Primary sarcopenia is associated with elevated spontaneous NET formation

Abstract

Introduction: Sarcopenia is a frequent complication of liver cirrhosis, but it can also occur independently as a result of any underlying cause. The immune system plays an important role in the pathogenesis of both liver cirrhosis and sarcopenia. Neutrophil function, including neutrophil extracellular trap (NET) formation, is linked to chronic inflammation; however, it has not been extensively studied in patients with sarcopenia. Here, we aim to study if main neutrophil functions, such as phagocytosis, reactive oxygen species (ROS) production, and NET formation, are altered in patients with sarcopenia with or without liver cirrhosis. Methods: Neutrophils from 92 patients (52 patients with liver cirrhosis and sarcopenia, 25 patients with liver cirrhosis without sarcopenia, and 15 patients with sarcopenia without liver cirrhosis) and 10 healthy controls were isolated and stimulated with heat-inactivated E. coli (250 bacteria/cell), phorbol 12-myristate 13-acetate (PMA) (100 nM), or incubation medium in duplicates for 2 h at 37°C. Cells were fixed with paraformaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI). Pictures of 10 random fields of vision per slide were taken with an Olympus BX51 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) at 600x total magnification. The DNA Area and NETosis Analysis (DANA) algorithm was used to quantify the percentage of NET formation per patient. Phagocytosis and ROS production were assessed with the PhagotestTM kit and PhagoburstTM kit (Glycotope, Heidelberg, Germany) in 92 patients and 21 healthy controls, respectively. Results: Spontaneous NET formation was significantly elevated in patients with only sarcopenia compared to patients with cirrhosis and sarcopenia (p = 0.008) and healthy controls (p = 0.039). NET formation in response to PMA was significantly decreased in patients with cirrhosis (p = 0.007), cirrhosis and sarcopenia (p < 0.001), and sarcopenia (p = 0.002) compared to healthy controls. There was no significant difference in NET formation in response to E. coli between the groups. The DANA algorithm was successfully optimized and validated for assessment of clinical samples. There were no significant changes in neutrophil phagocytosis between patients' groups compared to healthy controls. A significantly lower percentage of neutrophils produced ROS in response to N-formylmethionine-leucyl-phenylalanine (fMLF) in patients compared to healthy controls. Discussion: Spontaneous NET formation might contribute to chronic inflammation and sarcopenia pathogenesis. This, however, does not result in the impairment of the NET formation function of neutrophils in response to a bacterial stimulus and, therefore, cannot be not linked with the increased risk of bacterial infections neither in sarcopenia nor in cirrhosis. The semi-automated NET formation analysis can be successfully implemented to analyze the vast amount of data generated within clinical studies. This approach opens up the possibilities to develop an NET formation-based biomarker in different diseases including sarcopenia and implement NET formation analysis into clinical settings. Phagocytosis and ROS production were not affected in patients with sarcopenia. Further research is needed to explore the mechanism of NET formation in patients with sarcopenia and its potential as a biomarker in sarcopenia.

Keywords: NET formation; chronic inflammation; cirrhosis; neutrophils; sarcopenia.

