International Immunopharmacology 20 (2014) 24–32

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Hemin inhibits NLRP3 inflammasome activation in sepsis-induced acute lung injury, involving heme oxygenase-1 Yun-peng Luo a,1, Lei Jiang a,1, Kai Kang a, Dong-sheng Fei a, Xiang-lin Meng a, Chuan-chuan Nan a, Shang-ha Pan b, Ming-ran Zhao c, Ming-yan Zhao a,⁎ a b c

Department of ICU, The First Affiliated Hospital of Harbin Medical University, Harbin, China The Key Hepatosplenic Surgery Laboratory, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, China Department of Pediatrics, Child's Hospital of Harbin City, Harbin, China

a r t i c l e

i n f o

Article history: Received 22 October 2013 Received in revised form 22 January 2014 Accepted 14 February 2014 Available online 26 February 2014 Keywords: Heme oxygenase-1 NLRP3 inflammasome Sepsis Acute lung injury

a b s t r a c t NLRP3 inflammasome activation contributes to acute lung injury (ALI), accelerating caspase-1 maturation, and resulting in IL-1β and IL-18 over-production. Heme oxygenase-1 (HO-1) plays a protective role in ALI. This study investigated the effect of hemin (a potent HO-1 inducer) on NLRP3 inflammasome in sepsis-induced ALI. The sepsis model of cecal ligation and puncture (CLP) was used in C57BL6 mice. In vivo induction and suppression of HO-1 were performed by pretreatment with hemin and zinc protoporphyrin IX (ZnPP, a HO-1 competitive inhibitor) respectively. CLP triggered significant pulmonary damage, neutrophil infiltration, increased levels of IL-1β and IL-18, and edema formation in the lung. Hemin pretreatment exerted inhibitory effect on lung injury and attenuated IL-1β and IL-18 secretion in serum and lung tissue. In lung tissues, hemin downregulated mRNA and protein levels of NLRP3, ASC and caspase-1. Moreover, hemin reduced malondialdehyde and reactive oxygen species production, and inhibited NF-κB and NLRP3 inflammasome activity. Meanwhile, hemin significantly increased HO-1 mRNA and protein expression and HO-1 enzymatic activity. In contrast, no significant differences were observed between the CLP and ZnPP groups. Our study suggests that hemininhibited NLRP3 inflammasome activation involved HO-1, reducing IL-1β and IL-18 secretion and limiting the inflammatory response. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Sepsis is a condition resulting in a harmful systemic inflammatory response to an infection. Sepsis is a major cause of morbidity and mortality despite extensive research efforts and improvements in care [1]. Sepsis may lead to end-organ dysfunction, and septic patients are particularly at risk of developing acute lung injury (ALI) [2]. ALI is a common and severe pulmonary disease, and affects 10–15% of patients hospitalized in ICU. The most severe form of ALI, acute respiratory distress syndrome (ARDS), still has a high mortality despite progresses in ICU care. Sepsis remains the leading cause of ALI/ARDS [3]. The nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is a multiprotein complex that regulates the maturation of pro-inflammatory cytokines IL-1β and IL-18. It consists of NODAbbreviations: HO-1, heme oxygenase-1; NLRP3, nucleotide-binding domain (NOD)-like receptor protein 3; ZnPP, zinc protoporphyrin IX; ASC, apoptosisassociated speck-like protein containing caspase-1 activator domain (CARD); CLP, cecal ligation and perforation; MDA, malondialdehyde; ROS, reactive oxygen species. ⁎ Corresponding author at: Department of ICU, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China. E-mail address: [email protected] (M. Zhao). 1 Contributed equally to this work.

http://dx.doi.org/10.1016/j.intimp.2014.02.017 1567-5769/© 2014 Elsevier B.V. All rights reserved.

like receptor, NLRP3, the adaptor protein ASC [apoptosis-associated speck-like protein containing caspase-1 activator domain (CARD)] and caspase-1. Upon exogenous and endogenous stimuli, the NLRP3 inflammasome forms through activation of NLRP3 and recruitment of ASC and pro-caspase-1, resulting in caspase-1 activation and subsequently processing pro-IL-1β and pro-IL-18 into their active forms. The NLRP3 inflammasome can be activated by various stimulating factors such as bacteria, virus, fungi, and components of dying cells [4]. Dysregulated NLRP3 inflammasome activation participates in the pathogenesis of sepsis and ALI [5,6]. The inflammasome-associated cytokines belong to the IL-1 cytokine family, and propagate the acute inflammatory response. Heme oxygenase-1 (HO-1) is an inducible and rate-limiting enzyme in heme degradation, catalyzing heme cleavage to form carbon monoxide (CO), ferrous iron and biliverdin [7]. HO-1 can be induced by a variety of stimuli such as heat shock, cytokines, nitric oxide, endotoxin, and hyperoxia, all of which are produced in sepsis. Biliverdin is subsequently converted into bilirubin with significant anti-oxidant and anti-inflammatory properties. CO has a number of biological activities, especially anti-inflammatory features. HO-1 inhibits pulmonary inflammation and has beneficial effects on ALI and sepsis [8,9]. Hemin is a well-known HO-1 inducer, inhibiting oxidative stress-induced tissue

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

damage [10]. On the other hand, zinc protoporphyrin IX (ZnPP) is a HO1 inhibitor, and can inhibit the enzymatic activity of HO-1 [11]. Although the up-regulation of HO-1 and its role in the inflammatory response have been extensively studied, the association between HO-1 and the NLRP3 inflammasome remains unclear. Therefore, the aim of the present study was to investigate whether hemin inhibits NLRP3 inflammasome activation in sepsis-induced ALI and involves HO-1 pathway.

