Inflammation elevated IL-33 originating from the lung mediates inflammation in acute lung injury

Clinical Immunology 173 (2016) 32–43

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Inflammation elevated IL-33 originating from the lung mediates inflammation in acute lung injury Shi-hui Lin a,1, Juan Fu a,1, Chuan-jiang Wang a, Feng Gao a, Xuan-yun Feng a, Qiong Liu a, Ju Cao b, Fang Xu a,⁎ a b

Department of Emergency, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, PR China Department of Laboratory Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, PR China

a r t i c l e

i n f o

Article history: Received 3 December 2015 Received in revised form 23 July 2016 accepted with revision 24 October 2016 Available online 29 October 2016 Keywords: Acute respiratory distress syndrome Interleukin-33 Inflammation Cytokine

a b s t r a c t Excessive inflammatory reactions occur with acute respiratory distress syndrome (ARDS), however, the underlying mechanisms of ARDS remain incompletely understood. Here we investigated whether interleukin (IL)-33 was elevated in ARDS patients. Serum samples were obtained from 14 ARDS patients and 24 control healthy volunteers. ELISA was used to measure the concentrations of IL-33. Besides, we established pulmonary ARDS and extrapulmonary ARDS models in mice, and serum and lung tissue samples were collected for analyses. The results showed that serum IL-33 concentrations were significantly higher in pulmonary ARDS patients compared to controls. Also, the levels of IFN-γ and IL-2 were positively correlated with IL-33 levels. We also showed that there were increased IL-33 levels in both the serum and lungs in the pulmonary ARDS model. This was not the case, however, in the extrapulmonary ARDS model. Pulmonary inflammation and injury in the pulmonary ARDS model was reduced with IL-33 neutralizing antibody treatment. © 2016 Elsevier Inc. All rights reserved.

1. Introduction In 1967, Ashbaugh described acute respiratory distress syndrome (ARDS) and this definition was changed to the Berlin definition in 2012. The new definition for ARDS was acute respiratory failure in terms of acute onset, diffuse infiltrates on chest X-ray, hypoxia, and the absence of cardiac failure or pulmonary edema of cardiac origin [1,2]. ARDS is a frequent cause of ICU admission and has a high rate of morbidity and mortality [3]. The causes of ARDS have been segregated into direct (pneumonia, pulmonary contusion, inhalational injury, and near drowning) and indirect insults to the lungs (sepsis, trauma, pancreatitis, and hemorrhagic shock), hence ARDS has been classified as pulmonary and extrapulmonary ARDS, respectively [4]. Characterization of ARDS includes inflammation of the lung parenchyma, which leads to impaired gas exchange with attendant systemic release of inflammatory mediators [5]. Up-regulation of adhesion molecules and chemokines and an imbalance in inflammation/anti-inflammation are critical for the development and progression of ARDS. Although extensive research has revealed the

Abbreviations: ARDS, acute respiratory distress syndrome; ELISA, enzyme-linked immunosorbent assay; ICU, intensive care unit; PBS, phosphate buffered saline; H2O2, hydrogen peroxide; VFDs, ventilator-free days; RA, rheumatoid arthritis; AD, atopic dermatitis; IL-33, interleukin-33; COPD, chronic obstructive pulmonary disease; CLP, cecal ligation and puncture; H&E, hematoxylin and eosin; ANOVA, one-way analysis of variance; UC, ulcerative colitis; RA-SFs, rheumatoid arthritis synovial fibroblasts; MyD88, myeloid differentiation factor 88. ⁎ Corresponding author. E-mail address: [email protected] (F. Xu). 1 Both Shi-hui Lin and Juan Fu contributed equally to this work equal first author.

http://dx.doi.org/10.1016/j.clim.2016.10.014 1521-6616/© 2016 Elsevier Inc. All rights reserved.

possible underlying molecular mechanisms, the mortality of ARDS is still high. Therefore, there is an urgent need to find a new therapeutic target for ARDS in an effort to improve clinical outcomes. Interleukin (IL)-33 is a recently discovered member of the IL-1 cytokine family and it is constitutively expressed in the nucleus of vascular endothelial cells and epithelial cells of human barrier tissues (lung, skin, stomach) [6,7]. IL-33 “stored” in the nucleus will be released as an alarmin after endothelial and epithelial cell damage or necrosis and to alert the surrounding tissue and the immune system [7]. Once outside the cell, IL-33 mediates its cytokine-like effects via signaling through a heterodimeric receptor consisting of ST2 and IL-1R accessory protein (IL-1RAcP) [8]. The ST2 gene can produce two major products, a transmembrane form ST2 (ST2L) and a soluble secreted form ST2 (sST2). ST2L is considered to be the functional component for induction of IL33 bioactivities, while sST2 acts as a decoy receptor for IL-33 and sequester and neutralize IL-33 [9]. IL-33 has recently been shown to be a multi-effective cytokine [6]. For example, IL-33 exerts a pro-inflammatory effect in some inflammatory diseases, such as allergic asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA) and so on [10–12]. IL-33, however, is host-protective against parasitic infections and reduces atherosclerosis [13,14]. IL-33 has been reported to mainly participate in Th2-related responses, but recent studies have suggested it also can promote Th1 immune response [15]. In recent years, a growing number of studies found that IL-33 plays an important role in the development of lung inflammation. Kondo et al. reported that the exogenous administration of IL-33 causes severe airway inflammation, goblet cell hyperplasia, and airway hyper-responsiveness in mice [16]. A recent study demonstrated that over-expression

