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Presence of neutrophil extracellular traps and citrullinated histone H3 in the bloodstream of critically ill patients.

Hirose T, Hamaguchi S, Matsumoto N, Irisawa T, Seki M, Tasaki O, Hosotsubo H, Yamamoto N, Yamamoto K, Akeda Y, Oishi K, Tomono K, Shimazu T - PLoS ONE (2014)

Bottom Line: Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3.The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation.NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.

ABSTRACT
Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5-76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in "the presence of bacteria in tracheal aspirate" group (11/22, 50.0%) than in "the absence of bacteria in tracheal aspirate" group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.

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Representative images of immunostaining using anti-CD66b antibody in the blood smear sample from a critically ill patient.Triple staining by DAPI, anti-CD66b antibody, and anti-citrullinated histone H3 was performed using the blood smear sample obtained from a critically ill patient. A. The CD66b-positive cells were subjected to citrullination of histone H3 in their nuclei. Citrullination of histone H3 was not detected in the CD66b-negative cell (arrow). B. Arrow indicates the occurrence of citrullination of histone H3 in a neutrophil that had immunoreactivity against CD66b. Arrowheads indicate NETs stained with CD66b, whose appearance was of a string-like structure extending from the cell body. Asterisk indicates a neutrophil that was beginning to release NETs from its ruptured cell body. Interestingly, freshly produced NETs (asterisk) held immunoreactivity against citrullination of histone H3. In contrast, elongated NETs (arrowheads) were not stained with anti- citrullinated histone H3 antibody. Blue, DAPI; Red, CD66b; Green, citrullinated histone H3. (Magnification ×400). Scale bar; 50 µm.
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pone-0111755-g001: Representative images of immunostaining using anti-CD66b antibody in the blood smear sample from a critically ill patient.Triple staining by DAPI, anti-CD66b antibody, and anti-citrullinated histone H3 was performed using the blood smear sample obtained from a critically ill patient. A. The CD66b-positive cells were subjected to citrullination of histone H3 in their nuclei. Citrullination of histone H3 was not detected in the CD66b-negative cell (arrow). B. Arrow indicates the occurrence of citrullination of histone H3 in a neutrophil that had immunoreactivity against CD66b. Arrowheads indicate NETs stained with CD66b, whose appearance was of a string-like structure extending from the cell body. Asterisk indicates a neutrophil that was beginning to release NETs from its ruptured cell body. Interestingly, freshly produced NETs (asterisk) held immunoreactivity against citrullination of histone H3. In contrast, elongated NETs (arrowheads) were not stained with anti- citrullinated histone H3 antibody. Blue, DAPI; Red, CD66b; Green, citrullinated histone H3. (Magnification ×400). Scale bar; 50 µm.

Mentions: In the preliminary experiments, string-like structure extending from the cell body, which was positive for DNA and histone, was exclusively also positive for neutrophil elastase (Fig. S4). Hence, we considered the extracellular component that is double-positive for DNA and H3 to be a NET. The production of NETs and the specific expression of the citrullination of histone H3 in neutrophils were confirmed using anti-CD66b antibody (Fig. 1). Diff-Quik staining revealed the presence of a variety of blood cells in the smears (Fig. S5).


Presence of neutrophil extracellular traps and citrullinated histone H3 in the bloodstream of critically ill patients.

Hirose T, Hamaguchi S, Matsumoto N, Irisawa T, Seki M, Tasaki O, Hosotsubo H, Yamamoto N, Yamamoto K, Akeda Y, Oishi K, Tomono K, Shimazu T - PLoS ONE (2014)

