Dynamic changes in immune cell subsets and their functional indicators in patients with urine-derived sepsis
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摘要: 目的 探索尿源性脓毒症患者免疫细胞亚群及其功能指标的动态变化，分析其应用价值。 方法 采用病例对照研究设计，选择2021年3月—2022年3月由解放军陆军特色医学中心战创伤医学中心重症医学科(ICU)连续收治的60例患者作为研究对象，其中脓毒症30例，非脓毒症30例。按照感染来源，将患者分为尿源性脓毒症患者组15例以及非尿源性脓毒症患者组15例。采集患者入ICU后第1、3、5天外周血，用流式细胞仪检测患者淋巴细胞及其亚群的比例、中性粒细胞、单核细胞，并检测各亚群人类白细胞抗原DR(human leucocyte antigen DR，HLA-DR)、程序性死亡受体1(programmed death-1，PD-1)以及CD64的表达变化，Wilcoxon秩和检验分析其在各组患者中的差异变化，Pearson相关性检验分析上述指标与脓毒症严重程度的相关性。 结果 尿源性脓毒症患者入ICU第1、3、5天的序贯器官衰竭评估(SOFA)评分、急性生理学和慢性健康状况评分Ⅱ(Acute Physiology and Chronic Health Assessment Ⅱ，APACHE Ⅱ)、降钙素原(procalcitonin，PCT)、C反应蛋白(C-reactive protein，CRP)、白细胞介素-6(Interleukin-6，IL-6)与非尿源性脓毒症患者比较差异均无统计学意义(P>0.05)。尿源性脓毒症患者在入ICU第1天的淋巴细胞(Lym)比例以及B淋巴细胞比例均显著低于非尿源性脓毒症患者[Lym：(4.50±2.84)% vs (8.32±6.50)%，B淋巴细胞：(47.88±23.82)% vs (72.33±21.85)%，P＜0.05]；而T淋巴细胞在第3天及第5天均显著升高[第3天：(64.75±9.78)% vs (53.70±16.77)%，第5天：(66.96±9.32)% vs (49.99±20.19)%，P＜0.05]。入ICU第5天中性粒细胞CD64指数在尿源性脓毒症患者中显著降低[(1.87±1.62)% vs (3.93±2.71)%，P＜0.05]。与非尿源性脓毒症患者相比较，尿源性脓毒症患者入ICU第1、3、5天PD-1在T细胞表达水平均显著降低[第1天：(16.30±5.47)% vs (21.56±6.99)%，第3天：(16.48±6.98)% vs (25.79±11.07)%，第5天：(15.75±8.12)% vs (24.32±11.64)%，P＜0.05]；PD-1在第1、3天CD4+T细胞的表达水平均显著降低[第1天：(17.11±6.97)% vs (23.42±7.67)%，第3天：(19.20±12.18)% vs (29.79±15.11)%，P＜0.05]；PD-1在第5天CD8+T细胞的表达水平显著降低[(13.70±5.54)% vs (21.71±11.72)%，P＜0.05]，PD-1在第3、5天调节性T细胞(regulatory T cell，Treg)的表达水平均显著降低[第3天：(24.88±15.06)% vs (37.07±15.14)%，第5天：(19.77±14.29)% vs (35.79±17.71)%，P＜0.05]。相关性分析显示，患者入ICU第3、5天中性粒细胞与相应时间的SOFA评分呈显著正相关(r＝0.593、0.584，P＜0.05)；第3天的中性粒细胞比例与APACHE Ⅱ评分呈显著正相关(r=0.753，P=0.003)，第3天的Treg细胞比例与SOFA评分呈显著正相关(r=0.622，P=0.018)；第5天淋巴细胞比例与SOFA评分呈显著负相关(r=-0.658，P=0.014)，在第3、5天与APACHE Ⅱ评分呈显著负相关(r＝-0.747、-0.624，P＜0.05)。 结论 免疫细胞及其亚群在不同感染来源的脓毒症患者中存在显著差异，尿源性脓毒症患者外周血各淋巴细胞亚群PD-1的表达水平显著降低，尿源性脓毒症患者的免疫细胞亚群的改变可用于评估脓毒症严重程度。Abstract: Objective To explore the dynamic changes of immune cell subpopulations and functional indicators in patients with urinary sepsis, and analyze their application value. Methods Using a case-control study design, 30 sepsis patients and 30 non sepsis patients who were continuously admitted to the Intensive Care Unit(ICU) of PLA Army Specialty Medical Center from March 2021 to March 2022 were selected as the study subjects. According to the source of infection, the patients were divided into a group of 15 patients with urinary sepsis and a group of 15 patients with non urinary sepsis. The peripheral blood of patients on the 1st, 3rd and 5th days after admission to the ICU was collected, and the proportion of neutrophils, monocytes and lymphocytes subsets in patients was detected by flow cytometry. The expression changes of HLA-DR, PD-1 and CD64 were detected in each