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Neutrophil extracellular traps contribute to the intestine damage in endotoxemic rats
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Neutrophil extracellular traps contribute to the intestine damage in endotoxemic rats Xin Gao, MD,a,1 Shuangying Hao, MD,b,1 Huiying Yan, MD,b Weiwei Ding, MD,a,* Kuanyu Li, PhD,b,** and Jieshou Li, MDa a
Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, China b Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu Province, China
article info
abstract
Article history:
Background: Sepsis is one of the most troublesome problems in critically ill patients and
Received 24 September 2014
often accompanied with multiple organ dysfunction and high mortality. Gut injury or
Received in revised form
dysfunction may contribute to the pathogenesis of sepsis. Neutrophil extracellular traps
1 December 2014
(NETs) do not only kill microorganisms but also damage host cells during inflammatory
Accepted 10 December 2014
response to infection. The aim of this study was to investigate whether NETs are capable of
Available online 19 December 2014
promoting the impairment of the gut in a rat model of lipopolysaccharide (LPS)-induced sepsis.
Keywords:
Methods: The sepsis model was induced in rats by intraperitoneal injection of LPS
Neutrophil extracellular traps
(10 mg/kg). All rats were divided into three groups as follows: 1) control group; 2) LPS group;
(NETs)
and 3) LPS þ DNase I group. The DNase I solution (10 mg/kg) was injected intravenously to
Sepsis
disrupt NETs 30 min after the LPS treatment. The animals were sacrificed at 3 h and 24 h
Endotoxemia
after LPS or saline challenge. The intestinal cell apoptosis was examined by detecting the
Apoptosis
level of cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end
Intestinal injury
labeling assays. The length and morphology of Villi were assessed histologically through hematoxylin and eosin stain. The levels of tumor necrosis factor-alpha and interleukin-10 in serum and intestine were detected by enzyme-linked immunosorbent assay. Intestinal injury was evaluated with Chiu scoring system. Results: A large number of neutrophils infiltrated were activated to release NETs in the intestine of LPS-induced septic rats. The disruption of NETs reduced the acute systemic inflammatory response and apoptosis of intestinal epithelial cells and alleviated histologic pathogenesis. Removal of NETs provided a beneficial effect on intestinal injury. Conclusions: This study demonstrates that the release of NETs may contribute to the intestinal damage during sepsis. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China. Tel./fax: þ86 025 8086 0005. ** Corresponding author. Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, 22 Hankou Road, Nanjing 210093, Jiangsu Province, China. Tel./fax: þ86 25 8359 4791. E-mail addresses: [email protected] (W. Ding), [email protected] (K. Li). 1 Equal contributors. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.12.019
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1.
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Introduction
Neutrophils are the most abundant type of leukocytes in mammals and play a key role in innate immune as a first line of defense against infection in the early sepsis phase. Besides phagocytosis and degranulation, a new mechanism was discovered for neutrophils to kill microorganisms through neutrophil extracellular traps (NETs) [1]. In response to interleukin (IL)-8, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate, the stimulated neutrophils release NETs via a novel cell death program that was proved distinct from apoptosis and necrosisdNETosis [1,2]. NETs, an essential component of the antimicrobial repertoire, are composed of DNA in association with histones and granular proteins, such as myeloperoxidase (MPO) and several cytoplasmic proteases [1]. NETs can trap microorganisms to prevent their dissemination from the initial infection site and provide a high local concentration of antimicrobials to kill them [1]. However, the uncontrolled or excessive release of NETs may also injure surrounding cells and contribute to disease pathophysiology [3], including transfusion-related acute lung injury [4,5], septic liver damage [6], and systemic lupus erythematosus [7]. Sepsis is the systemic inflammatory response syndrome caused by the infection [8]. And severe sepsis is always accompanied with organ dysfunction or tissue hypoperfusion [9]. Notably, gut injury or dysfunction frequently occurs in sepsis. Gut is not only the victim but also the prime mover during sepsis [10]. The gut injury or dysfunction can trigger bacterial translocation and the abundant release of gutderived inflammatory factors that reach the systemic circulation by lymphatic and portal pathways. These bacterial and nonbacterial factors may cause the development of the pathogenesis including epithelial lifting of villi and enterocyte necrosis and promote distant organ dysfunction [11]. This is the gut hypothesis of multiple organ dysfunction syndrome. In sepsis, particularly severe sepsis, the infection with an overwhelming inflammatory response mostly contributes to the pathogenesis of this syndrome. The overactive immune cells, especially neutrophils, and the secreted abundant proinflammatory mediators may cause organ impairment [12]. It was demonstrated that the increased infiltration of activated neutrophils in the intestinal tissue may effect intestinal tissue damage leading to bacterial translocation after burn [13]. But whether NETs contribute to the intestinal injury during sepsis has not yet been investigated. Thereby, we present an LPS-induced severe sepsis model in rats to investigate the effects of NETs on the impairment of the gut.
2.
Materials and methods
2.1.
Animals
Adult male SpragueeDawley rats weighing 200 30 g (from Experimental Animal Center, Jinling Hospital, Nanjing, China) were used for the experiments. All rats were maintained under special pathogen-free environment and had free access to food and water. All animal procedures were performed
under local and national ethical guidelines and were approved by the Institutional Animal Care and Use Committee at Jinling Hospital, Nanjing University.
2.2.
