Boonhok Rachasak, Senghoi Wilaiwan, Sangkanu Suthinee, Lim Chooi Ling, Pudla Matsayapan, Pereira Maria de Lourdes, Wilairatana Polrat, Mahboob Tooba, Rahman Md Atiar, Utaisincharoen Pongsak, Hiransai Poonsit, Nissapatorn Veeranoot
Department of Medical Technology, School of Allied Health Sciences, and Research Excellence Center for Innovation and Health Products (RECIHP), Walailak University, Nakhon Si Thammarat 80160, Thailand.
Department of Medical Technology, School of Allied Health Sciences, and Center of Excellence Research for Melioidosis and Microorganisms (CERMM), Walailak University, Nakhon Si Thammarat 80160, Thailand.
Scientifica (Cairo). 2025 Mar 12;2025:3430892. doi: 10.1155/sci5/3430892. eCollection 2025.
Noncanonical autophagy including unconventional protein secretion has been extensively studied. Our work focused on a leaderless IL-1β protein secretion from human macrophage in response to components, culture supernatant (CS) and cell lysate (CL), as well as its association with macrophage autophagy. Phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages were treated with components of pathogenic (ATCC50739) and nonpathogenic (ATCC30010) strains in vitro. The data showed that treatment resulted in low IL-1β secretion from macrophages. In addition, CL of both strains was able to upregulate autophagy-related (Atg) protein 8, an autophagy marker, whereas CS downregulated Atg8 expression. We further manipulated autophagy and found that autophagy induction by starvation diminished IL-1β secretion while autophagy inhibition by 3-methyladenine (3MA) increased IL-1β secretion. Interestingly, in the presence of components either under starvation or 3MA treatment, IL-1β secretion was significantly reduced. Transcriptional expression of other ATG genes, i.e., ATG6, ATG7, and ATG5, were investigated and showed that their mRNA expression was maintained at the basal level under CS or CL treatment. Inflammasome-related genes, NLRP3 and CASPASE1, were upregulated following 50739 CS treatment but not in 50739 CL-treated condition. However, both conditions were able to increase IL-1β mRNA expression. TEM micrographs revealed that 3MA treatment induced the formation of large vacuoles and accumulation of autophagosome at the edge of THP-1 macrophages. However, the number and size of their structures were declined in the presence of 50739 CS with 3MA. Furthermore, immunofluorescence staining demonstrated the association between Atg8/LC3 and IL-1β expression, where downregulation of Atg8 by 50739 CS led to the upregulation of IL-1β. Altogether, the data indicate that can manipulate macrophage autophagy, thereby controlling low IL-1β secretion. The expression of autophagy- and inflammasome-related genes also indicates multiple mechanisms in IL-1β secretion in response to components. However, further characterization of Atg proteins and investigations into other intracellular pathways or defense mechanisms are needed to fully understand the unconventional secretion of IL-1β in macrophages. This knowledge could eventually lead to the development of innovative therapeutic strategies against infection by modulating autophagy or macrophage responses.
包括非常规蛋白质分泌在内的非经典自噬已得到广泛研究。我们的工作聚焦于人类巨噬细胞在病原体成分、培养上清液（CS）和细胞裂解物（CL）刺激下无信号肽的白细胞介素-1β（IL-1β）蛋白分泌，以及其与巨噬细胞自噬的关联。用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）诱导的THP-1巨噬细胞在体外接受致病性（ATCC50739）和非致病性（ATCC30010）菌株的成分处理。数据显示，该处理导致巨噬细胞分泌的IL-1β水平较低。此外，两种菌株的CL均能上调自噬相关（Atg）蛋白8（一种自噬标志物）的表达，而CS则下调Atg8的表达。我们进一步对自噬进行调控，发现饥饿诱导自噬会减少IL-1β的分泌，而3-甲基腺嘌呤（3MA）抑制自噬则会增加IL-1β的分泌。有趣的是，在饥饿或3MA处理的情况下，病原体成分的存在会显著降低IL-1β的分泌。对其他自噬相关基因（即ATG6、ATG7和ATG5）的转录表达进行了研究，结果表明在CS或CL处理下它们的mRNA表达维持在基础水平。炎性小体相关基因NLRP3和CASPASE1在ATCC50739菌株的CS处理后上调，但在ATCC50739菌株的CL处理条件下未上调。然而，两种情况均能增加IL-1β的mRNA表达。透射电子显微镜照片显示，3MA处理诱导THP-1巨噬细胞边缘形成大液泡并积累自噬体。然而，在3MA存在的情况下，ATCC50739菌株的CS会使这些结构的数量和大小减少。此外，免疫荧光染色显示Atg8/微管相关蛋白轻链3（LC3）与IL-1β表达之间存在关联，其中ATCC50739菌株的CS对Atg8的下调导致IL-1β的上调。总之，数据表明病原体成分可调控巨噬细胞自噬，从而控制低水平的IL-1β分泌。自噬和炎性小体相关基因的表达也表明在对病原体成分的反应中，IL-1β分泌存在多种机制。然而，需要对Atg蛋白进行进一步表征，并研究其他细胞内途径或防御机制，以全面了解巨噬细胞中IL-1β的非常规分泌。这一知识最终可能会通过调节自噬或巨噬细胞反应，推动针对病原体感染的创新治疗策略的开发。