2. Materials and methods 2.1. Animals Male C57BL6 mice (body weight 20–22 g, 6–8 weeks old, purchased from Beijing HFK Bioscience CO., Ltd., Beijing, China), were housed in a light- and temperature-controlled room (21–23 °C; 12 h cycle), with free access to food and water. All experimental procedures were approved by the ethical committee of the Harbin Medical College (Heilongjiang, China), and conducted in accordance with all state regulations.

2.2. Animal model of sepsis Under sodium pentobarbital (50 mg/kg) anesthesia, sepsis was induced through cecal ligation and perforation (CLP), as previously described, with minor modifications [12]. Briefly, the abdomen was incised and the cecum was exposed. The distal half of the cecum was ligated, and then punctured through-and-through once using a 22G needle after the cecal content was gently pushed toward the distal cecum. A small amount of feces was extruded from both the mesenteric and antimesenteric penetration holes to ensure patency. Then, the cecum was repositioned into the abdominal cavity, and the incision was closed with 6.0 surgical sutures. Sham-operated mice underwent the same procedure, including opening of the peritoneum and exposing the cecum, but without ligation and needle perforation of the cecum. After surgery, mice were resuscitated by injecting 1 mL of physiologic saline solution subcutaneously. Mice were then returned to their cages and provided food and water ad libitum. Animals were reanesthetized 12 h after CLP induction. The upper part of the left lung was used for lung wet/dry (W/D) weight ratio estimation. The lower part of left lung was fixed in 4% paraformaldehyde for histology and immunohistochemistry. The right lung was snap-frozen in liquid nitrogen, and stored at − 80 °C for enzyme-linked immunosorbent assay (ELISA), myeloperoxidase (MPO) activity, real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and western blot analysis.

2.3. Experimental protocols Hemin (Sigma, St. Louis, MO, USA, 28 mg/kg), a HO-1 inducer, and ZnPP (Sigma, St Louis, MO, USA, 28 mg/kg), a HO-1 inhibitor, were prepared under subdued light by dissolving the compound in 1 mL of 0.1 N NaOH, adjusting the pH to 7.4 with 1 N HCl, and diluting the solution to the final volume with normal saline (NS) [13]. Mice received intraperitoneal administration with hemin or ZnPP 12 h before CLP induction, and an equal volume of NS was administered to the control animals intraperitoneally. The dose and injection time of ZnPP and hemin treatment we used in the present study were based on previous reports and our preliminary studies [8,14]. Mice were randomly divided into four groups: vehicle-treated sham; vehicle + CLP; hemin + CLP; and ZnPP + CLP. All mice were sacrificed 12 h after CLP. No animal died before being sacrificed.

25

2.4. Lung wet/dry weight ratio Lung edema was estimated by determining lung W/D weight ratios. The fresh upper part of the left lung was weighed and dried in an oven at 80 °C for at least 24 h, then weighed again when it was dry, to calculate the lung W/D weight ratio. 2.5. Histology Left lung paraffin sections (5 μm) were stained with hematoxylin– eosin (H&E) and evaluated using an optical microscope (Olympus Optical, Tokyo, Japan). Pathological severity response of the lung was determined using a previously described semiquantitative scoring system [15], which includes alveolar congestion, hemorrhage, aggregation of neutrophil or leukocyte infiltration, and thickness of the alveolar wall, each graded on a zero (absent) to four points (extensive) scale. 2.6. Measurements of cytokines in serum and lung tissue Serum and lung homogenate levels of cytokines IL-1β and IL-18 were analyzed using commercially available ELISA kits (R&D Systems, Abingdon, Oxon, UK), according to the manufacturer's instructions. Blood was coagulated for 1 h at room temperature and the serum was separated by centrifugation at 4000 rpm for 10 min in a tabletop centrifuge. Tissues (10 mg) from the right lung were homogenized in PBS containing proteinase inhibitor, and centrifuged at 12,000 rpm for 30 min in a refrigerated tabletop centrifuge to obtain protein extracts. 2.7. MPO activity In brief, a part of the frozen right lung tissue was thawed and homogenized in 1 mL of 0.5% hexadecyltrimethylammonium bromide. The sample was then freeze-thawed, and the MPO activity of the supernatant was measured as previously described [16]. The enzyme activity was assayed by measuring absorbance changes in the redox reaction of H2O2 by spectrophotometry at 450 nm. Results were expressed as MPO units per gram of tissue. 2.8. Immunohistochemistry Lung tissue sections (5 μm) were stained with rabbit antibodies against NLRP3 (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) overnight at 4 °C and incubated with alkaline phosphatase (AP)-conjugated goat anti-rabbit Ig-G (Biosynthesis Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature. The AP activity was revealed in brown using DAB (Beijing Biosynthesis Biotechnology Co., Ltd.). 2.9. Real-time RT-PCR analysis Total RNA from a portion of the right lung was extracted using the UltrapureRNA Kit (CoWin Biotech, Beijing, China) and reverse transcribed into first-strand cDNA synthesis using the HiFi-MMLV cDNA Kit (CoWin Biotech). Ultra SYBR Mixture (with ROX) for SYBR Green (CoWin Biotech) and Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) were used for realtime PCR. The following primers were used: NLRP3: forward 5′-TCT GAC CTC TGT GCT CAA AAC CAA C-3′ and reverse 5′-TGA GGT GAG GCT GCA GTT GTC TAA T-3′; ASC: forward 5′-ACT CAT TGC CAG GGT CAC AGA AGT G-3′ and reverse 5′-GCT TCC TCA TCT TGT CTT GGC TGG T-3′; caspase-1: forward:5′-ACT GAC TGG GAC CCT CAA GTT TTG C-3′ and reverse 5′-GGC AAG ACG TGT ACG AGT GGT TGT A-3′; and HO-1: forward 5′-CGA ATG AAC ACT CTG GAG ATG ACA C-3′ and reverse 5′CCT CTG ACG AAG TGA CGC CAT CTG T-3′. Each gene expression was normalized to β-actin mRNA and calculated relatively to naïve sham mice using the comparative CT method.