Experimental design
All rats were randomly divided into three groups as follows: 1) control group; 2) LPS group; and 3) LPS þ DNase I group. In the LPS and LPS þ DNase I groups, sepsis was induced in rats by an intraperitoneal injection of LPS (from Escherichia coli 055:B5; SigmaeAldrich, St. Louis, MO; 10 mg/kg) dissolved in saline, whereas an equal volume of saline was injected in the control group. DNase I (SigmaeAldrich, 10 mg/kg) dissolved in saline was injected via the tail vein 30 min after the LPS challenge for LPS þ DNase I group or an equal volume of saline for control and LPS groups. At 3 and 24 h after LPS or saline solution injection, the blood of anesthetized rats was collected via the inferior vena cava, and the intestines (ileum) were removed and stored in liquid nitrogen for later assays. There were six rats in every group at each time point.
2.3.
Immunofluorescence
The activated neutrophils can decondense the chromatin to release NETs. The histones citrullinated by peptidylarginine deiminase 4 mediate chromatin decondensation, which are extruded with DNA during NETs formation [14]. Thus, the CitH3DNA complexes were chosen as the marker of NETs [15]. To identify NETs and neutrophils in the rat intestine, optimum cutting temperature-embedded frozen ileum was sectioned (7 mm). After blocked with the immunol staining blocking buffer (Beyotime, Haimen, China), the sections were incubated with antibody against Cit-Histone 3 or MPO (Abcam, Cambridge, MA) and then with species-specific secondary antibodies coupled with Alexa Fluor 488 Dyes. DNA was stained using 40 ,6-diamidino-2-phenylindole. The sections were observed under the confocal laser-scanning microscope (FluoView FV10i; Olympus Corporation, Tokyo , Japan).
2.4.
MPO-DNA enzyme-linked immunosorbent assay
To quantify NET levels in rats by a serological method, we used a capture enzyme-linked immunosorbent assay (ELISA) as descripted previously [5]. Antibody against MPO in 75 mL (5 mg/mL) was coated onto 96-well plates overnight at 4 C. The coating solution was then removed and 200 mL of incubation buffer (bottle 5 in Cell Death Detection ELISA kit; Roche, Mannheim, Germany) was added to each well for incubation for 30 min at room temperature. After three times wash with 300 mL of wash buffer, 40 mL of the sample were added to each well with 60 mL of incubation buffer. The plate was incubated for 2 h at room temperature. After three washes (300 mL each), 100 mL of incubation buffer containing a peroxidase-labeled antieDNA monoclonal antibody (dilution 1:10) were added to each well. After three washes (300 mL each), 100 mL of peroxidase substrate (ABTS) were added. Absorbance at a wavelength of 405 nm was measured after 30-min incubation at room temperature in the dark. Values for soluble NETs formation are expressed as units relative to the control group.
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2.5.
Western blot analysis
Caspase-3 is a master executioner during apoptosis [16]. Procaspase-3 (32 kDa), which normally presents in cytoplasm, may be cleaved into 17 kDa and 12 kDa subunits leading to assembly of the active heterotetrameric enzyme in apoptosis. A Western blot analysis for cleaved caspase-3 was performed to assess cell apoptosis in the gut. Tissues were lysed in lysis buffer containing 40 mM Tis-HCl (pH 7.4), 150 mM NaCl, 1% NP40, and protease inhibitor cocktail tablets (Roche) and kept on ice for 30 min. Isolated total proteins were subjected to electrophoresis at 100 V using a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a 0.2-mm pore-size nitrocellulose membrane (PALL, New York, NY). The membranes were incubated with 1:500 dilution of rat antiecaspase-3 (Cell Signaling Technology, Beverly, MA) and 1:1000 rat antie GAPDH (Bioworld, Louis Park, MN) overnight at 4 C. The washed membranes were incubated with 1:1000 dilution of a secondary antibody for 1 h. Signal was detected with horseradish peroxidaseeconjugated immunoglobulin G (IgG) using enhanced chemiluminescence detection reagents (Amersham International, Buckinghamshire, United Kingdom). Blot bands were quantified by densitometry with ImageJ software (ImageJ; NIH, Bethesda, Maryland).
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performed with the In Situ Cell Death Detection kit (Roche) according to the manufacturer’s instructions. Apoptotic cells were determined by counting cells in 10 randomly selected fields of each ileum section. Counting was done by an observer blinded to the experiment. The apoptotic index was expressed as the number of apoptotic cells per 100 cells.
2.7.
Morphologic analysis of intestine
The samples collected for histologic analysis were fixed for 24 h in 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and sectioned (4 mm). The slides were stained with hematoxylin and eosin and observed under a light microscope. Histologic alteration was quantified according to the Chiu score system [17]. All sections were evaluated by two blinded examiners.
2.8. Detection of tumor necrosis factor-alpha and IL-10 by ELISA Tumor necrosis factor-alpha (TNF-a) and IL-10 levels in the serum were determined using rat TNF-a and IL-10 ELISA kits (Multiscience, Hangzhou, China) according to the manufacturer’s instructions. Concentrations were calculated from the standard curve made with recombinant rat TNF-a and IL-10. The lower detection limit of TNF-a was 7.14 pg/mL and IL-10 was 0.48 pg/mL.
2.6. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assays
2.9.
To evaluate cell apoptosis in the gut, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was
Statistical analyses were performed using GraphPad Prism for Windows version 5. Data are expressed as mean standard
Statistical analyses
Fig. 1 e Neutrophils infiltrated intestine to release NETs in the intestine of endotoxemic rat at 24 h after LPS injection. (A) A rat intraperitoneally injected with LPS (10 mg/kg) to induce sepsis showed MPO positive (green) cell infiltration in intestine. Scale bar: 20 mm. (B) NETs were formed after treatment with LPS. Arrowheads indicate Cit-H3epositive (green) NET-like structures. Scale bar: 50 mm. (Color version of the figure is available online.)