S. Lin et al. / Clinical Immunology 173 (2016) 32–43 Table 1 Characteristics of the study population.

Characteristic

ARDS (n = 49)

Controls (n = 28)

Age(years) Male/female gender, no. Smoker Major surgery Multiple trauma Diabetic ketoacidosis Pregnancy induced hypertension Organic phosphorus poisoning Transfusion Pancreatitis Pneumonia Pulmonary contusion PaO2/FiO2 ratio APACHE II score Ventilator Free Days ICU free days Survival

51 ± 13 30/19 26(53.1%) 7(14.3%) 8(16.3%) 2(4.1%) 3(6.1%) 1(2.1%) 5(10.2%) 4(8.2%) 13(26.5%) 6(12.2%) 142.5 ± 49.7 17.6 ± 6.4 9.1 ± 6.7 11.8 ± 6.2 45(91.8%)

49 ± 8 18/10 11(39.3%) – – – – – – – – – 422.6 ± 79.2 – – – 28(100%)

a) Data as a percentage of patients or mean ± SEM. b) ARDS acute respiratory distress syndrome. c) APACHE II Acute Physiology and Chronic Health Evaluation II.

of endogenous IL-33 has been shown to lead to spontaneous pulmonary inflammation in mIL-33 transgenic mice [17]. Another studies demonstrated that over-expression of sST2 can attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice [18,19]. Bajwa et al. reported that the concentration of plasma sST2 is significantly higher in ARDS patients compared to controls, and the level of plasma sST2 is associated with worse outcome in ARDS [20]. In addition, there are studies found that intratracheal administration of LPS to induce acute lung injury in mice resulted in a higher concentration of IL-33 in lung tissue and bronchoalveolar lavage fluid (BALF) [21,22]. In the rat model of ventilator-induced lung injury, the expression of IL-33 in lung tissue was also increased significantly [23]. Based on these findings, it has been established that IL-33 plays a pro-inflammatory role in the development of ALI/ARDS. However, Mato et al. reported that the expression of IL-33 in serum and BALF didn't increase in ARDS patients [24]. This finding is inconsistent with the above research results. By reviewing a large amount of literatures and analyzing the above research, we found that all the animal studies is to use direct lung injury model (LPS or ventilator-induced lung injury model) to study the role of IL-33 in ALI/ARDS, and there is no one animal research to use indirect lung injury model to study the role of IL-33 in ALI/ARDS. Because different etiologies of ARDS may have different underlying mechanisms, IL-33 may only play

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a role in pulmonary ARDS, but not in the extrapulmonary ARDS. However, all kinds of ARDS patients are included in the research of Mato, they didn't evaluate the expression of IL-33 in pulmonary ARDS and extrapulmonary ARDS patients, respectively. This is probably one of the reason why their study results are inconsistent with the other research results. In order to confirm our speculation, we collected the serum of pulmonary ARDS and extrapulmonary ARDS patients respectively and measured and analyzed the concentrations of serum IL-33. We also assessed whether or not there is a difference in IL-33 levels between pulmonary and extrapulmonary ARDS based on LPS-induced direct and CLP-induced indirect lung injury models. Besides, we also determined whether treatment with anti-IL-33 neutralizing antibodies reduced lung inflammatory reactions in a lung injury model. 2. Materials and methods 2.1. Study population Forty-nine ARDS patients who were receiving standard intensive care and mechanical ventilation support were randomly enrolled from the intensive care unit (ICU) at The First Affiliated Hospital of Chongqing Medical University between 2013 and 2014. Diagnosis of all patients was in accord with the criteria of the Berlin definition [2]. ARDS was observed after sepsis, multiple trauma, major surgery, transfusion, organic phosphorus poisoning, pneumonia, pancreatitis, or pulmonary contusion. Patients who had massive transfusion or hemofiltration within the preceding 24 h, immunosuppressive or immunoenhancing therapy, or chronic lung diseases were excluded from the study. All study patients were admitted to the ICU while in the acute phase of the disease (disease onset within 24 h). Blood collection was obtained from all patients immediately upon admission to the ICU. Healthy volunteers (n = 28) without lung diseases who donated serum served as controls. The study protocol was approved by the Clinical Research Ethics Committee of The First Affiliated Hospital of Chongqing Medical University. Informed consent was obtained from all participants according to the Declaration of Helinski. 2.2. Human serum cytokine measurements Blood was drawn by venipuncture from patients and controls. The blood was centrifuged at 1000 g at 4 °C for 10 min immediately after collection. The serum was then aliquoted into small volumes and kept frozen at − 80 °C until analysis. Serum of fourteen patients with ARDS (pulmonary ARDS, n = 8; extrapulmonary ARDS, n = 6) and twentyfour healthy volunteers were randomly selected and detected the levels of cytokines. An enzyme-linked immunosorbent assay (ELISA) kit (R&D