26

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

2.10. Western blot analysis Proteins (80 or 120 μg) from each right lung homogenate were loaded onto 10–12% polyacrylamide gels for electrophoresis. Proteins were then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dried milk for 2 h at room temperature to reduce nonspecific binding, and washed with PBS containing 0.1% Tween-20 (PBST). Bands were detected using polyclonal antibodies against NLRP3 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase-1 p10 (1:500; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), HO-1 (1:1000, Cell Signaling Technology, Beverly, MA, USA), ASC (1:1000, Cell Signaling Technology, Beverly, MA, USA) and monoclonal antibody against mouse β-actin (Sigma, St. Louis, MO, USA). The binding of all antibodies was detected using an ECL detection system (Thermo Fisher Scientific, Waltham, MA, USA).

were equilibrated for 30 min at 37 °C in PBS. PBS containing DHE (5 μmol/L) was applied to each tissue section and incubated for 30 min in a light-protected and humidified chamber at 37 °C. Oxidized DHE was detected by fluorescence microscopy (TE2000, Nikon, Tokyo, Japan; excitation 488 nm, emission 610 nm). 2.16. Statistical analysis Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Comparison of continuous variables among the four groups was performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test for multiple comparisons. P b 0.05 was considered statistically significant. 3. Results

2.11. Co-immunoprecipitation and immunoblotting analysis

3.1. Hemin improves the severity of acute lung injury

Lung tissue lysates were immunoprecipitated with anti-ASC antibodies (1:100, Cell Signaling Technology, Beverly, MA, USA). Immunoprecipitated proteins were eluted with 5 × sodium dodecyl sulfate (SDS) loading buffer, separated using SDS gels, and transferred onto a polyvinylidene difluoride membrane, on which the western blot was performed.

H&E-stained lung tissue sections obtained from hemin-pretreated mice were compared with the other groups to determine if hemin improves sepsis-induced lung injury. CLP-induced lung injury showed diffuse pathological changes characterized by alveolar congestion and inflammatory cells infiltration into the airspace. However, these changes were inhibited in the hemin group (Fig. 1C). Evaluation of pathological severity of the lung, as performed by independently scoring four parameters, showed a significant decrease in total histopathological score in the hemin group compared with the CLP or ZnPP groups (Fig. 1E). In agreement with this pathological analysis, the W/D ratio of lung tissue showed a similar result (Fig. 1F). These results suggest that hemin-pretreated mice were resistant to CLPinduced ALI.

2.12. Caspase-1 activity assay Caspase-1 enzymatic activity was tested using a colorimetric assay kit (R&D system, Minneapolis, MN). Lung tissue proteins were extracted with cold protein lysis buffer. Tissue lysate (50 μL) was added to 50 μL of caspase-1 reaction buffer in a 96-well flat bottom microplate. Caspase-1 substrate (WEHD-pNA, 200 mM) was added to each sample, followed by 2 h of incubation at 37 °C. Caspase-1 activity was measured at a wavelength of 405 nm. 2.13. HO-1 activity assay HO-1 activity was measured by bilirubin generation, as previously described [14,17]. Lung tissues were homogenized on ice in 1.15% (w/v) KCl containing protease inhibitors and centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was further centrifuged at 100,000 g for 60 min at 4 °C and was then incubated with mouse liver cytosol (3 mg), a source of biliverdin reductase, hemin (20 μM), glucose-6phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 μ), and NADPH (0.8 mM) for 1 h at 37 °C in the dark. The produced bilirubin was extracted with chloroform. Optical density was measured at 465–530 nm and enzyme activity was calculated as picomoles of bilirubin formed per minute per milligram of proteins.