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diarrhea 1 h after the LPS challenge. These symptoms last for at least 12 h after the LPS injection. The loss of body weight was obviously observed in LPS and LPS-DNase I groups, but there was no significant difference between these two groups in weight loss 24 h after the LPS injection (data not shown).
3.2. NETs were present in the intestine during endotoxemia
Fig. 2 e The injection of LPS promoted NETs formation, and DNase I treatment disrupted NETs. NETs formation was quantified (MPO-DNA ELISA) in rat serum and presented as mean ± standard error of the mean (n > 4 per group). *P < 0.05, **P < 0.01.
error of the mean. Data obtained from multiple groups were analyzed using one-way analysis of variance followed by NewmaneKeuls post-test. Data were considered to be statistically significant at P < 0.05.
3.
Results
3.1.
General observations
All the rats treated with LPS presented multiple symptoms, such as lethargy, idleness, lagging in response, and stop drinking. Typically, the rats began to show different degrees of
A number of neutrophils were observed 24 h after the LPS injection in the gut of the LPS-induced rats by immunofluorescence (Fig. 1A). The scattered NETs were observed in the ileum mucosa and submucosa of LPS-treated rats (Fig. 1B). However, the CitH3-DNA complexes, a marker of NETs, were not found in the sections from control and LPS-DNase I groups (figure not shown). Local levels of NETs were difficult to be quantified objectively by microscopy because they tend to be diffusely scattered and/or tethered to neutrophils [18]. Therefore, a modified capture ELISA was performed to detect serum NETs-associated MPO-DNA complexes, which was reported to be able to reflect the NETs levels in vivo [5]. The level of the complexes in LPS-induced endotoxemic rats was significantly elevated 24 h after the LPS injection, whereas the levels of the complexes were reduced by http://abbr.dict. cn/intravenousinjection/i_2Ev_2E intravenous injection of DNase I (Fig. 2). The results suggest that LPS-induced formation of NETs was disrupted by DNase I.
3.3. The disruption of NETs reduced intestinal cell apoptosis To reveal the effect of LPS-induced NETs on apoptosis of intestinaleepithelial cells, cleaved caspase-3 was measured.
Fig. 3 e Apoptosis of intestinaleepithelial cells was initiated by the LPS-induced NETs. The levels of cleaved caspase-3 in intestines increased at 24 h not 3 h after LPS injection, and the disruption of NETs inhibited the increase. Values are presented as means ± standard error of the mean (n > 4 per group), *P < 0.05.
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Fig. 4 e LPS-induced apoptosis of intestinal cells was confirmed by terminal deoxynucleotidyl transferase dUTP nick-end labeling stain, and removal of NETs reversed the effect. The rats from the LPS group presented more terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive (green) cells than rats from the control and LPS D DNase I groups per 3200 field. Values of apoptosis index are present as means ± standard error of the mean (n [ 6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001. (Color version of the figure is available online.)
Although there were no significant differences of the levels of intestinal-cleaved caspase-3 between the three groups 3 h after the LPS challenge, the levels of cleaved caspase-3 remarkably increased 24 h after the LPS challenge compared with those in the control group (Fig. 3). The disruption of NETs decreased the level of cleaved caspase-3 at this time point (Fig. 3). The correlation between the levels of cleaved caspase3 and NETs implies that the NETs formation in the intestine
might contribute to pathologic intestinal cell apoptosis resulting from the LPS challenge. To see if LPS-induced apoptotic process reached the late phase, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was performed to mark these cells in situ, in which DNA was nicked during relatively late apoptosis. The results showed that LPS treatment increased the number of total apoptotic cells 24 h after the LPS challenge, and the
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Fig. 5 e The LPS-induced injury of ileum was protected mildly by removal of NETs. The injury was assessed and quantified via Chiu scoring system (see Methods) at 24 h after LPS or saline injection. Subepithelial space development, congestion, and inflammatory cells in the lamina propria were observed in the LPS group, whereas light improvement of these changes was revealed in DNase I group. Values of Chiu score are present as means ± standard error of the mean (n [ 6 per group), **P < 0.01. (Color version of the figure is available online.)
disruption of NETs by DNase I reduced the number of apoptotic cells (Fig. 4), confirming that NETs are involved in LPS-induced intestinal cell apoptosis.
3.4. NETs formation contributed to the deleterious histologic changes of intestinal tissue To further evaluate the consequence of NETs formation, a histologic examination was carried out. No obvious villous injury was observed in the control group. However, the LPS challenge induced the development of subepithelial Gruenhagen space and vacuolization at the villus tip and the congestion and inflammatory cell infiltration in the lamina propria in the LPS group. DNase I infusion modestly decreased these pathologic changes (Fig. 5). The intestinal tissue injury was quantified with hematoxylin and eosin-stained ileac sections from all rats via Chiu scoring system. The results showed that the mean injury score in the LPS group significantly increased compared with that in the control group, whereas the disruption of NETs by DNase I partially but not significantly reduced the injury score 24 h after the LPS administration (Fig. 5, quantification panel).