Representative images of immunostaining using anti-CD66b antibody in the blood smear sample from a critically ill patient.Triple staining by DAPI, anti-CD66b antibody, and anti-citrullinated histone H3 was performed using the blood smear sample obtained from a critically ill patient. A. The CD66b-positive cells were subjected to citrullination of histone H3 in their nuclei. Citrullination of histone H3 was not detected in the CD66b-negative cell (arrow). B. Arrow indicates the occurrence of citrullination of histone H3 in a neutrophil that had immunoreactivity against CD66b. Arrowheads indicate NETs stained with CD66b, whose appearance was of a string-like structure extending from the cell body. Asterisk indicates a neutrophil that was beginning to release NETs from its ruptured cell body. Interestingly, freshly produced NETs (asterisk) held immunoreactivity against citrullination of histone H3. In contrast, elongated NETs (arrowheads) were not stained with anti- citrullinated histone H3 antibody. Blue, DAPI; Red, CD66b; Green, citrullinated histone H3. (Magnification ×400). Scale bar; 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230949&req=5

pone-0111755-g001: Representative images of immunostaining using anti-CD66b antibody in the blood smear sample from a critically ill patient.Triple staining by DAPI, anti-CD66b antibody, and anti-citrullinated histone H3 was performed using the blood smear sample obtained from a critically ill patient. A. The CD66b-positive cells were subjected to citrullination of histone H3 in their nuclei. Citrullination of histone H3 was not detected in the CD66b-negative cell (arrow). B. Arrow indicates the occurrence of citrullination of histone H3 in a neutrophil that had immunoreactivity against CD66b. Arrowheads indicate NETs stained with CD66b, whose appearance was of a string-like structure extending from the cell body. Asterisk indicates a neutrophil that was beginning to release NETs from its ruptured cell body. Interestingly, freshly produced NETs (asterisk) held immunoreactivity against citrullination of histone H3. In contrast, elongated NETs (arrowheads) were not stained with anti- citrullinated histone H3 antibody. Blue, DAPI; Red, CD66b; Green, citrullinated histone H3. (Magnification ×400). Scale bar; 50 µm.
Mentions: In the preliminary experiments, string-like structure extending from the cell body, which was positive for DNA and histone, was exclusively also positive for neutrophil elastase (Fig. S4). Hence, we considered the extracellular component that is double-positive for DNA and H3 to be a NET. The production of NETs and the specific expression of the citrullination of histone H3 in neutrophils were confirmed using anti-CD66b antibody (Fig. 1). Diff-Quik staining revealed the presence of a variety of blood cells in the smears (Fig. S5).

Bottom Line: Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3.The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation.NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Traumatology and Acute Critical Medicine, Osaka University Graduate School of Medicine, Osaka, Japan.

ABSTRACT
Neutrophil extracellular traps (NETs), a newly identified immune mechanism, are induced by inflammatory stimuli. Modification by citrullination of histone H3 is thought to be involved in the in vitro formation of NETs. The purposes of this study were to evaluate whether NETs and citrullinated histone H3 (Cit-H3) are present in the bloodstream of critically ill patients and to identify correlations with clinical and biological parameters. Blood samples were collected from intubated patients at the time of ICU admission from April to June 2011. To identify NETs, DNA and histone H3 were visualized simultaneously by immunofluorescence in blood smears. Cit-H3 was detected using a specific antibody. We assessed relationships of the presence of NETs and Cit-H3 with the existence of bacteria in tracheal aspirate, SIRS, diagnosis, WBC count, and concentrations of IL-8, TNF-α, cf-DNA, lactate, and HMGB1. Forty-nine patients were included. The median of age was 66.0 (IQR: 52.5-76.0) years. The diagnoses included trauma (7, 14.3%), infection (14, 28.6%), resuscitation from cardiopulmonary arrest (8, 16.3%), acute poisoning (4, 8.1%), heart disease (4, 8.1%), brain stroke (8, 16.3%), heat stroke (2, 4.1%), and others (2, 4.1%). We identified NETs in 5 patients and Cit-H3 in 11 patients. NETs and/or Cit-H3 were observed more frequently in "the presence of bacteria in tracheal aspirate" group (11/22, 50.0%) than in "the absence of bacteria in tracheal aspirate" group (4/27, 14.8%) (p<.01). Multiple logistic regression analysis showed that only the presence of bacteria in tracheal aspirate was significantly associated with the presence of NETs and/or Cit-H3. The presence of bacteria in tracheal aspirate may be one important factor associated with NET formation. NETs may play a pivotal role in the biological defense against the dissemination of pathogens from the respiratory tract to the bloodstream in potentially infected patients.

Show MeSH
Related in: MedlinePlus