Lactoferrin-DNA complex detection to evaluate plasma neutrophil extracellular traps of patients with severe influenza A (H1N1)

doi:10.3877/cma.j.issn.1674-1358.2019.02.007

Abstract

Abstract:

Objective

To establish a relative quantitative assay for lactoferrin-DNA complex, and to assess the levels of neutrophil extracellular traps (NETs)in plasma samples from patients with severe influenza A (H1N1) and other pathogen infectious diseases.

Methods

Plasma samples with clear DNA content were serially diluted as a standard, and the level of lactoferrin-DNA complex in plasma was determined by Enzyme-linked immunosorbent assay (ELISA). The lactoferrin-DNA ELISA method was used to compare the difference of plasma lactoferrin-DNA complex between 19 patients with severe influenza A (H1N1) and 19 blood donors (control group).

Results

Level of plasma lactoferrin-DNA complex of patients in severe influenza A (H1N1) was positively correlated with that of cfDNA (cell-free DNA) and histone-DNA complex, respectively (r = 0.6763, P < 0.0001; r = 0.756, P < 0.0001). The plasma lactoferrin-DNA complex content in patients with severe influenza A (H1N1) was 2.082 (1.169, 5.021) μg/ml, which was significantly higher than that of the controls [0.233 (0.170, 0.376) μg/ml, P < 0.0001], and positively correlated with Acute Physiological and Chronic Health Evaluation Ⅱ score (APACHE Ⅱ score) (r = 0.7379, P = 0.0005).

Conclusion

Plasma lactoferrin-DNA complex level could be used to assess NETs level in patients with severe influenza A (H1N1).

Key words: Severe influenza A (H1N1), Lactoferrin-DNA complex, Neutrophil extracellular traps, Enzyme-linked immunosorbent assay

Xiaoyu Yang, yue Zhang, Weili Mai, Rui Li, Liuluan Zhu, Junyan Han, Hui Zeng. Lactoferrin-DNA complex detection to evaluate plasma neutrophil extracellular traps of patients with severe influenza A (H1N1)[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2019, 13(02): 122-127.

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References 25

[1]	Caudrillier A, Kessenbrock K, Gilliss BM, et al. Platelets induce neutrophil extracellular traps in transfusion-related acute lung injury[J]. J Clin Invest,2012,122(7):2661-2671.
[2]	Lefrancais E, Mallavia B, Zhuo H, et al. Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury[J]. JCI Insight,2018,3(3):e98178.
[3]	Czaikoski PG, Mota JM, Nascimento DC, et al. Neutrophil extracellular traps induce organ damage during experimental and clinical sepsis[J]. PLoS One,2016,11(2):e148142.
[4]	Sorensen OE, Borregaard N. Neutrophil extracellular traps--the dark side of neutrophils[J]. J Clin Invest,2016,126(5):1612-1620.
[5]	Zhu L, Liu L, Zhang Y, et al. High level of neutrophil extracellular traps correlates with poor prognosis of severe influenza A infection[J]. J Infect Dis,2018,217(3):428-437.
[6]	Papayannopoulos V. Neutrophil extracellular traps in immunity and disease[J]. Nat Rev Immunol,2018,18(2):134-147.
[7]	Hampson P, Dinsdale RJ, Wearn CM, et al. Neutrophil dysfunction, immature granulocytes, and cell-free DNA are early biomarkers of sepsis in burn-injured patients: A prospective observational cohort study[J]. Ann Surg,2017,265(6):1241-1249.
[8]	Vong L, Sherman PM, Glogauer M. Quantification and visualization of neutrophil extracellular traps (NETs) from murine bone marrow-derived neutrophils[M]//Allen IC. Methods in Molecular Biology: Mouse Models of Innate Immunity. New Jersey: Humana Press,2013,1031:41-50.
[9]	van der Strate BW, Beljaars L, Molema G, et al. Antiviral activities of lactoferrin[J]. Antiviral Res,2001,52(3):225-239.
[10]	Wakabayashi H, Oda H, Yamauchi K, et al. Lactoferrin for prevention of common viral infections[J]. J Infect Chemother,2014,20(11):666-671.
[11]	Sivanandham R, Brocca-Cofano E, Krampe N, et al. Neutrophil extracellular trap production contributes to pathogenesis in SIV-infected nonhuman primates[J]. J Clin Invest,2018,128(11):5178-5183
[12]	钟南山，李兰娟，王辰, 等. 甲型H1N1流感诊疗方案(2009年第3版)[J]. 中华医学杂志,2009,89(45):559.
[13]	Paules C, Subbarao K. Influenza[J]. Lancet,2017,390(10095):697-708.
[14]	Mantovani A, Cassatella MA, Costantini C, et al. Neutrophils in the activation and regulation of innate and adaptive immunity[J]. Nat Rev Immunol,2011,11(8):519-531.
[15]	Mayadas TN, Cullere X, Lowell CA. The multifaceted functions of neutrophils[J]. Annu Rev Pathol,2014,9:181-218.
[16]	Matthay MA, Ware LB, Zimmerman GA. The acute respiratory distress syndrome[J]. J Clin Invest,2012,122(8):2731-2740.
[17]	Porto BN, Stein RT. Neutrophil Extracellular Traps in Pulmonary Diseases: Too Much of a Good Thing?[J]. Front Immunol,2016,7:311.
[18]	Narayana MA, Narasaraju T, Rai P, et al. In vivo and in vitro studies on the roles of neutrophil extracellular traps during secondary pneumococcal pneumonia after primary pulmonary influenza infection[J]. Front Immunol,2013,4:56.
[19]	Narasaraju T, Yang E, Samy RP, et al. Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis[J]. Am J Pathol,2011,179(1):199-210.
[20]	Maruchi Y, Tsuda M, Mori H, et al. Plasma myeloperoxidase-conjugated DNA level predicts outcomes and organ dysfunction in patients with septic shock[J]. Crit Care,2018,22(1):176.
[21]	朱鏐娈，张玥，李国力, 等. 建立体外诱导和检测中性粒细胞胞外诱捕网的方法[J/CD]. 中华实验和临床感染病杂志(电子版), 2015(3):405-408.
[22]	Hamaguchi S, Akeda Y, Yamamoto N, et al. Origin of circulating free DNA in sepsis: analysis of the CLP mouse model[J]. Mediat Inflamm,2015,2015:1-9.
[23]	Bronkhorst AJ, Aucamp J, Pretorius PJ. Cell-free DNA: Preanalytical variables[J]. Clin Chim Acta,2015,450:243-253.
[24]	Yoo DG, Floyd M, Winn M, et al. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes[J]. Immunol Lett,2014,160(2):186-194.
[25]	Knaus WA, Draper EA, Wagner DP, et al. APACHE Ⅱ: a severity of disease classification system[J]. Crit Care Med,1985,13(10):818-829.