subpopulation, Wilcoxon rank sum test was used to analyze the difference between patients in each group. Pearson correlation test was used to analyze its correlation with the severity of sepsis patients. Results There was no significant difference in SOFA score, APACHE Ⅱ score, PCT, CRP, IL-6 between patients with urinary sepsis and non urinary sepsis patients on the 1st, 3rd, and 5th day of admission to the ICU(P>0.05). The proportion of lymphocytes and B cells on the first day of ICU admission in patients with urine-induced sepsis were significantly lower than those in patients with non-urinary sepsis(Lym: [4.50±2.84]% vs [8.32±6.50]%, B cells: [47.88±23.82]% vs [72.33±21.85]%, P < 0.05). However, T lymphocytes significantly elevated on both day 3 and 5(D3: [64.75±9.78]% vs [53.70±16.77]%, D5: [66.96±9.32]% vs [49.99±20.19]%, P < 0.05). Neutrophil CD64 index on day 5 of ICU admission significantly reduced in patients with urine-derived sepsis([1.87±1.62]% vs [3.93±2.71]%, P < 0.05). Compared with patients with non-urinary sepsis, the expression levels of PD-1 in T cells on the 1st, 3rd and 5th days of admission in patients with urinary sepsis significantly reduced(D1: [16.30±5.47]% vs [21.56±6.99]%, D3: [16.48±6.98]% vs [25.79±11.07]%, D5: [15.75±8.12]% vs [24.32±11.64]%, P < 0.05). The expression levels of PD-1 in CD4+ T cells on the first and third days significantly reduced(D1: [17.11±6.97]% vs [23.42±7.67]%, D3: [19.20±12.18]% vs [29.79±15.11]%, P < 0.05). The expression level of PD-1 in CD8+ T cells on day 5 significantly reduced([13.70±5.54]% vs [21.71±11.72]%, P < 0.05), and the expression level of PD-1 in Treg cells on days 3 and 5 significantly reduced(D3: [24.88±15.06]% vs [37.07±15.14]%, D5: [19.77±14.29]% vs [35.79±17.71]%, P < 0.05). Correlation analysis showed that neutrophils on the 3rd and 5th days of ICU admission were positively correlated with the SOFA score at the corresponding time(r＝0.593 and 0.584, P < 0.05). The proportion of neutrophils on day 3 was positively correlated with the APACHE Ⅱscore(r=0.753, P=0.003), and the proportion of Treg cells on day 3 was positively correlated with the SOFA score(r=0.622, P=0.018). The proportion of lymphocytes was significantly negatively correlated with SOFA score on day 5(r=-0.658, P=0.014), and on days 3 and 5, it was negatively correlated with APACHE Ⅱ score(r＝-0.747, -0.624, both P < 0.05). Conclusion Lymphocytes and their subsets differ significantly in patients with sepsis of different origins, and the expression levels of PD-1 in various lymphocyte subsets in peripheral blood of patients with urinary sepsis significantly reduced. Thus, and changes in immune cell subsets in patients with urine-derived sepsis can be used to assess disease severity.
