動物模式闡明HLJ1缺失對內毒素引發敗血症之影響

Mouse model elucidates the impact of HLJ1 deficiency on immunomodulation during endotoxin-induced sepsis

羅偉嘉(Wei-Jia Luo)

指導教授 ： 蘇剛毅

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/博士(2022年)

https://doi.org/10.6342/NTU202203334

摘要

敗血症被定義為由宿主對感染的反應失調引起的危及生命的器官功能障礙，在重症監護患者中敗血症是最常見的死因，而全身性的細菌感染常是造成敗血性休克的主因，當細菌感染時，其膜上的脂多醣(LPS)主要透過活化TLR4受器，導致大量多種細胞激素分泌進而活化免疫系統。其中IL-12釋放後可透過活化自然殺手細胞，進而促進γ干擾素(IFN-γ) 釋放，先前研究指出IL-12與IFN-γ激素與器官衰竭及敗血性休克息息相關，因此針對阻斷IL-12與IFN-γ路徑是否可用以治療敗血症為一重要課題。HLJ1為熱休克蛋白40家族中成員之一，大量研究指出HLJ1蛋白在人類肺癌中為抑癌基因，我們利用HLJ1基因剔除小鼠探討HLJ1蛋白的生理功能，並發現其具有內質網壓力引發之脂質代謝異常與脂肪肝，除此之外HLJ1缺失導致小鼠對致肝癌劑較敏感，顯示其在維持肝臟功能恆定中扮演重要角色，然而HLJ1如何在肝臟中調控細菌脂多糖引發之發炎與敗血性休克仍不清楚。
本研究中我們初步給予小鼠不同劑量LPS刺激，發現HLJ1基因剔除小鼠存活顯著較野生型小鼠佳，且肝腎受損程度較低。利用細胞激素微陣列分析與中和抗體抑制細胞激素，發現此現象是由於HLJ1基因剔除小鼠體內IFN-γ減少所致，利用單細胞定序分析肝臟免疫細胞轉錄組，發現IFN-γ缺失小鼠自然殺手細胞、巨噬細胞、及樹突細胞內的IFN-γ相關訊息路徑活化異常，進一步利用流式細胞儀分析脾臟免疫細胞，我們確認了HLJ1缺失導致自然殺手細胞分泌較少IFN-γ。若是以CLP手術模擬活菌感染，仍可發現HLJ1基因剔除小鼠的IFN-γ表現量下降且器官受損較少，並且給予抗生素可使其活得較野生型小鼠佳。接下來利用中和抗體抑制細胞激素，發現IFN-γ減少是導因於IL-12表現量下降，因此我們將HLJ1缺失的巨噬細胞移植到野生型小鼠體內，發現其可降低IL-12及 IFN-γ表現並且小鼠存活明顯改善，顯示HLJ1在巨噬細胞中調控活體內IL-12生成與敗血性死亡。若分離巨噬細胞進行體外培養後給予LPS刺激，可發現HLJ1缺失細胞內與細胞外之IL-12蛋白量皆較對照組低。為了探討此分子機制，我們在293T細胞中過表達人類IL-12次單元IL-12p35，同時降低HLJ1表現量，結果發現HLJ1的減少導致錯誤折疊的IL-12p35蛋白同二聚體(homodimer)堆積，使得自然折疊的IL-12p35單體所佔的比例下降，IL-12p35單體的缺乏可能導致其與IL-12p40的異二聚化(heterodimerization)程度下降，最後使有正常功能的IL-12p70異二聚體 (heterodimer)無法合成與分泌。
總結來說， HLJ1在巨噬細胞中幫助IL-12蛋白折疊與釋放，使得自然殺手細胞表達過量IFN-γ，進而引發敗血症導致的器官受損與死亡。此研究結果說明未來針對HLJ1可以發展新穎的敗血症的治療策略，例如抑制HLJ1的藥物可搭配抗生素以減緩敗血症導致的死亡，而除了敗血症外，以HLJ1為治療標的的策略同樣可應用於治療存在IL-12與IFN-γ活化的免疫與發炎疾病。

關鍵字

敗血症 ； 脂多醣 ； 熱休克蛋白 ； 單細胞定序 ； 介白素12 ； 異二聚化

並列摘要

Sepsis is described as life-threatening organ dysfunction owing to a dysregulated host response to infection, and is a growing health problem remains to be solved since it is the single most encountered cause of mortality in intensive care patients. When infection occurs, LPS as an endotoxin initiates immune response and multiple cytokine production. Recently, HSP40 has emerged as a key factor in both innate and adaptive immunity, whereas the role of HLJ1, a molecular chaperone in HSP40 family, in modulating endotoxin–induced sepsis severity is still unclear.
In the study, we investigate the impact of HLJ1 on LPS-induced sepsis with HLJ1 whole-body knockout mice. During LPS-induced endotoxemia, HLJ1 knockout mice display significantly alleviated vital organ damage and mortality when compared with wild-type mice. With cytokine array analysis and cytokine neutralization experiments, we found HLJ1 amplifies IFN-γ-dependent mortality in vivo. We take adventage of single-cell RNA sequencing and characterize liver nonparenchymal cell transcriptome under LPS stimulation, and show that HLJ1 deletion affected IFN-γ-related gene signatures in macrophages, dendritic cells and NK cells. Splenocytes analyzed with intracellular staining validate diminished production of IFN-γ in HLJ1-deficient NK cells instead of CD4+ or CD8+ T cells. In addition, during CLP-induced sepsis with live bacterial infection, HLJ1-deleted mice show reduced IFN-γ expression and alleviated organ injury. CLP-associated mortality rate decreases as well when HLJ1 knockout mice receive systemic antibiotics.
By using ELISA analysis, we found LPS-treated mice exhibit reduced serum levels of IL-12 after HLJ1 deletion. IL-12 neutralization experiment demonstrates that diminished IL-12 contributing to dampened production of IFN-γ and subsequent improved survival. Transplantation of wild-type macrophages into HLJ1 knockout mice significantly elevates serum levels of IL-12 as well as IFN-γ and confers lethality, indicating HLJ1 functions mainly in macrophages upon LPS stimulation. On the other hand, adoptive transfer of HLJ1-deleted macrophages into LPS-treated mice results in reduced IL-12 as well as IFN-γ levels and protects the mice from IFN-γ–dependent mortality. In the context of molecular mechanisms, HLJ1 protein is significantly induced in macrophages, where it converts misfolded IL-12p35 homodimers to monomers, maintaining bioactive IL-12p70 heterodimerization and secretion. Finally, we found HLJ1 can be detected in serum and its amount is positively correlated to serum IL-12 when LPS-induced sepsis occurs.
In summary, HLJ1 in macrophages causes IFN-γ–dependent septic lethality by promoting IL-12 heterodimerization, suggesting HLJ1-targeting agent has therapeutic potential and provides novel strategy for inflammatory diseases involving activated IL-12/IFN-γ-axis. In addition, HLJ1 deletion results in reduced CLP-induced mortality when mice are treated with antibiotics, implying combined therapy provides novel treatment regimens for sepsis. Finally, HLJ1 might be a serum biomarker for patient selection as its expression correlated to serum levels of IL-12.

並列關鍵字

Sepsis ； Lipopolysaccharide ； Heat shock protein ； Single-cell RNA sequencing ； IL-12 ； heterodimerization

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