3.2. NLRP3 inflammasome-regulated cytokines are attenuated by hemin Serum and lung homogenate levels of IL-1β and IL-18 were tested to determine if NLRP3 inflammasome-dependent cytokines were reduced by hemin. CLP induced significant increases in IL-1β and IL-18 levels (Fig. 2). We observed a notable reduction in serum and lung tissue levels of IL-1β and IL-18 in hemin-pretreated mice (Fig. 2). These results indicate that hemin may protect mice against sepsisinduced ALI by inhibiting NLRP3 inflammasome activation. 3.3. CLP induced recruitment of inflammatory cells and elevation of NLRP3 is repressed in the hemin group

Nuclear protein extracts were obtained from lung tissues using a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA), according to the manufacturer's instructions. The p65 DNA binding activity was measured using the TransAM NF-κB Kit (Active Motif, Carlsbad, CA, USA).

Lung tissue sections were immunostained with anti-NLRP3 to locate NLRP3 in the lung and to determine if hemin reduces the positively stained cells. We observed that the NLRP3 protein was mainly expressed in the inflammatory cells (i.e. macrophages, neutrophils). There were fewer positively stained cells in lung tissues of the hemin group compared with the CLP or ZnPP group (Fig. 3E). MPO activity, indicating accumulation of neutrophils in the lung, showed a similar result (Fig. 3F). These results suggest that NLRP3 inflammasome activation may accelerate inflammatory cells infiltration in CLPinduced ALI, and that hemin may alleviate the infiltration by NLRP3 inflammasome inhibition.

2.15. Lung tissue malondialdehyde (MDA) and ROS determination

3.4. NLRP3, ASC, caspase-1 mRNA expression in lung are inhibited by hemin

Malondialdehyde (MDA) levels in lung tissue were determined using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's instructions. The MDA content was expressed as nanomoles per milligram of proteins. The oxidative fluorescent dye dihydroethidium was used to evaluate in situ production of reactive oxygen species (ROS) from lung tissue. Lung cryosections (10 μm) were placed on microscope slides, and

Relative NLRP3, ASC, and caspase-1 mRNA levels in lung tissues were detected to determine if NLRP3 inflammasome was inhibited by hemin. CLP significantly increased NLRP3, ASC and caspase-1 mRNA expression compared with sham-operated mice. Hemin pretreatment suppressed the up-regulation of these mRNAs, while ZnPP pretreatment enhanced these mRNA expressions (Fig. 4). Relative HO-1 mRNA levels were significantly up-regulated by hemin

2.14. NF-κB transcription factor assay

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

27

Fig. 1. Effects of hemin on septic lung injury in mice. Representative H&E sections of lung (×200). Tissue morphology was analyzed 12 h after cecal ligation and puncture (CLP) induction. Sham-operated animals served as negative controls (sham) (A). Mice were pretreated with vehicle (normal saline) (CLP) (B), hemin (hemin + CLP) (C) or ZnPP (ZnPP + CLP) (D) 12 h before CLP induction. Semi-quantitative lung injury analysis was evaluated in H&E-stained lung tissue sections. Total histopathological score was expressed as summed scores of four independent parameters: alveolar congestion, hemorrhage, aggregation of neutrophil or leukocyte infiltration, and thickness of the alveolar wall (E). The W/D ratio of lung is also shown (F). Results are presented as mean ± standard deviation (SD) (n = 7 in each group). *P b 0.05 vs. the sham group; #P b 0.05 vs. the CLP group.

compared with the CLP and ZnPP groups. These results show that hemin inhibited NLRP3 inflammasome expression at the transcriptional level and involved the HO-1 pathway.

3.5. NLRP3, ASC, caspase-1 protein expression are suppressed in the hemin group Proteins NLRP3, ASC, and caspase-1 in lung tissue were immunoblotted to further determine if NLRP3 inflammasome expressions were down-regulated by hemin at translational levels. We observed marked up-regulations of NLRP3, ASC and caspase-1 protein levels in CLPinduced lung injury. Hemin pretreatment decreased these protein expressions significantly compared with CLP group or ZnPP group (Fig. 5). HO-1 protein expression was markedly enhanced by hemin compared with the CLP and ZnPP groups. These results show that hemin inhibits NLRP3 inflammasome protein expression, and involved HO-1 induction.

3.6. Interaction between NLRP3 and ASC was inhibited by hemin To determine whether the interaction of NLRP3 with ASC was affected by hemin, lung tissue lysate was subjected to immunoprecipitation with ASC. The amount of NLRP3 associated with ASC was increased by CLP treatment. This increase was inhibited by hemin, while it was not enhanced by ZnPP (Fig. 6A) These results show that the activity of NLRP3 inflammasome may be suppressed by hemin.

3.7. Caspase-1 activity was inhibited by hemin To further determine whether NLRP3 inflammasome activity was inhibited by hemin, caspase-1 enzymatic activity was measured in lung tissues. Caspase-1 activity was higher in the CLP group, but hemin pretreatment inhibited CLP-enhanced caspase-1 activity.

28

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

Fig. 2. Effects of hemin on the NLRP3 inflammasome-regulated cytokine release in the mice model of CLP-induced acute lung injury (ALI). Levels of IL-1β and IL-18 in serum (A, B) and lung tissues (C, D) were determined by ELISA. Results are presented as mean ± standard deviation (SD) (n = 7 in each group). *P b 0.05 vs. the sham group; #P b 0.05 vs. the CLP group.

