活性氧，直接毒殺真菌。半乳糖凝集素-3則會抑制Syk的活化，造成毒殺能力下降。共免疫沉降法和螢光染色顯示胞內的半乳糖凝集素-3可直接和Syk作用。而半乳糖凝集素-3基因剔除的小鼠，比起一般小鼠，在全身性白色念珠菌感染後體內真菌數量較低、腎臟病變程度也減緩、死亡率顯著下降。進一步以靜脈注射方式將嗜中性顆粒球送入感染的小鼠體內則證明了半乳糖凝集素-3是藉由調控嗜中性顆粒球的功能而抑制宿主抗真菌之能力。不僅是對實驗用菌株，比較了從病患血液中取得的臨床菌株，我們發現半乳糖凝集素-3也可以抑制嗜中性顆粒球毒殺這些臨床菌株的能力。人類嗜中性顆粒球在半乳糖凝集素-3拮抗劑處理後或以siRNA轉染的方式減少半乳凝集素-3基因表現後，其負責毒殺真菌的活性氧生成量也顯著上升。我們的研究提出了抑制嗜中性顆粒球胞內的半乳糖凝集素-3是一種可行的全身性白色念珠菌治療方式。

Galectin-3 (Gal3), belonging to the galectin family, has an evolutionarily conserved C-terminal carbohydrate-recognition domain and a unique N-terminal peptide. While it has been reported to modulate host response to autoimmune diseases, cancer metastasis, bacterial and parasite infections, and fibrosis, there are few studies address its role in fungal infection. Fungal infection as a hidden killer for human beings on the Earth leads to more than 50% mortality when disseminated even in patients with anti-fungal treatment. How to control its spread and provide a promising therapeutic strategy are universal issues. My studies on dimorphic Histoplasma capsulatum infection and opportunistic Candida albicans infection reveal the importance of endogenous gal3 in innate cell response to fungi. While most people live in endemic area infected by Histoplasma without any symptoms, its infection easily disseminates in immuno-depressed or immunocompromised patients and leads to death. The clearance of pulmonary Histoplasma infection is dependent on both Th1 and Th17 responses. My study shows that gal3 negatively regulated bone marrow-derived and splenic dendritic cell IL-17A-axis cytokine production. The level of IL-17A production by both CD4 T cells and neutrophils was higher in gal3-/- mice after systemic Histoplasma infection. IL-17A alone or combined with IFN-γ activates macrophage to inhibit the growth of intracellular Histoplasma. Gal3-/- mice had lower fungal burden in the spleen compared to gal3+/+ mice. Neutralizing IL-17A increased fungal burden in gal3-/- mice to a level comparable to that in gal3+/+ mice, while neutralizing IFN-γ increased fungal burden in both gal3+/+ and gal3-/- mice. Adoptive transfer of dendritic cells showed that cell intrinsic gal3 in dendritic cells negatively regulated host IL-17A-axis cytokine production and fungal clearance. In this study, we demonstrated a negative role of intrinsic gal3 in dendritic cell IL-17A-axis cytokine production as well as in fungal clearance. Candida albicans is an opportunistic dimorphic fungus, which is a commensal in the mucosa surface and skin in most humans. Invasive candidiasis is the first leading overall health-associated infection as well as bloodstream infection in intensive care units in Taiwan. Neutrophils are the major effector cells to clear fungal infection. We show that unstimulated human and mouse neutrophils express galectin-3 intracellularly, and it becomes detectable on the cell surface after stimulation by opsonized Candida. Upon CR3 engagement with opsonized Candida, galectin-3 downregulates Syk-mediated ROS production and subsequent killing of fungus. Co-immunoprecipitation and immunofluorescence staining reveal that cytosolic gal3 physically interacts with Syk. Moreover, galectin-3 deficiency ameliorates systemic candidiasis by reducing fungal burden, renal pathology, and mortality. Adoptive transfer experiments demonstrate that cell intrinsic gal3 negatively regulates neutrophil effector function in candidiasis. Additionally, the effect of galectin-3 on neutrophil anti-Candida function and host resistance to candidiasis is tested in clinical isolates. Treatment with galectin-3 antagonist or siRNA enhances human neutrophil ROS production. Our work raises the possibility that blocking gal3 in neutrophils may be a promising therapeutic strategy for treating systemic candidiasis.