3.5. Disruption of NETs diminished LPS-induced acute inflammation The proinflammatory cytokines TNF-a and anti-inflammatory cytokine IL-10 were determined in serum and intestinal
tissues 3 h and 24 h after the LPS challenge by ELISA (Fig. 6). Compared with the rats in the control group, serum TNF-a and IL-10 were both dramatically increased in the LPS-treated rats 3 h after the LPS challenge. At 24 h, serum TNF-a remarkably dropped in both LPS-treated groups (Fig. 6A), whereas there was a delay in the changes of serum IL-10 (Fig. 6C). Interestingly, the treatment of DNase I partially diminished the increase of TNF-a 3 h after the LPS challenge (Fig. 6A), suggesting that the disruption of NETs may provide a beneficial effect of a low systemic inflammatory response in the endotoxemic rats. Unexpectedly, local cytokines including both TNF-a and IL-10 did not significantly change except the mild but significant increase of TNF-a 3 h after the LPS challenge (Fig. 6B and D).
4.
Discussion
Our results showed that many neutrophils infiltrated and activated to release NETs in the intestinal tissue during LPS-induced endotoxemia. This study shows that application of DNase I leads to a reduction of MPO-DNA complexes associated NETs in vivo. In this study, removal of NETs provided the protective effects, such as diminishing the inflammation, reducing the apoptosis of intestinal epithelial cells, partially, but not significantly, and reversing the destructive histologic change of the intestinal villi.
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Fig. 6 e Disruption of NETs lowered systemic proinflammatory response in the endotoxemic rats. The levels of the local and systemic cytokines 3 and 24 h were detected after the LPS challenge. (A) Serum TNF-a; (B) local TNF-a; (C) serum IL-10 (D); and local IL-10. Values are presented as mean ± standard error of the mean (n [ 6 per group).*P < 0.05, **P < 0.01, and ***P < 0.001.
The intestinal damage and dysfunction are often present in septic patients. The increase of apoptotic activity in the gut after endotoxin infusion is associated with increased gut macromolecular permeability [19]. The increased intestinal permeability is believed to be with subsequent release of bacteria, endotoxin, and inflammatory factors into the systemic circulation, which lead to the systemic inflammatory response syndrome and multiple organ dysfunction syndrome [10,11]. Based on our results, the mechanism of intestinal damage and dysfunction in sepsis is, at least, partially explained by the activation of neutrophils and formation of NETs in the intestine. How NETs cause the injury may be complex. First, the direct effect of NETs to induce cell apoptosis was proposed. Mounting evidence has been shown that NETs and their components can directly induce cell death and tissue injury, such as histone [4,15,20] and MPO [21]. Therefore, the systemic disruption of NETs may diminish the local concentration of NETs and their components in the intestine, which result in decreasing the cytotoxicity of NETs. Second, NETs may impair the microvascular of the gut indirectly contributing to the damage. NETs were found to injure vascular endothelial cells leading to the increase of microvascular permeability, which would cause tissue edema and oxygenation impairment [4]. In addition, NETs can induce exposure of normal extravascular tissue factor to blood [4], and NETs or their components are able to activate platelets, further promote an excessive coagulopathy and thrombosis, and even induce disseminated intravascular coagulation [22e25]. The microvascular dysfunction can cause
organ damage resulting from oxygenation disorders [26,27]. Finally, inflammatory cytokines are vital mediators in sepsisinduced intestinal barrier dysfunction [28]. On the other hand, it is believed that NETs exert a proinflammatory role in sepsis [29], which is consistent with our results. Our study showed that the disruption of NETs decreased the systemic inflammation. In addition, dsDNA, which is the main component of NETs, is also involved in damage-associated molecular patterns activating immune response [30]. However, the detailed and exact mechanism how NETs contribute to the gut or other organs’ damage during sepsis is not clear, which needs to be further investigated. Both a previous study and our results support that NETs formation contribute to organ damage and dysfunction in sepsis. Therefore, disruption or removal of NETs or their cytotoxic components would benefit for host survival in sepsis. Active protein-C, which can degrade histones and prevent their cytotoxicity [20], was regarded as one of the most potential treatments [31]. However, disruption of NETs may impair host immune defenses and cause bacteria spreading throughout the body [6,32]. Thus, effective antimicrobial treatment should be needed if the therapy targeting to NETs is considered. Besides, more recently, circulating free DNA derived from NETs was thought to be a possible marker for the injury severity and prediction of inflammatory second hit and sepsis [33]. Thus, the quantification of NETs or their components may be a valuable tool to predict the development of organ dysfunction, intestinal injury in this case, during sepsis.
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Conclusions
In conclusion, we observed that the activated and infiltrated neutrophils released NETs in the intestinal tissue in the rat model of LPS-induced sepsis and NETs enhanced intestinal cell apoptosis. The disruption of NETs by DNase I treatment decreased intestinal apoptosis activity and systemic acute inflammatory response to protect the intestine from injury. These data support the hypothesis that NETs formation in the intestine contributes to intestinal damage during sepsis.
Acknowledgment This work was financially supported by the National Natural Science Foundation of China (grant nos.81300278). Authors’ contributions: X.G., S.H., W.D., K.L., and J.L. designed the research. X.G., S.H., and H.Y. performed the research. X.G., S.H., H.Y., and K.L. analyzed the data. X.G., W.D., K.L., and J.L. wrote the article.