ZnPP did not increase caspase-1 activity in the CLP group (Fig. 6B). These results show that NLRP3 inflammasome activation induced by CLP may be inhibited by hemin. 3.8. HO-1 enzyme activity is inhibited by ZnPP We observed that HO-1 was slightly up-regulated by ZnPP pretreatment at the transcriptional and translational levels (Figs. 4A, 5). HO-1 activity assay was performed to determine if HO-1 activity was successfully inhibited by ZnPP. We found that administration of hemin increased HO-1 enzyme activity in mice lung tissues, and that HO-1 enzyme activity was significantly decreased using ZnPP pretreatment (Fig. 7A). 3.9. NF-κB activity was inhibited by hemin NF-κB p65 transcriptional factor was assayed to determine whether NF-κB activity was inhibited by hemin. NF-κB activity was significantly increased compared with the sham group. However, hemin pretreatment inhibited CLP-induced NF-κB activity in the lung. ZnPP did not increase NF-κB activity compared with the CLP group (Fig. 7B). These results show that hemin inhibited the NLRP3 inflammasome expression possibly by down-regulating NF-κB activity. 3.10. MDA and ROS were reduced by hemin Determination of lung MDA and ROS levels revealed that hemin pretreatment could reduce MDA and ROS levels in lung tissues compared with the CLP group, but that ZnPP did not increase their levels compared with the CLP group (Fig. 7C, D). These results show that hemin inhibited the activity of NLRP3 inflammasome, possibly by down-regulating ROS levels.

4. Discussion Our study shows that hemin played important functional roles in the protection against sepsis-induced ALI by NLRP3 inflammasome regulation. After a septic insult, hemin pretreatment inhibited NLRP3 inflammasome activation, inflammatory cells infiltration in the lung, and significantly down-regulated lung injury scores. Hemin is believed to be an inducer of HO-1, and our results show that the expression and enzymatic activity of HO-1 were induced by hemin. We may then conclude that the effect of hemin is partly mediated via HO-1 induction, in accordance with our previous studies [8,14], in which preinduction of HO-1 with hemin was associated with alleviated sepsisinduced lung injury. However, administration of ZnPP and hemin abolished the protective effects of HO-1 [13]. In the lung, alveolar macrophages are the most abundant innate immune cells, which usually generate excessive quantities of IL-1β in ARDS patients [18]. HO-1 expression in macrophages mediates potent anti-inflammatory effects, possibly by restraining them from inducing lung injury, and by regulating their role in the inflammatory response [19]. IL-1β induces surface abnormalities [20], and alveolar epithelial and vascular endothelial permeability [21], resulting in alveolar edema which is a key component of lung injury. IL-6 levels in sepsis are also sensitive to IL-1β, and infusion of IL-1β in humans led to increased IL-6 plasma levels in a dose-dependent manner [22]. Inhibition of the IL-1 pathway improved ALI scores in animal models [23], but most ARDS patients did not get much benefit from IL-1 receptor antagonist treatment [24]. It may be effective to treat sepsis or ARDS patients by inhibiting more than one pro-inflammatory pathway. Lung tissue injury can be alleviated by genetic deletion or chemical blockade of IL-18 in ALI. Elevated IL-18 concentrations are related to morbidity and mortality in patients with ARDS [6,25]. NLRP3 inflammasome-dependent cytokines IL-33 and HMGB1 also play important roles in sepsis-induced ALI. IL-33 belongs to the IL-1

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

29

Fig. 3. Effects of hemin on the NLRP3 inflammasome in the lung of the CLP-induced ALI mice model. NLRP3 expression was determined by immunohistochemistry (×400). Sections from CLP lungs were highly positively stained for NLRP3. Scattered positive cytoplasmic staining was noted in the inflammatory cells (B). In the hemin pretreated group, only rare cells were stained for NLRP3 (C). In the ZnPP group, the stained cells were markedly increased (D). NLRP3-positive cells were marked (arrows). Results were scored semi-quantitatively by averaging the number of stained cells per field (×400 magnification) (E). MPO activity in the lung (F). Results are presented as mean ± standard deviation (SD) (n = 5–7 in each group). *P b 0.05 vs. the sham group; #P b 0.05 vs. the CLP group.

cytokine family, which also contains IL-1β and IL-18, and NRLP3 inflammasome activation catalyzes its maturation and release. IL-33 increases inflammatory response in the lung [26]. High mobility group box 1 (HMGB1) is a late mediator of lethal systemic inflammation in sepsis. It has been demonstrated that NLRP3 and ASC/caspase-1/IL-1β signaling promotes HMGB1 induction and release [27,28]. NLRP3 inflammasome activation in macrophages influences the secretion of these pro-inflammatory mediators, as well as alveolar permeability [29]. Our study shows that sepsis-induced IL-1β and IL-18 elevation in serum and lung tissues were markedly suppressed by hemin. It has been reported that HO-1 can down-regulate IL-33 and HMGB1 to protect against lung tissue injury [9,26]. These results suggest that the anti-inflammatory effects of hemin may result from its ability to repress NLRP3 inflammasome activation through HO-1, in turn inhibiting caspase-1 maturation and secretion of IL-1β and IL-18. NLRP3 inflammasome inhibition or deletion in ALI may be an effective strategy to decrease caspase-1 activation and improve sepsis-induced ALI [15]. Neutrophil infiltration is a critical characteristic of sepsis-induced ALI and has detrimental effects on lungs during sepsis. However, neutrophil accumulation in the lung can be inhibited by hemin, as

