HMGB1/IL-1β complexes in plasma microvesicles modulate immune responses to burn injury

Modulating immune responses to sepsis and trauma remain one of the most difficult challenges in modern medicine. Large burn injuries (LBI) are a severe form of trauma associated with sepsis, immune impairment, and mortality. Immune dysfunction after LBI is complex, involving both enhanced and impaired immune activation. The release of Damage-Associated Molecular Patterns (DAMPs), such as HMGB1, and cytokines (e.g. IL-1β) creates an environment of immune dysfunction often leading to end organ failure and death. Both HMGB1 and IL-1β have been found to play critical roles in sepsis and post-burn immune dysfunction. HMGB1 and IL-1β have been shown previously to form potent complexes in vitro. We recently identified the presence of HMGB1/IL-1β heterocomplexes in human tissue. We now find HMGB1/IL-1β complexes in human and mouse plasma, and identify a synergistic role of HMGB1/IL-1β complexes in post-burn immune dysfunction. In both humans and mice, we found that HMGB1 was enriched in plasma microvesicles (MVs) after LBI. HMGB1 was found form complexes with IL-1β. Using flow cytometry of mouse plasma MVs, we identified an increase in an HMGB1+/IL-1β+ MVs. Using co-IP, HMGB1 was found to bind the pro-form of IL-1β in mouse and human plasma. Pro-IL-1β, which is traditionally considered inactive, became active when complexed with HMGB1. Human THP-1 monocytes treated with HMGB1-pro-IL-1β complexes showed increased transcription of LBI associated cytokines IL-6 and IFNβ along with suppression of iNOS, mimicking findings associated with LBI. These findings identify that HMGB1/IL-1β complexes released after burn injuries can modulate immune responses, and microvesicles are identified as a novel reservoir for these immune mediators. These complexes might serve as novel immune targets for the treatment of systemic immune responses due to LBI or other causes of sepsis.

调控脓毒症与创伤后的免疫应答，仍是现代医学领域最具挑战性的难题之一。大面积烧伤（Large Burn Injuries, LBI）属于严重创伤类型，常伴随脓毒症、免疫功能受损及死亡风险升高。大面积烧伤后出现的免疫功能紊乱机制复杂，兼具免疫激活过度与免疫应答低下两种表型。损伤相关分子模式（Damage-Associated Molecular Patterns, DAMPs，如高迁移率族蛋白B1（HMGB1））与细胞因子（如白细胞介素-1β（IL-1β））的释放，会构建免疫功能紊乱的微环境，最终常诱发多器官衰竭乃至死亡。已有研究证实，HMGB1与IL-1β在脓毒症及烧伤后免疫功能紊乱中均发挥关键调控作用。既往研究已证明，二者在体外可形成具有强生物活性的复合物。本团队此前已在人体组织中检测到HMGB1/IL-1β异源复合物。本次研究进一步在人类与小鼠血浆中发现了该复合物，并明确了HMGB1/IL-1β复合物在烧伤后免疫功能紊乱中的协同调控作用。在人类与小鼠模型中，我们均观察到大面积烧伤后，血浆微囊泡（microvesicles, MVs）内HMGB1的含量显著富集，且HMGB1可与IL-1β形成复合物。通过对小鼠血浆微囊泡进行流式细胞术分析，我们发现HMGB1+/IL-1β+ 微囊泡的比例显著升高。采用免疫共沉淀（co-IP）实验，我们证实HMGB1可在小鼠与人类血浆中结合IL-1β的前体形式（pro-IL-1β）。传统观点认为pro-IL-1β不具有生物活性，但当其与HMGB1结合形成复合物后，可被激活并发挥功能。用HMGB1-pro-IL-1β复合物处理人THP-1单核细胞后，可观察到与大面积烧伤相关的细胞因子IL-6及干扰素-β（IFNβ）的转录水平上调，同时诱导型一氧化氮合酶（iNOS）的表达受到抑制，这一表型与大面积烧伤后的免疫状态一致。本研究明确了烧伤后释放的HMGB1/IL-1β复合物可调控机体免疫应答，并证实微囊泡是这类免疫介质的新型储存载体。这类复合物有望成为针对大面积烧伤或其他病因引发脓毒症的全身性免疫应答的新型免疫治疗靶点。