Disclosure No conflict of interest to be claimed.
references
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Neutrophil extracellular traps contribute to the intestine damage in endotoxemic rats Xin Gao, MD,a,1 Shuangying Hao, MD,b,1 Huiying Yan, MD,b Weiwei Ding, MD,a,* Kuanyu Li, PhD,b,** and Jieshou Li, MDa a
Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu Province, China b Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, Nanjing, Jiangsu Province, China
article info
abstract
Article history:
Background: Sepsis is one of the most troublesome problems in critically ill patients and
Received 24 September 2014
often accompanied with multiple organ dysfunction and high mortality. Gut injury or
Received in revised form
dysfunction may contribute to the pathogenesis of sepsis. Neutrophil extracellular traps
1 December 2014
(NETs) do not only kill microorganisms but also damage host cells during inflammatory
Accepted 10 December 2014
response to infection. The aim of this study was to investigate whether NETs are capable of
Available online 19 December 2014
promoting the impairment of the gut in a rat model of lipopolysaccharide (LPS)-induced sepsis.
Keywords:
Methods: The sepsis model was induced in rats by intraperitoneal injection of LPS
Neutrophil extracellular traps
(10 mg/kg). All rats were divided into three groups as follows: 1) control group; 2) LPS group;
(NETs)
and 3) LPS þ DNase I group. The DNase I solution (10 mg/kg) was injected intravenously to
Sepsis
disrupt NETs 30 min after the LPS treatment. The animals were sacrificed at 3 h and 24 h
Endotoxemia
after LPS or saline challenge. The intestinal cell apoptosis was examined by detecting the
Apoptosis
level of cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick-end
Intestinal injury
labeling assays. The length and morphology of Villi were assessed histologically through hematoxylin and eosin stain. The levels of tumor necrosis factor-alpha and interleukin-10 in serum and intestine were detected by enzyme-linked immunosorbent assay. Intestinal injury was evaluated with Chiu scoring system. Results: A large number of neutrophils infiltrated were activated to release NETs in the intestine of LPS-induced septic rats. The disruption of NETs reduced the acute systemic inflammatory response and apoptosis of intestinal epithelial cells and alleviated histologic pathogenesis. Removal of NETs provided a beneficial effect on intestinal injury. Conclusions: This study demonstrates that the release of NETs may contribute to the intestinal damage during sepsis. ª 2015 Elsevier Inc. All rights reserved.
* Corresponding author. Research Institute of General Surgery, Jinling Hospital, School of Medicine, Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu Province, China. Tel./fax: þ86 025 8086 0005. ** Corresponding author. Jiangsu Key Laboratory for Molecular Medicine, Medical School of Nanjing University, 22 Hankou Road, Nanjing 210093, Jiangsu Province, China. Tel./fax: þ86 25 8359 4791. E-mail addresses: [email protected] (W. Ding), [email protected] (K. Li). 1 Equal contributors. 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.12.019
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Introduction
Neutrophils are the most abundant type of leukocytes in mammals and play a key role in innate immune as a first line of defense against infection in the early sepsis phase. Besides phagocytosis and degranulation, a new mechanism was discovered for neutrophils to kill microorganisms through neutrophil extracellular traps (NETs) [1]. In response to interleukin (IL)-8, lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate, the stimulated neutrophils release NETs via a novel cell death program that was proved distinct from apoptosis and necrosisdNETosis [1,2]. NETs, an essential component of the antimicrobial repertoire, are composed of DNA in association with histones and granular proteins, such as myeloperoxidase (MPO) and several cytoplasmic proteases [1]. NETs can trap microorganisms to prevent their dissemination from the initial infection site and provide a high local concentration of antimicrobials to kill them [1]. However, the uncontrolled or excessive release of NETs may also injure surrounding cells and contribute to disease pathophysiology [3], including transfusion-related acute lung injury [4,5], septic liver damage [6], and systemic lupus erythematosus [7]. Sepsis is the systemic inflammatory response syndrome caused by the infection [8]. And severe sepsis is always accompanied with organ dysfunction or tissue hypoperfusion [9]. Notably, gut injury or dysfunction frequently occurs in sepsis. Gut is not only the victim but also the prime mover during sepsis [10]. The gut injury or dysfunction can trigger bacterial translocation and the abundant release of gutderived inflammatory factors that reach the systemic circulation by lymphatic and portal pathways. These bacterial and nonbacterial factors may cause the development of the pathogenesis including epithelial lifting of villi and enterocyte necrosis and promote distant organ dysfunction [11]. This is the gut hypothesis of multiple organ dysfunction syndrome. In sepsis, particularly severe sepsis, the infection with an overwhelming inflammatory response mostly contributes to the pathogenesis of this syndrome. The overactive immune cells, especially neutrophils, and the secreted abundant proinflammatory mediators may cause organ impairment [12]. It was demonstrated that the increased infiltration of activated neutrophils in the intestinal tissue may effect intestinal tissue damage leading to bacterial translocation after burn [13]. But whether NETs contribute to the intestinal injury during sepsis has not yet been investigated. Thereby, we present an LPS-induced severe sepsis model in rats to investigate the effects of NETs on the impairment of the gut.
2.
Materials and methods
2.1.
Animals
Adult male SpragueeDawley rats weighing 200 30 g (from Experimental Animal Center, Jinling Hospital, Nanjing, China) were used for the experiments. All rats were maintained under special pathogen-free environment and had free access to food and water. All animal procedures were performed
under local and national ethical guidelines and were approved by the Institutional Animal Care and Use Committee at Jinling Hospital, Nanjing University.
2.2.