shown in the present study. In terms of the relationship between NLRP3 inflammasome and neutrophil recruitment, it has been reported that neutrophil recruitment to thermally-injured liver surface can be blunted by NLRP3 or ASC gene knockout [30]. NLRP3-deficient mice are also resistant to hyperoxia-induced ALI because of decreased neutrophil infiltration [15]. Hemin may inhibit neutrophils recruitment in ALI through NLRP3 inflammasome inhibition. To investigate the effect of hemin on NLRP3 inflammasome, we observed that hemin down-regulated the components of NLRP3 inflammasome expression in lung tissue. NLRP3 inflammasome is activated in a two-step manner. In the first step, TLR4 agonist induces NLRP3, pro-caspase-1, pro-IL-1β and pro-IL-18 expressions through the NF-κB pathway [31]. Sepsis is usually caused by Gram-negative bacteria infection and involves inflammatory cytokines mediated by endotoxins such as LPS. LPS is the most common TLR4 agonist in polymicrobial sepsis. After LPS binding to TLR4, monocytes and macrophages in the lung are activated, and release inflammatory cytokines. LPS activates NF-κB through both MyD88-dependent and -independent pathways, resulting in the induction of NLRP3 protein expression. It has been demonstrated that NF-κB plays an important role in NLRP3 in LPS-induced NLRP3 expression and its associated pro-mediators [31].

30

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

Fig. 4. Effects of hemin on NLRP3, ASC and caspase-1 mRNA expression in lung tissues in the CLP-induced ALI mice model. HO-1 (A), NLRP3 (B), ASC (C) and caspase-1 (D) mRNA expressions were determined by real-time RT-PCR. β-Actin was used as an internal control. Results are presented as mean ± standard deviation (SD) (n = 7 in each group). *P b 0.05 vs. the sham group; #P b 0.05 vs. the CLP group.

HO-1 and its catalyzed product CO have been reported to negatively regulate TLR signaling pathway, to inhibit NF-κB activation in various cells and tissues [32]. In the present study, we observed that NF-κB activity in ALI was decreased by hemin. Therefore, hemin may inhibit NLRP3 inflammasome expression by involving the HO1 pathway. After this priming phase, NLRP3 inflammasome can be activated by reactive oxygen species (ROS), mitochondrial DNA (mtDNA) or other stimuli [33,34]. A recent study demonstrated that increased ROS promoted NLRP3 expression at the transcriptional level, but not NLRP3 activation [35]. To further evaluate the influence of hemin on NLRP3 inflammasome activation, we observed that hemin repressed

the binding of NLRP3 with ASC and caspase-1 activity. Meanwhile, hemin reduced MDA and ROS production in lung tissue. Mitochondrial damage is well recognized in sepsis [36]. Overproduction of ROS and mtDNA released from damaged mitochondria both participate in the pathogenesis of sepsis [37]. HO-1 is an important antioxidant enzyme that modulates intracellular ROS levels. This effect may depend on its derived biliverdin and bilirubin that are considered to be physiologically important defenses against ROS [38]. It has also been reported to protect mitochondria from damage and to facilitate mitochondria biogenesis [39], significantly suppressing ROS generation and oxidative stress [32,40]. Hemin may inhibit NLRP3 inflammasome activation involving HO-1 pathway.

Fig. 5. Effects of hemin on NLRP3, ASC and caspase-1 protein expression in the mice model of CLP-induced ALI. HO-1, NLRP3, ASC and caspase-1 p10 protein expressions were determined by western blot. Protein expression was normalized to β-actin. Results are presented as mean ± standard deviation (SD) (n = 3–5 in each group). *P b 0.05 vs. the sham group; #P b 0.05 vs. the CLP group.

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

31

the formation of active NLRP3 inflammasome complex in inflammatory cells could not be directly observed. ZnPP also failed to significantly exacerbate lung injury and inflammatory response, which may be due to its incomplete or competitive inhibition. Our study shows that HO-1 protein expression and HO-1 enzymatic activity were slightly elevated in the CLP group. Thus, it may be very difficult to aggravate CLP-induced lung injury and inflammation for a competitive enzymatic inhibitor. ZnPP may reverse the effect of hemin on HO-1 at a higher level of enzymatic activity [13]. We did not use HO-1 knockout mice or an in vitro knockout model to directly study the impact of HO-1 on NLRP3 inflammasome, and there is still a possibility that hemin affects the NLRP3 inflammasome through other independent effects. Nevertheless, our study yielded important new insights about the anti-inflammatory mechanisms of hemin in sepsis-induced ALI. In summary, hemin protects against sepsis-induced ALI through NLRP3 inflammasome inhibition, reducing IL-1β and IL-18 secretion and restricting the inflammatory response. HO-1 is involved in the effect of hemin on NLRP3 inflammasome. However, the mechanisms of interaction between HO-1 and NLRP3 inflammasome remain to be clarified. A better understanding of the interrelated processes involved in ALI might reveal new therapeutic strategies to prevent and/or treat this life-threatening condition. Fig. 6. Effects of hemin on NLRP3 interaction with ASC and caspase-1 activity in lung tissue. The association of NLRP3 and ASC was detected by immunoprecipitation (IP) using antiASC antibodies followed by western blotting (WB) of NLRP3 and ASC (A). Caspase-1 enzymatic activity was measured using a colorimetric assay kit (B). Results are presented as mean ± standard deviation (SD) (n = 3–7 in each group). *P b 0.05 vs. the sham group; # P b 0.05 vs. the CLP group.