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Key words:
- urinary sepsis /
- immune cell subsets /
- functional indicators /
- dynamic changes /
- correlation analysis
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表 1 脓毒症患者与非脓毒症患者基线资料
例(%)，X±S，M(Q1，Q3) 指标 非脓毒症(30例) 脓毒症(30例) 尿源性(15例) 非尿源性(15例) P值 性别(男性) 21(70.00) 8(53.33) 11(73.33) 0.256 年龄/岁 56.93±9.82 56.27±10.92 57.60±8.92 0.717 ICU住院时间/d 12.07±0.09 16.13±13.43 14.93±9.83 0.782 SOFA评分 第1天 6.27±3.79 7.00±4.11 6.53±3.85 0.751 第3天 5.55±3.34 7.29±4.20 8.07±5.11 0.658 第5天 4.89±3.54 6.85±4.90 6.93±5.30 0.967 APACHE Ⅱ评分 第1天 17.53±4.64 16.80±6.29 17.93±4.73 0.582 第3天 16.45±4.80 14.77±5.00 16.60±4.79 0.332 第5天 16.64±5.36 14.23±4.82 16.64±5.33 0.230 PCT/(ng/mL) 第1天 0.41(0.17，3.40) 21.63(4.02，68.15) 8.65(2.71，62.42) 0.539 第3天 0.81(0.25，5.20) 5.87(1.94，29.50) 9.69(2.38，36.46) 0.813 第5天 0.33(0.13，1.74) 2.91(1.16，2.91) 4.16(1.29，10.98) 0.728 CRP/(mg/mL) 第1天 79.88±62.91 156.69±70.56 150.98±81.21 0.839 第3天 106.21±77.56 136.61±77.78 134.64±80.09 0.949 第5天 71.87(31.00，140.53) 153.52(22.74，196.10) 77.73(38.93，180.92) 0.424 IL-6/(ng/L) 第1天 86.60(44.16，407.00) 211.90(61.26，575.92) 133.60(80.56，614.05) 0.744 第3天 58.63(27.12，113.20) 150.95(31.15，341.58) 87.00(44.58，194.35) 0.579 第5天 47.35(20.12，65.88) 52.64(19.56，188.25) 70.86(27.70，152.50) 0.721 住院28 d死亡率 7(23.33) 4(26.67) 5(33.33) 0.690 
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表 2 尿源性脓毒症与非尿源性脓毒症患者免疫细胞亚群比例
%，X±S 指标 非脓毒症(30例) 脓毒症(30例) 尿源性(15例) 非尿源性(15例) P值 Mon 第1天 5.31±2.87 2.92±2.31 4.19±2.49 0.157 第3天 5.57±2.24 3.16±1.83 4.13±2.76 0.275 第5天 6.74±2.31 5.72±4.33 5.25±3.30 0.756 Neu 第1天 83.31±11.78 87.49±13.38 86.92±7.03 0.884 第3天 85.49±8.24 89.04±4.14 85.98±10.51 0.318 第5天 82.60±7.40 82.38±12.79 83.73±12.73 0.789 Lym 第1天 9.02±6.46 4.50±2.84 8.32±6.50 0.046 第3天 8.05±6.09 5.79±3.79 6.99±5.53 0.505 第5天 9.15±6.01 8.42±7.40 6.35±4.14 0.386 CD3+ 第1天 67.67±13.33 56.87±15.26 51.77±17.70 0.405 第3天 67.38±14.49 64.75±9.78 53.70±16.77 0.038 第5天 69.83±11.68 66.96±9.32 49.99±20.19 0.012 CD4+ 第1天 57.74±13.36 59.96±10.07 58.55±10.80 0.726 第3天 57.55±14.07 67.26±11.50 62.33±10.08 0.231 第5天 61.69±13.73 65.35±14.09 64.76±9.19 0.903 CD8+ 第1天 35.05±12.17 36.06±11.54 35.07±10.11 0.811 第3天 34.16±11.32 28.54±10.24 31.38±9.91 0.440 第5天 30.12±10.92 30.76±13.20 31.00±8.83 0.959 Treg 第1天 7.39±5.80 8.42±2.58 7.19±3.26 0.258 第3天 7.64±3.53 8.16±3.54 7.94±4.41 0.885 第5天 7.50±3.88 9.11±4.38 8.21±2.89 0.553 CD3-CD19+ 第1天 53.68±17.23 57.66±26.72 56.76±27.66 0.929 第3天 50.52±15.93 50.91±21.29 63.98±22.83 0.122 第5天 52.99±16.74 47.88±23.82 72.33±21.85 0.014 NK细胞 第1天 41.99±16.99 37.00±23.99 40.02±26.55 0.747 第3天 38.20±21.94 35.54±18.73 32.45±23.24 0.696 第5天 32.42±23.01 34.51±21.11 23.75±22.34 0.228