Experimental design
All rats were randomly divided into three groups as follows: 1) control group; 2) LPS group; and 3) LPS þ DNase I group. In the LPS and LPS þ DNase I groups, sepsis was induced in rats by an intraperitoneal injection of LPS (from Escherichia coli 055:B5; SigmaeAldrich, St. Louis, MO; 10 mg/kg) dissolved in saline, whereas an equal volume of saline was injected in the control group. DNase I (SigmaeAldrich, 10 mg/kg) dissolved in saline was injected via the tail vein 30 min after the LPS challenge for LPS þ DNase I group or an equal volume of saline for control and LPS groups. At 3 and 24 h after LPS or saline solution injection, the blood of anesthetized rats was collected via the inferior vena cava, and the intestines (ileum) were removed and stored in liquid nitrogen for later assays. There were six rats in every group at each time point.
2.3.
Immunofluorescence
The activated neutrophils can decondense the chromatin to release NETs. The histones citrullinated by peptidylarginine deiminase 4 mediate chromatin decondensation, which are extruded with DNA during NETs formation [14]. Thus, the CitH3DNA complexes were chosen as the marker of NETs [15]. To identify NETs and neutrophils in the rat intestine, optimum cutting temperature-embedded frozen ileum was sectioned (7 mm). After blocked with the immunol staining blocking buffer (Beyotime, Haimen, China), the sections were incubated with antibody against Cit-Histone 3 or MPO (Abcam, Cambridge, MA) and then with species-specific secondary antibodies coupled with Alexa Fluor 488 Dyes. DNA was stained using 40 ,6-diamidino-2-phenylindole. The sections were observed under the confocal laser-scanning microscope (FluoView FV10i; Olympus Corporation, Tokyo , Japan).
2.4.
MPO-DNA enzyme-linked immunosorbent assay
To quantify NET levels in rats by a serological method, we used a capture enzyme-linked immunosorbent assay (ELISA) as descripted previously [5]. Antibody against MPO in 75 mL (5 mg/mL) was coated onto 96-well plates overnight at 4 C. The coating solution was then removed and 200 mL of incubation buffer (bottle 5 in Cell Death Detection ELISA kit; Roche, Mannheim, Germany) was added to each well for incubation for 30 min at room temperature. After three times wash with 300 mL of wash buffer, 40 mL of the sample were added to each well with 60 mL of incubation buffer. The plate was incubated for 2 h at room temperature. After three washes (300 mL each), 100 mL of incubation buffer containing a peroxidase-labeled antieDNA monoclonal antibody (dilution 1:10) were added to each well. After three washes (300 mL each), 100 mL of peroxidase substrate (ABTS) were added. Absorbance at a wavelength of 405 nm was measured after 30-min incubation at room temperature in the dark. Values for soluble NETs formation are expressed as units relative to the control group.
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2.5.
Western blot analysis
Caspase-3 is a master executioner during apoptosis [16]. Procaspase-3 (32 kDa), which normally presents in cytoplasm, may be cleaved into 17 kDa and 12 kDa subunits leading to assembly of the active heterotetrameric enzyme in apoptosis. A Western blot analysis for cleaved caspase-3 was performed to assess cell apoptosis in the gut. Tissues were lysed in lysis buffer containing 40 mM Tis-HCl (pH 7.4), 150 mM NaCl, 1% NP40, and protease inhibitor cocktail tablets (Roche) and kept on ice for 30 min. Isolated total proteins were subjected to electrophoresis at 100 V using a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a 0.2-mm pore-size nitrocellulose membrane (PALL, New York, NY). The membranes were incubated with 1:500 dilution of rat antiecaspase-3 (Cell Signaling Technology, Beverly, MA) and 1:1000 rat antie GAPDH (Bioworld, Louis Park, MN) overnight at 4 C. The washed membranes were incubated with 1:1000 dilution of a secondary antibody for 1 h. Signal was detected with horseradish peroxidaseeconjugated immunoglobulin G (IgG) using enhanced chemiluminescence detection reagents (Amersham International, Buckinghamshire, United Kingdom). Blot bands were quantified by densitometry with ImageJ software (ImageJ; NIH, Bethesda, Maryland).
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performed with the In Situ Cell Death Detection kit (Roche) according to the manufacturer’s instructions. Apoptotic cells were determined by counting cells in 10 randomly selected fields of each ileum section. Counting was done by an observer blinded to the experiment. The apoptotic index was expressed as the number of apoptotic cells per 100 cells.
2.7.
Morphologic analysis of intestine
The samples collected for histologic analysis were fixed for 24 h in 4% paraformaldehyde in phosphate-buffered saline, embedded in paraffin, and sectioned (4 mm). The slides were stained with hematoxylin and eosin and observed under a light microscope. Histologic alteration was quantified according to the Chiu score system [17]. All sections were evaluated by two blinded examiners.
2.8. Detection of tumor necrosis factor-alpha and IL-10 by ELISA Tumor necrosis factor-alpha (TNF-a) and IL-10 levels in the serum were determined using rat TNF-a and IL-10 ELISA kits (Multiscience, Hangzhou, China) according to the manufacturer’s instructions. Concentrations were calculated from the standard curve made with recombinant rat TNF-a and IL-10. The lower detection limit of TNF-a was 7.14 pg/mL and IL-10 was 0.48 pg/mL.
2.6. Terminal deoxynucleotidyl transferase dUTP nick-end labeling assays
2.9.