Acknowledgments

This study used a CLP-induced ALI model to investigate how hemin affects NLRP3 inflammasome through HO-1. Despite the strong negative correlation between HO-1 and NLRP3 inflammasome in lung tissue, a direct causal interaction could not be confirmed because of the limitations of the present in vivo study. The influence of hemin on

This work was supported by the National Natural Scientific Foundation of China (Grant No. 81171785), the Natural Scientific Foundation of Heilongjiang Province (Grant No. ZJYO704-02), the Scientific Research Fund of Heilongjiang Provincial Education Department (Grant No. 12521214) and the Youth Scientific Foundation of Heilongjiang

Conflict of interest The authors declare that they have no conflict of interest.

Fig. 7. Effects of hemin on HO-1 enzymatic activity, NF-κB activity, and MDA and ROS production in lung tissue. HO-1 activity in lung tissue (A). NF-κB transcriptional activity in lung tissue (B). MDA and ROS levels in lung tissue (C, D). Results are presented as mean ± standard deviation (SD) (n = 7 in each group). *P b 0.05 vs. the sham group; #P b 0.05 vs. the CLP group.

32

Y. Luo et al. / International Immunopharmacology 20 (2014) 24–32

Province (Grant No. QC2012C014). The authors sincerely thank Dr. Huayang Pan, Dr. Bowen Dong, Dr. You Zhou and Dr. Xiang Fang for their technical support in our experiments.

References [1] Cohen J. The immunopathogenesis of sepsis. Nature 2002;420:885–91. [2] Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003;348:1546–54. [3] Fein AM, Calalang-Colucci MG. Acute lung injury and acute respiratory distress syndrome in sepsis and septic shock. Crit Care Clin 2000;16:289–317. [4] Strowig T, Henao-Mejia J, Elinav E, Flavell R. Inflammasomes in health and disease. Nature 2012;481:278–86. [5] Mao K, Chen S, Chen M, Ma Y, Wang Y, Huang B, et al. Nitric oxide suppresses NLRP3 inflammasome activation and protects against LPS-induced septic shock. Cell Res 2013;23:201–12. [6] Dolinay T, Kim YS, Howrylak J, Hunninghake GM, An CH, Fredenburgh L, et al. Inflammasome-regulated cytokines are critical mediators of acute lung injury. Am J Respir Crit Care Med 2012;185:1225–34. [7] Ryter SW, Choi AM. Heme oxygenase-1/carbon monoxide: novel therapeutic strategies in critical care medicine. Curr Drug Targets 2010;11:1485–94. [8] Fei D, Meng X, Kang K, Nan C, Zhao M, Pan S, et al. Heme oxygenase-1 modulates thrombomodulin and activated protein C levels to attenuate lung injury in cecal ligation and puncture-induced acute lung injury mice. Exp Lung Res 2012;38:173–82. [9] Gong Q, Yin H, Fang M, Xiang Y, Yuan CL, Zheng GY, et al. Heme oxygenase-1 upregulation significantly inhibits TNF-alpha and Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol 2008;8:792–8. [10] An L, Liu CT, Qin XB, Liu QH, Liu Y, Yu SY. Protective effects of hemin in an experimental model of ventilator-induced lung injury. Eur J Pharmacol 2011;661:102–8. [11] Lee TS, Chau LY. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med 2002;8:240–6. [12] Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. Immunodesign of experimental sepsis by cecal ligation and puncture. Nat Protoc 2009;4:31–6. [13] Kim SJ, Eum HA, Billiar TR, Lee SM. Role of heme oxygenase 1 in TNF/TNF receptormediated apoptosis after hepatic ischemia/reperfusion in rats. Shock 2013;39:380–8. [14] Fei D, Meng X, Zhao M, Kang K, Tan G, Pan S, et al. Enhanced induction of heme oxygenase-1 suppresses thrombus formation and affects the protein C system in sepsis. Transl Res 2012;159:99–109. [15] Fukumoto J, Fukumoto I, Parthasarathy PT, Cox R, Huynh B, Ramanathan GK, et al. NLRP3 deletion protects from hyperoxia-induced acute lung injury. Am J Physiol Cell Physiol 2013;305:C182–9. [16] Hasan Z, Palani K, Rahman M, Zhang S, Syk I, Jeppsson B, et al. Rho-kinase signaling regulates pulmonary infiltration of neutrophils in abdominal sepsis via attenuation of CXC chemokine formation and mac-1 expression on neutrophils. Shock 2012;37:282–8. [17] Kang K, Nan C, Fei D, Meng X, Liu W, Zhang W, et al. Heme oxygenase 1 modulates thrombomodulin and endothelial protein C receptor levels to attenuate septic kidney injury. Shock 2013;40:136–43. [18] Jacobs RF, Tabor DR, Burks AW, Campbell GD. Elevated interleukin-1 release by human alveolar macrophages during the adult respiratory distress syndrome. Am Rev Respir Dis 1989;140:1686–92. [19] Otterbein LE, Soares MP, Yamashita K, Bach FH. Heme oxygenase-1: unleashing the protective properties of heme. Trends Immunol 2003;24:449–55. [20] Hybertson BM, Lee YM, Cho HG, Cho OJ, Repine JE. Alveolar type II cell abnormalities and peroxide formation in lungs of rats given IL-1 intratracheally. Inflammation 2000;24:289–303.

