經由系統性發炎至多重器官衰竭的嶄新觀點來探討敗血症患者預後生物指標

Explore prognostic factors in sepsis patients from systemic inflammation to novel insights of multi-organ dysfunction

鐘桂彬(Kuei-Pin Chung)

指導教授 ： 余忠仁

共同指導教授 ： 郭錦樺(Ching-Hua Kuo)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2019年)

https://doi.org/10.6342/NTU201904291

摘要

緒論
敗血症是由微生物感染所引起的臨床症候群，病態生理的主要特徵是異常的系統性免疫反應。敗血症的治療沒有太大的進展，同時，不同臨床試驗間，結果並不完全一致。這可能是因為敗血症患者族群的異質性所導致。因此，我們需要進一步探索和敗血症併發之急性器官功能障礙相關的預後生物指標，以更精準的定義和依疾病嚴重度來分類敗血症患者族群。
研究目的
在本論文中，我們將聚焦於敗血症所引起的免疫功能障礙以及系統性代謝途徑的改變，發現可以於敗血症患者臨床照護應用的潛在預後生物指標。
研究材料與方法
我們進行前瞻性研究，召募轉入內科加護病房，診斷為敗血症合併急性器官功能障礙的成人患者(年齡20歲以上)。對於脂肪酸β氧化反應的目標代謝體研究，我們排除罹患有慢性腎病和肝硬化的患者。敗血症患者若周邊血淋巴球數目小於0.5 × 10^3/μL，則定義為併發嚴重周邊血淋巴球低下。我們同時收集周邊血單核細胞，以萃取核醣核酸(RNA)或是進行流式細胞儀分析。患者血中代謝物濃度則使用高效能液相層析質譜儀進行分析。
結果
我們發現發生嚴重淋巴球低下的敗血症患者，血中腫瘤壞死因子α、介白素6、8和10的濃度較高，並且有較高的28天死亡率(校正後風險比值1.262; 95%信賴區間 1.482~8.416, P = 0.004; 經由考克斯比例風險模式計算)。敗血症引發之嚴重淋巴球低下會伴隨血中介白素15濃度輕度而顯著的升高，但是不會合併血中介白素7濃度的顯著變化，同時也會伴隨周邊血單核細胞的BCL2 信使核醣核酸(messenger RNA, mRNA)的表現量顯著較低。我們發現死亡的敗血症患者其血中效應CD4+ T淋巴球的比例顯著較高，但是，並沒有發現任何和院內感染的風險相關的淋巴球族群或是表面標記的變化。
系統代謝體分析研究結果指出，脂肪酸β氧化反應的相關代謝物，可能是潛在敗血症預後相關的生物指標。經由目標代謝體平台，我們在衍生族群中發現，敗血症患者血中短鏈和中鏈肉毒鹼濃度和敗血症所引起之肝腎功能障礙、血小板低下和高乳酸血症有顯著的正相關。然而，僅有乙酰肉毒鹼的血中濃度是和患者血中不同細胞激素濃度有顯著的正相關，並且和陽性血液培養、以及患者28天存活有顯著的相關。我們於驗證族群中確認衍生族群中的分析結果，並且發現敗血症患者若血中乙酰肉毒鹼濃度大於6000 ng/mL，會有顯著較高的28天死亡率(風險比值5.293, 95%信賴區間2.340~11.975, P < 0.001, 經由考克斯比例風險模式計算)。
結論
總結上述，我們發現嚴重周邊血淋巴球低下，以及CD4+ T淋巴球的活化，和嚴重敗血症的死亡率有顯著的相關性。同時，我們也確認敗血症患者血中乙酰肉毒鹼濃度反映急性器官功能障礙和系統性發炎的嚴重度，以及和陽性血液培養的相關性，並可作為敗血症時的預後生物指標。

關鍵字

敗血症 ； 介白素7 ； 介白素15 ； 淋巴球低下 ； 乙酰肉毒鹼 ； 肉鹼 ； 多重器官衰竭 ； 菌血症 ； 死亡率

並列摘要

Introduction
Sepsis is a clinical syndrome caused by micro-organism infection, leading to the hallmark of dysregulated systemic immune responses. The progress of sepsis treatment is lacking, and the results from clinical trials were inconsistent, probably due to heterogeneity of sepsis population. Therefore, it is warranted to explore prognostic biomarkers related to sepsis-related organ dysfunction, for precise definition and risk stratification of sepsis patients.
Aims
In this thesis, we focused on sepsis-related immune dysfunction and metabolic alternations, and explored the potential prognostic biomarkers for application in clinical care of sepsis patients.
Materials and Methods
This prospective project enrolled adult patients ( 20 years of age) admitted to medical intensive care units for sepsis with acute organ dysfunction. For metabolomic analyses targeting at fatty acid β-oxidation, patients with chronic kidney disease or cirrhosis will be excluded. Severe lymphopenia was defined as a lymphocyte count < 0.5 × 10^3/μL. Peripheral blood mononuclear cells (PBMCs) were isolated for RNA extraction or for flow cytometric analyses. The levels of plasma metabolites were measured using ultra-high performance liquid chromatography-mass spectrometry after metabolite extraction.
Results
We found that sepsis-related severe lymphopenia was associated with significantly higher plasma levels of tumor necrosis factor α, interleukin (IL)6, IL8, and IL10, and was also independently associated with 28-day mortality (adjusted hazard ratio 1.262; 95% confidence interval 1.482~8.416, P = 0.004 by Cox proportional hazard model). In patients with severe lymphopenia, the levels plasma IL15, but not IL7, were modestly but significantly increased, and BCL2 mRNA expression in PBMCs is significantly decreased. We subsequently found that sepsis non-survivors had significantly increased percentages of effector CD4+ T lymphocytes at admission, but none of the lymphocytic features we evaluated were related to the risk of nosocomial infection.
Systemic metabolomic profiling suggested that metabolites related to fatty acid β-oxidation may be prognostic biomarkers in sepsis. Through targeted metabolomic analyses, we found in the derivation cohort that increased plasma levels of short- and medium-chain acylcarnitines were significantly associated with hepatobiliary dysfunction, renal dysfunction, thrombocytopenia, and hyperlactatemia. However, only plasma acetylcarnitine levels significantly correlated with various plasma cytokine concentrations, and were also associated with blood culture positivity and 28-day mortality risk. The findings in the deviration cohort were confirmed in the independent validation cohort, and showed that patients with plasma acetylcarnitine levels more than 6000 ng/mL had significantly increased 28-day mortality (hazard ratio 5.293, 95% confidence interval 2.340~11.975, P < 0.001 by Cox proportional hazard model).
Conclusion
In conclusion, severe lymphopenia and increased activation of CD4+ T lymphocytes were associated with increased mortality in patients with severe sepsis. We further confirmed that plasma acetylcarnitine can reflect the severity of organ dysfunction, inflammation, and infection in sepsis, and can serve as a prognostic biomarker for mortality prediction.

並列關鍵字

sepsis ； interleukin 7 ； interleukin 15 ； lymphopenia ； acetylcarnitine ； carnitine ； multiple organ failure ； bacteremia ； mortality

參考文獻

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