To evaluate cell apoptosis in the gut, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was
Statistical analyses were performed using GraphPad Prism for Windows version 5. Data are expressed as mean standard
Statistical analyses
Fig. 1 e Neutrophils infiltrated intestine to release NETs in the intestine of endotoxemic rat at 24 h after LPS injection. (A) A rat intraperitoneally injected with LPS (10 mg/kg) to induce sepsis showed MPO positive (green) cell infiltration in intestine. Scale bar: 20 mm. (B) NETs were formed after treatment with LPS. Arrowheads indicate Cit-H3epositive (green) NET-like structures. Scale bar: 50 mm. (Color version of the figure is available online.)
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diarrhea 1 h after the LPS challenge. These symptoms last for at least 12 h after the LPS injection. The loss of body weight was obviously observed in LPS and LPS-DNase I groups, but there was no significant difference between these two groups in weight loss 24 h after the LPS injection (data not shown).
3.2. NETs were present in the intestine during endotoxemia
Fig. 2 e The injection of LPS promoted NETs formation, and DNase I treatment disrupted NETs. NETs formation was quantified (MPO-DNA ELISA) in rat serum and presented as mean ± standard error of the mean (n > 4 per group). *P < 0.05, **P < 0.01.
error of the mean. Data obtained from multiple groups were analyzed using one-way analysis of variance followed by NewmaneKeuls post-test. Data were considered to be statistically significant at P < 0.05.
3.
Results
3.1.
General observations
All the rats treated with LPS presented multiple symptoms, such as lethargy, idleness, lagging in response, and stop drinking. Typically, the rats began to show different degrees of
A number of neutrophils were observed 24 h after the LPS injection in the gut of the LPS-induced rats by immunofluorescence (Fig. 1A). The scattered NETs were observed in the ileum mucosa and submucosa of LPS-treated rats (Fig. 1B). However, the CitH3-DNA complexes, a marker of NETs, were not found in the sections from control and LPS-DNase I groups (figure not shown). Local levels of NETs were difficult to be quantified objectively by microscopy because they tend to be diffusely scattered and/or tethered to neutrophils [18]. Therefore, a modified capture ELISA was performed to detect serum NETs-associated MPO-DNA complexes, which was reported to be able to reflect the NETs levels in vivo [5]. The level of the complexes in LPS-induced endotoxemic rats was significantly elevated 24 h after the LPS injection, whereas the levels of the complexes were reduced by http://abbr.dict. cn/intravenousinjection/i_2Ev_2E intravenous injection of DNase I (Fig. 2). The results suggest that LPS-induced formation of NETs was disrupted by DNase I.
3.3. The disruption of NETs reduced intestinal cell apoptosis To reveal the effect of LPS-induced NETs on apoptosis of intestinaleepithelial cells, cleaved caspase-3 was measured.
Fig. 3 e Apoptosis of intestinaleepithelial cells was initiated by the LPS-induced NETs. The levels of cleaved caspase-3 in intestines increased at 24 h not 3 h after LPS injection, and the disruption of NETs inhibited the increase. Values are presented as means ± standard error of the mean (n > 4 per group), *P < 0.05.
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Fig. 4 e LPS-induced apoptosis of intestinal cells was confirmed by terminal deoxynucleotidyl transferase dUTP nick-end labeling stain, and removal of NETs reversed the effect. The rats from the LPS group presented more terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive (green) cells than rats from the control and LPS D DNase I groups per 3200 field. Values of apoptosis index are present as means ± standard error of the mean (n [ 6 per group). *P < 0.05, **P < 0.01, and ***P < 0.001. (Color version of the figure is available online.)
Although there were no significant differences of the levels of intestinal-cleaved caspase-3 between the three groups 3 h after the LPS challenge, the levels of cleaved caspase-3 remarkably increased 24 h after the LPS challenge compared with those in the control group (Fig. 3). The disruption of NETs decreased the level of cleaved caspase-3 at this time point (Fig. 3). The correlation between the levels of cleaved caspase3 and NETs implies that the NETs formation in the intestine
might contribute to pathologic intestinal cell apoptosis resulting from the LPS challenge. To see if LPS-induced apoptotic process reached the late phase, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay was performed to mark these cells in situ, in which DNA was nicked during relatively late apoptosis. The results showed that LPS treatment increased the number of total apoptotic cells 24 h after the LPS challenge, and the
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Fig. 5 e The LPS-induced injury of ileum was protected mildly by removal of NETs. The injury was assessed and quantified via Chiu scoring system (see Methods) at 24 h after LPS or saline injection. Subepithelial space development, congestion, and inflammatory cells in the lamina propria were observed in the LPS group, whereas light improvement of these changes was revealed in DNase I group. Values of Chiu score are present as means ± standard error of the mean (n [ 6 per group), **P < 0.01. (Color version of the figure is available online.)
disruption of NETs by DNase I reduced the number of apoptotic cells (Fig. 4), confirming that NETs are involved in LPS-induced intestinal cell apoptosis.
3.4. NETs formation contributed to the deleterious histologic changes of intestinal tissue To further evaluate the consequence of NETs formation, a histologic examination was carried out. No obvious villous injury was observed in the control group. However, the LPS challenge induced the development of subepithelial Gruenhagen space and vacuolization at the villus tip and the congestion and inflammatory cell infiltration in the lamina propria in the LPS group. DNase I infusion modestly decreased these pathologic changes (Fig. 5). The intestinal tissue injury was quantified with hematoxylin and eosin-stained ileac sections from all rats via Chiu scoring system. The results showed that the mean injury score in the LPS group significantly increased compared with that in the control group, whereas the disruption of NETs by DNase I partially but not significantly reduced the injury score 24 h after the LPS administration (Fig. 5, quantification panel).