[21] Ganter MT, Roux J, Miyazawa B, Howard M, Frank JA, Su G, et al. Interleukin-1beta causes acute lung injury via alphavbeta5 and alphavbeta6 integrin-dependent mechanisms. Circ Res 2008;102:804–12. [22] Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996;87:2095–147. [23] Alexander HR, Doherty GM, Venzon DJ, Merino MJ, Fraker DL, Norton JA. Recombinant interleukin-1 receptor antagonist (IL-1ra): effective therapy against gram-negative sepsis in rats. Surgery 1992;112:188–93 [discussion 93–4]. [24] Fisher Jr CJ, Dhainaut JF, Opal SM, Pribble JP, Balk RA, Slotman GJ, et al. Recombinant human interleukin 1 receptor antagonist in the treatment of patients with sepsis syndrome. Results from a randomized, double-blind, placebo-controlled trial. Phase III rhIL-1ra Sepsis Syndrome Study Group. JAMA 1994;271:1836–43. [25] Makabe H, Kojika M, Takahashi G, Matsumoto N, Shibata S, Suzuki Y, et al. Interleukin-18 levels reflect the long-term prognosis of acute lung injury and acute respiratory distress syndrome. J Anesth 2012;26:658–63. [26] Yin H, Li X, Yuan B, Zhang B, Hu S, Gu H, et al. Heme oxygenase-1 ameliorates LPSinduced acute lung injury correlated with downregulation of interleukin-33. Int Immunopharmacol 2011;11:2112–7. [27] Kamo N, Ke B, Ghaffari AA, Shen XD, Busuttil RW, Cheng G, et al. ASC/caspase-1/IL1beta signaling triggers inflammatory responses by promoting HMGB1 induction in liver ischemia/reperfusion injury. Hepatology 2013;58:351–62. [28] Willingham SB, Allen IC, Bergstralh DT, Brickey WJ, Huang MT, Taxman DJ, et al. NLRP3 (NALP3, cryopyrin) facilitates in vivo caspase-1 activation, necrosis, and HMGB1 release via inflammasome-dependent and -independent pathways. J Immunol 2009;183:2008–15. [29] Kolliputi N, Galam L, Parthasarathy PT, Tipparaju SM, Lockey RF. NALP-3 inflammasome silencing attenuates ceramide-induced transepithelial permeability. J Cell Physiol 2012;227:3310–6. [30] McDonald B, Pittman K, Menezes GB, Hirota SA, Slaba I, Waterhouse CC, et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 2010;330:362–6. [31] Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 2009;183:787–91. [32] Wang XM, Kim HP, Nakahira K, Ryter SW, Choi AM. The heme oxygenase-1/carbon monoxide pathway suppresses TLR4 signaling by regulating the interaction of TLR4 with caveolin-1. J Immunol 2009;182:3809–18. [33] Zhou R, Tardivel A, Thorens B, Choi I, Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol 2010;11:136–40. [34] Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S, et al. Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity 2012;36:401–14. [35] Bauernfeind F, Bartok E, Rieger A, Franchi L, Nunez G, Hornung V. Cutting edge: reactive oxygen species inhibitors block priming, but not activation, of the NLRP3 inflammasome. J Immunol 2011;187:613–7. [36] Brealey D, Brand M, Hargreaves I, Heales S, Land J, Smolenski R, et al. Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 2002;360:219–23. [37] Garrabou G, Moren C, Lopez S, Tobias E, Cardellach F, Miro O, et al. The effects of sepsis on mitochondria. J Infect Dis 2012;205:392–400. [38] Matsumoto H, Ishikawa K, Itabe H, Maruyama Y. Carbon monoxide and bilirubin from heme oxygenase-1 suppresses reactive oxygen species generation and plasminogen activator inhibitor-1 induction. Mol Cell Biochem 2006;291:21–8. [39] MacGarvey NC, Suliman HB, Bartz RR, Fu P, Withers CM, Welty-Wolf KE, et al. Activation of mitochondrial biogenesis by heme oxygenase-1-mediated NF-E2-related factor-2 induction rescues mice from lethal Staphylococcus aureus sepsis. Am J Respir Crit Care Med 2012;185:851–61. [40] Nakahira K, Kim HP, Geng XH, Nakao A, Wang X, Murase N, et al. Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts. J Exp Med 2006;203:2377–89.