探討內溶小體離子通道TRPML2在巨噬細胞M1極化與白色念珠菌的交互作用

Abstract

巨噬細胞，特別是M1表型，對於維持體內恆定以及在病原感染期間提供宿主防禦扮演關鍵角色。鈣訊號在巨噬細胞極化過程中具有核心調控功能，尤其在誘導M1 表型的過程中扮演重要角色。雖然過往研究已廣泛探討細胞外鈣離子攝取與內質網釋放之機制，但內溶小體(endolysosome)作為細胞內最主要的酸性鈣離子儲庫其內的鈣離子在巨噬細胞極化中是否存在關聯則相對未明。因此，本研究聚焦於兩種內溶小體陽離子通道--TPC2與TRPML2，這兩者主要表現在單核吞噬細胞系統中。我們以THP-1衍生之巨噬細胞作為模型，系統性地分析其向M1型極化過程中的變化，並探討內溶小體離子通道在抗白色念珠菌(Candida albicans)免疫中的角色。 首先，我們觀察到THP-1細胞自圓形的M0巨噬細胞轉變為具胞質延伸不規則形狀的M1巨噬細胞。接著我們發現，在極化早期(LPS與IFN-γ處理6小時)，TRPML2而非TPC2出現短暫性表現上升。另外，我們也透過內溶小體膜片鉗技術(endolysosomal patch-clamp)記錄證實，M0與M1巨噬細胞中皆具功能性TPC2電流，與基因表現結果一致。我們進一步以鈣指示劑(Oregon Green 488 Dye)測定內溶小體相對鈣含量後發現，M1巨噬細胞的鈣含量明顯高於M0，此現象在野生型(WT)和TPC2敲除細胞中都可以觀察到，然而在TRPML2敲除細胞中此差異消失。此外，使用TRPML2激動劑ML2-SA2處理後可提升細胞內鈣含量，而抑制劑ML-SI3處理則未顯著改變鈣含量。其次，在極化過程中加TPC2或TRPML2的專一性激動劑以增加通道活性，不論是在低濃度(1 μM)或是較高濃度(10 μM)的刺激下，都並未提升TNF-α與IL-6的表現，這些結果說明單純透過這些通道釋放鈣離子不足以驅動促發炎細胞激素的轉錄表現。最後，我們發現TRPML2的表現量在野生型及ece1Δ突變株的白色念珠菌感染中均於感染後期顯著上升，顯示TRPML2可能參與抗真菌免疫反應。總結而言，本研究揭示了TRPML2在調控內溶小體鈣動態與抗真菌免疫中的潛在功能，並強調其作為調節巨噬細胞功能狀態的重要性。

Macrophages, particularly the M1 phenotype, are essential for maintaining homeostasis and host defense during pathogen infection. Calcium signaling plays a pivotal role in macrophage polarization, especially in the transition to the M1 phenotype. While previous studies have extensively explored extracellular calcium uptake and calcium release from the endoplasmic reticulum (ER), the role of lysosomes, as primary intracellular acidic calcium reservoirs, remains poorly understood. In this study, we focus on two endolysosomal cation channels, TPC2 and TRPML2, which are predominantly expressed in the mononuclear phagocyte system. Using THP-1-derived macrophage as a model, we aim to perform a systematic analysis of macrophage polarization to M1 phenotype and investigate the role of endolysosomal ion channel in antifungal immunity against Candida albicans. First, we observed that THP-1 cells underwent a morphological change from rounded M0 to irregular M1 macrophages. We also demonstrated that TRPML2, but not TPC2, was transiently upregulated at the early stage of M1 polarization (LPS and IFN-γ treated for 6 hours). Patch-clamp recordings further confirmed the functional presence of TPC2-like currents in both M0 and M1 macrophages, consistent with gene expression data. Notably, we revealed that relative calcium content in endolysosome was significantly elevated in M1 macrophages compared to M0, a difference abolished in TRPML2 knockout cells but preserved in TPC2 knockouts. In addition, pharmacological activation of TRPML2 with ML2-SA2 elevated calcium content, whereas its inhibition with ML-SI3 showed no significant difference. Secondary, our results showed that increasing TPC2 or TRPML2 channel activity by applying channel-specific agonists during polarization failed to enhance the expression of TNF-α and IL-6, suggesting that calcium efflux via these channels alone is insufficient to drive polarization in term of pro-inflammatory cytokine transcription. Third, we found that TRPML2 expression was significantly induced upon both WT and ece1Δ mutant strain Candida albicans prolonged infection, indicating that TRPML2 play a potential role in response to antifungal immunity. To sum up, these findings highlight a previously underappreciated role of TRPML2 in calcium regulation and antifungal immunity and suggest its potential as a modulator of macrophage functional states.