3.5. Disruption of NETs diminished LPS-induced acute inflammation The proinflammatory cytokines TNF-a and anti-inflammatory cytokine IL-10 were determined in serum and intestinal
tissues 3 h and 24 h after the LPS challenge by ELISA (Fig. 6). Compared with the rats in the control group, serum TNF-a and IL-10 were both dramatically increased in the LPS-treated rats 3 h after the LPS challenge. At 24 h, serum TNF-a remarkably dropped in both LPS-treated groups (Fig. 6A), whereas there was a delay in the changes of serum IL-10 (Fig. 6C). Interestingly, the treatment of DNase I partially diminished the increase of TNF-a 3 h after the LPS challenge (Fig. 6A), suggesting that the disruption of NETs may provide a beneficial effect of a low systemic inflammatory response in the endotoxemic rats. Unexpectedly, local cytokines including both TNF-a and IL-10 did not significantly change except the mild but significant increase of TNF-a 3 h after the LPS challenge (Fig. 6B and D).
4.
Discussion
Our results showed that many neutrophils infiltrated and activated to release NETs in the intestinal tissue during LPS-induced endotoxemia. This study shows that application of DNase I leads to a reduction of MPO-DNA complexes associated NETs in vivo. In this study, removal of NETs provided the protective effects, such as diminishing the inflammation, reducing the apoptosis of intestinal epithelial cells, partially, but not significantly, and reversing the destructive histologic change of the intestinal villi.
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Fig. 6 e Disruption of NETs lowered systemic proinflammatory response in the endotoxemic rats. The levels of the local and systemic cytokines 3 and 24 h were detected after the LPS challenge. (A) Serum TNF-a; (B) local TNF-a; (C) serum IL-10 (D); and local IL-10. Values are presented as mean ± standard error of the mean (n [ 6 per group).*P < 0.05, **P < 0.01, and ***P < 0.001.
The intestinal damage and dysfunction are often present in septic patients. The increase of apoptotic activity in the gut after endotoxin infusion is associated with increased gut macromolecular permeability [19]. The increased intestinal permeability is believed to be with subsequent release of bacteria, endotoxin, and inflammatory factors into the systemic circulation, which lead to the systemic inflammatory response syndrome and multiple organ dysfunction syndrome [10,11]. Based on our results, the mechanism of intestinal damage and dysfunction in sepsis is, at least, partially explained by the activation of neutrophils and formation of NETs in the intestine. How NETs cause the injury may be complex. First, the direct effect of NETs to induce cell apoptosis was proposed. Mounting evidence has been shown that NETs and their components can directly induce cell death and tissue injury, such as histone [4,15,20] and MPO [21]. Therefore, the systemic disruption of NETs may diminish the local concentration of NETs and their components in the intestine, which result in decreasing the cytotoxicity of NETs. Second, NETs may impair the microvascular of the gut indirectly contributing to the damage. NETs were found to injure vascular endothelial cells leading to the increase of microvascular permeability, which would cause tissue edema and oxygenation impairment [4]. In addition, NETs can induce exposure of normal extravascular tissue factor to blood [4], and NETs or their components are able to activate platelets, further promote an excessive coagulopathy and thrombosis, and even induce disseminated intravascular coagulation [22e25]. The microvascular dysfunction can cause
organ damage resulting from oxygenation disorders [26,27]. Finally, inflammatory cytokines are vital mediators in sepsisinduced intestinal barrier dysfunction [28]. On the other hand, it is believed that NETs exert a proinflammatory role in sepsis [29], which is consistent with our results. Our study showed that the disruption of NETs decreased the systemic inflammation. In addition, dsDNA, which is the main component of NETs, is also involved in damage-associated molecular patterns activating immune response [30]. However, the detailed and exact mechanism how NETs contribute to the gut or other organs’ damage during sepsis is not clear, which needs to be further investigated. Both a previous study and our results support that NETs formation contribute to organ damage and dysfunction in sepsis. Therefore, disruption or removal of NETs or their cytotoxic components would benefit for host survival in sepsis. Active protein-C, which can degrade histones and prevent their cytotoxicity [20], was regarded as one of the most potential treatments [31]. However, disruption of NETs may impair host immune defenses and cause bacteria spreading throughout the body [6,32]. Thus, effective antimicrobial treatment should be needed if the therapy targeting to NETs is considered. Besides, more recently, circulating free DNA derived from NETs was thought to be a possible marker for the injury severity and prediction of inflammatory second hit and sepsis [33]. Thus, the quantification of NETs or their components may be a valuable tool to predict the development of organ dysfunction, intestinal injury in this case, during sepsis.
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Conclusions
In conclusion, we observed that the activated and infiltrated neutrophils released NETs in the intestinal tissue in the rat model of LPS-induced sepsis and NETs enhanced intestinal cell apoptosis. The disruption of NETs by DNase I treatment decreased intestinal apoptosis activity and systemic acute inflammatory response to protect the intestine from injury. These data support the hypothesis that NETs formation in the intestine contributes to intestinal damage during sepsis.
Acknowledgment This work was financially supported by the National Natural Science Foundation of China (grant nos.81300278). Authors’ contributions: X.G., S.H., W.D., K.L., and J.L. designed the research. X.G., S.H., and H.Y. performed the research. X.G., S.H., H.Y., and K.L. analyzed the data. X.G., W.D., K.L., and J.L. wrote the article.
Disclosure No conflict of interest to be claimed.
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