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表 3 尿源性脓毒症与非尿源性脓毒症患者免疫细胞亚群功能指标的表达水平
X±S 指标 非脓毒症(30例) 脓毒症(30例) 尿源性(15例) 非尿源性(15例) P值 Mon HLA-DR 第1天 30.17±21.42 12.62±5.75 13.90±10.03 0.637 第3天 29.56±20.03 12.92±6.00 13.04±7.93 0.965 第5天 29.61±18.90 14.43±11.96 10.31±6.64 0.304 Neu CD64 index 第1天 1.62±1.17 3.09±1.99 4.48±4.33 0.274 第3天 1.25±1.10 3.14±2.76 2.79±1.62 0.689 第5天 1.75±2.15 1.87±1.62 3.93±2.71 0.036 Lym PD-1 第1天 10.76±6.97 12.07±3.33 12.59±5.57 0.762 第3天 10.49±6.50 14.90±8.01 15.26±7.29 0.899 第5天 10.48±6.44 14.23±6.02 13.50±6.01 0.764 CD3+PD-1 第1天 14.50±8.92 16.30±5.47 21.56±6.99 0.029 第3天 13.90±8.00 16.48±6.98 25.79±11.07 0.014 第5天 13.88±9.04 15.75±8.12 24.32±11.64 0.048 CD4+PD-1 第1天 16.65±9.43 17.11±6.97 23.42±7.67 0.029 第3天 16.45±8.45 19.20±12.18 29.79±15.11 0.047 第5天 16.06±11.02 20.21±9.68 25.96±13.55 0.232 CD8+PD-1 第1天 13.74±10.61 13.78±5.64 16.92±7.52 0.206 第3天 12.77±9.49 15.50±9.37 20.97±9.87 0.138 第5天 12.24±7.24 13.70±5.54 21.71±11.72 0.044 Treg PD-1 第1天 21.11±12.94 25.74±14.58 29.70±14.19 0.457 第3天 18.95±9.75 24.88±15.06 37.07±15.14 0.039 第5天 19.55±12.90 19.77±14.29 35.79±17.71 0.023 注：Neu CD64 index=(中性/淋巴)/(单核/中性)。
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表 4 各细胞亚群与尿源性脓毒症严重程度的相关性分析
指标 SOFA评分 APACHE Ⅱ评分 指标 SOFA评分 APACHE Ⅱ评分 第1天 第3天 第5天 第1天 第3天 第5天 第1天 第3天 第5天 第1天 第3天 第5天 Neu CD8+T r 0.267 0.593 0.584 -0.084 0.753 0.468 r -0.166 -0.362 -0.094 -0.337 -0.468 -0.197 P值 0.336 0.025 0.036 0.756 0.003 0.107 P值 0.589 0.203 0.759 0.260 0.107 0.518 Mon Treg r 0.195 0.055 -0.313 -0.023 -0.055 -0.312 r 0.453 0.622 -0.092 0.099 0.429 -0.200 P值 0.487 0.853 0.298 0.936 0.857 0.300 P值 0.090 0.018 0.765 0.727 0.144 0.513 Lym NK细胞 r 0.162 -0.493 -0.658 -0.029 -0.747 -0.624 r 0.135 -0.112 -0.005 0.227 0.010 0.051 P值 0.565 0.073 0.014 0.917 0.003 0.023 P值 0.632 0.703 0.987 0.416 0.975 0.870 CD3+T CD3-CD19+ r -0.328 -0.295 -0.349 0.032 -0.128 -0.319 r -0.120 0.240 0.239 -0.167 0.204 0.120 P值 0.233 0.306 0.243 0.911 0.676 0.288 P值 0.671 0.390 0.431 0.552 0.505 0.695 CD4+T r 0.169 0.435 0.146 0.314 0.536 0.255 P值 0.547 0.120 0.634 0.255 0.059 0.401
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