FIGURE 1

Elevated spontaneous NET formation and decreased NET formation in response to PMA in sarcopenia. (A–J) Neutrophils were isolated from the peripheral blood of patients and healthy controls, NET formation was induced with RPMI (spontaneous NET formation) (A, D, E, I), E.coli (100 uL of heat-inactivated E. coli BL21 5 × 108 CFU/mL per 500 uL of 4 × 105 neutrophils/mL) (B, F, H, I), or 100 nM PMA (C, G, I, J) for 2 h at 37°C. The cells were fixed and stained with DAPI. Cell images taken with a fluorescent microscope were analyzed using the DANA algorithm. The NET formation percentage was quantified as NET-like structures divided by the total number of neutrophils. (A) Spontaneous NET formation (% of neutrophils producing NET) in cirrhosis patients (n = 25), cirrhosis and sarcopenia patients (n = 52), sarcopenia patients (n = 15), and healthy controls (n = 10). (B) NET formation in response to E.coli (% of neutrophils producing NET) in cirrhosis patients (n = 24), cirrhosis and sarcopenia patients (n = 51), sarcopenia patients (n = 14), and healthy controls (n = 10). (C) NET formation in response to PMA (% of neutrophils producing NET) in cirrhosis patients (n = 25), cirrhosis and sarcopenia patients (n = 52), sarcopenia patients (n = 15), and healthy controls (n = 10). (D) Spontaneous NET formation (% of neutrophils producing NET) in patients with HCC (n = 38) and patients without HCC (n = 39). (E) Spontaneous NET formation (% of neutrophils producing NET) in male (n = 42) and female (n = 10) patients from the cirrhosis and sarcopenia group. (F) NET formation in response to E. coli (% of neutrophils producing NET) in male (n = 42) and female (n = 9) patients from the cirrhosis and sarcopenia group. (G) NET formation in response to PMA (% of neutrophils producing NET) in male (n = 42) and female (n = 10) patients from the cirrhosis and sarcopenia group. (H) NET formation in response to E. coli (% of neutrophils producing NET) in patients with HCC (n = 38) and patients without HCC (n = 37) (I) Representative pictures of NET formation in neutrophils from patients’ groups and healthy controls after staining with DAPI. (J) NET formation in response to PMA (% of neutrophils producing NET) in patients with HCC (n = 38) and patients without HCC (n = 39). Truncated violin plots show the frequency distribution of measured parameters; the broken line indicates median, and dotted lines indicate quartiles; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (A–C); Mann–Whitney U test (D, E); unpaired t-test (F, G, H, J). NET: neutrophil extracellular trap; DANA: DNA Area and NETosis Analysis; PMA: phorbol 12-myristate 13-acetate; RPMI: Roswell Park Memorial Institute; DAPI: 4′,6-diamidino-2-phenylindole; HCC: hepatocellular carcinoma.

FIGURE 2

Validation and optimization of the DANA algorithm. (A–D) Neutrophils were isolated from peripheral blood of patients and healthy controls, NET formation was induced with RPMI (spontaneous NET formation) (B), E.coli (250 bacteria/cell) (C), or 100 nM PMA (D) for 2 h at 37°C. The cells were fixed and stained with DAPI. Cell images taken with a fluorescent microscope were analyzed using the DANA algorithm and by eye. The NET formation percentage was quantified as NET-like structures divided by the total number of neutrophils. (A) Examples of images before and after ROI segmentation by DANA ImageJ/Fiji plugin. (B) Bland–Altman test to compare counting of spontaneous NET formation by eye and DANA in the same samples (n = 10), bias (SD) = 4.38 (16.33), 95% limits of agreement = −27.62; 36.39. (C) Bland–Altman test to compare counting of NET formation in response to E.coli by eye and DANA in the same samples (n = 10), bias (SD) = -2.4 (9.77), 95% limits of agreement = −21.54; 16.74. (D) Bland–Altman test to compare counting of NET formation in response to PMA by eye and DANA in the same samples (n = 10), bias (SD) = -8.59 (14.01), 95% limits of agreement = −36.05; 18.87. DANA: DNA Area and NETosis Analysis; PMA: phorbol 12-myristate 13-acetate; RPMI: Roswell Park Memorial Institute; NET: neutrophil extracellular trap; ROI: region of interest; SD: standard deviation.

FIGURE 3

Neutrophil phagocytosis and ROS production are not affected in sarcopenia. (A–J) Peripheral venous blood from patients and healthy controls was collected, and the Phagotest^® kit and Phagoburst^® kit (Glycotope, Heidelberg, Germany) were used to assess neutrophil phagocytosis and ROS production, according to the manufacturer’s instructions. (A, B, F, G) Phagocytosis of E. coli (4 × 107 bacteria/100 µL blood). (A, F) Neutrophil phagocytic capacity normalized to the E. coli batch. (B, G) Percentage of non-phagocytic neutrophils. (C–E, H–J) ROS production. Percentage of neutrophils, which produced ROS (C, H) without any stimulus or after 10 min of (D, I) fMLF (0.8 µM) or (E, J) E. coli (2-4x107 bacteria/100 µL blood) stimulation. (A–E) Cirrhosis n = 21, cirrhosis and sarcopenia n = 39, sarcopenia n = 12, healthy controls n = 21; (F–I) cirrhosis and sarcopenia group male n = 33, female = 6. Truncated violin plots show the frequency distribution of measured parameters; the broken line indicates median, and dotted lines indicate quartiles; Kruskal–Wallis test with Dunn’s test (A–E); Mann–Whitney U test (F–J). ROS: reactive oxygen species; fMLF: N-formyl-met-leu-phe.