临床资料		对照组（19例）	甲型H1N1重症流感患者（19例）	统计量	P值
年龄（岁）^a		37（31，65）	33（25，69）	Z =－1.125	0.261
男性[例（%）]		10（52.6）	12（63.2）	χ² = 0.432	0.511
基础疾病[例（%）]		?	?	?	?
?	呼吸系统疾病	1（5.3）	1（5.3）	χ² = 0.000	1.000
?	高血压	2（10.5）	7（36.8）	χ² = 2.330	0.127
?	糖尿病	0（0.0）	5（26.3）	χ² = 3.683	0.055
?	心脏病	0（0.0）	2（10.5）	χ² = 0.528	0.468
?	自身免疫性疾病	1（5.3）	0（0.0）	χ² = 0.000	1.000
?	肝脏疾病	0（0.0）	1（5.3）	χ² = 0.000	1.000
?	妊娠	0（0.0）	0（0.0）	—	—
病原微生物培养阳性[例（%）]		?	?	?	?
?	细菌	0（0.0）	10（52.6）	χ² = 10.993	0.001
?	真菌	0（0.0）	4（21.1）	χ² = 2.515	0.113
?	细菌和真菌	0（0.0）	2（10.5）	χ² = 0.528	0.468
住院时间^a		0（0，0）	8（4，13）	—	—
入院前发热天数^a		0（0，0）	3（2，7）	—	—
抗病毒治疗[例（%）]		0（0.0）	19（100.0）	χ² = 34.105	< 0.001
抗菌药物治疗[例（%）]		0（0.0）	19（100.0）	χ² = 34.105	0
糖皮质激素治疗[例（%）]		0（0.0）	5（26.3）	χ² = 3.683	0.055
免疫调节治疗[例（%）]		0（0.0）	0（0.0）	—	—
中性粒细胞计数（× 10⁹/L）^a		3.1（2.9，3.6）	8.5（4.2，16.9）	Z =－4.306	< 0.001
APACHE Ⅱ评分^a		—	12（8，17）	—	—
SOFA评分^a		—	8（4，9）	—	—

临床资料		对照组（19例）	甲型H1N1重症流感患者（19例）	统计量	P值
年龄（岁）^a		37（31，65）	33（25，69）	Z =－1.125	0.261
男性[例（%）]		10（52.6）	12（63.2）	χ² = 0.432	0.511
基础疾病[例（%）]		?	?	?	?
?	呼吸系统疾病	1（5.3）	1（5.3）	χ² = 0.000	1.000
?	高血压	2（10.5）	7（36.8）	χ² = 2.330	0.127
?	糖尿病	0（0.0）	5（26.3）	χ² = 3.683	0.055
?	心脏病	0（0.0）	2（10.5）	χ² = 0.528	0.468
?	自身免疫性疾病	1（5.3）	0（0.0）	χ² = 0.000	1.000
?	肝脏疾病	0（0.0）	1（5.3）	χ² = 0.000	1.000
?	妊娠	0（0.0）	0（0.0）	—	—
病原微生物培养阳性[例（%）]		?	?	?	?
?	细菌	0（0.0）	10（52.6）	χ² = 10.993	0.001
?	真菌	0（0.0）	4（21.1）	χ² = 2.515	0.113
?	细菌和真菌	0（0.0）	2（10.5）	χ² = 0.528	0.468
住院时间^a		0（0，0）	8（4，13）	—	—
入院前发热天数^a		0（0，0）	3（2，7）	—	—
抗病毒治疗[例（%）]		0（0.0）	19（100.0）	χ² = 34.105	< 0.001
抗菌药物治疗[例（%）]		0（0.0）	19（100.0）	χ² = 34.105	0
糖皮质激素治疗[例（%）]		0（0.0）	5（26.3）	χ² = 3.683	0.055
免疫调节治疗[例（%）]		0（0.0）	0（0.0）	—	—
中性粒细胞计数（× 10⁹/L）^a		3.1（2.9，3.6）	8.5（4.2，16.9）	Z =－4.306	< 0.001
APACHE Ⅱ评分^a		—	12（8，17）	—	—
SOFA评分^a		—	8（4，9）	—	—

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