Fig. 1. (a) An ELISA was used to measure serum IL-33 levels in ARDS patients (n = 14) and healthy controls (n = 24). (b) Serum IL-33 levels in pulmonary ARDS patients (n = 8) and extrapulmonary ARDS patients (n = 6). Non-parametric Mann–Whitney test. Statistical analysis was used to assess the differences in IL-33 levels. ** P b 0.01, ***P b 0.001 when compared between groups denoted by horizontal lines.

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Fig. 2. Correlation analysis between serum IL-33 and other cytokines levels in ARDS patients (n = 14). (a) Correlation between serum IFN-γ and IL-33 levels in ARDS patients. (b) Correlation between serum IL-33 and IL-2 levels in patients with ARDS. (c)-(k) Correlation between serum IL-33 and TNF-α, IL-6, IL-1β, CXCL1, CXCL8, CXCL10, IL-4, IL-5, IL-13 levels in ARDS patients. A non-parametric Spearman rank correlation test was used for correlation analyses.

Systems, Inc., Minneapolis, MN, USA) was used to measure serum levels of IL-33 according to the manufacturer's instructions. TNF-α, IL-1β, IL-2, IL-6, IFN-γ, CXCL1, CXCL8, CXCL10 and Th2 cytokines (IL-4, IL-5, IL-13) levels were determined using a Human Cytokine/Chemokine Magnetic Bead Panel Kit (Merck Millipore, Darmstadt, Germany).

2.3. Animals C57BL/6 mice that were male and 8–12 weeks old were purchased from the Laboratory Animal Center of Chongqing Medical University. Prior to experiments, the mice were acclimatized to the new environment for 7 days at 22 °C with a free access to water and food and with a 12 h light/dark cycle. The study procedures involving animals were carefully reviewed and approved by the Institutional Animal Care and Use Committee's guidelines at the Chongqing Medical University.

2.4. Cecal ligation and puncture (CLP)-induced lung inflammation/injury model CLP was performed as previously described [25]. Intraperitoneal administration of chloral hydrate (3.5%) was used to anesthetize the mice. The mice were positioned in the dorsal position. After shaving and aseptic preparation of the surgical site, a ventral midline incision (1 cm) was made to allow exteriorization of the cecum. The cecum was found and penetrated with a 21-guage needle and 3–0 silk suture 75% from the tip. After puncturing, the cecum was pinched to produce a small amount of feces, and then the cecum was returned to the abdominal cavity. Then, the abdominal incision was closed. The same surgical laparotomy was performed on the sham-operated control mice after anesthesia, and the cecum was pulled through and manipulated as described above, the cecum was not punctured or ligated. Immediately after surgery, animals were resuscitated with subcutaneously injected saline (50 ml/kg). Mice

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Fig. 2 (continued).

were then sacrificed at 6 h, 12 h or 24 h after the CLP or initial sham procedure and lung tissues were obtained for analysis. 2.5. A model of LPS-induced lung inflammation/injury

IgG (Bioss, Beijing, China) 30 min before the LPS challenge. It is reported that the airway inflammatory response in mice was significantly reduced by the dose of anti-IL-33 polyclonal antibodies [27]. Blood and lung tissues were then collected at pre-determined time points.

C57BL/6 mice were anesthetized by intraperitoneal injection of chloral hydrate (3.5%) and then experimental mice were administrated 50 μg of LPS intranasally (Escherichia coli, serotype 055:B5; Sigma-Aldrich, St. Louis, MO, USA) in 50 μl phosphate buffered saline (PBS) to induce lung injury. The mice in the control group were given 50 μl of PBS intranasally without LPS, as described previously [26]. Six, 12, and 24 h after the LPS or PBS injection, mice were euthanized, and blood and lung tissues were harvested for analysis.

2.7. Estimating pulmonary edema

2.6. Treatment with anti-IL-33 antibody

2.8. Histopathology

Mice received an intraperitoneal injection of 100 mg of polyclonal goat anti-mouse IL-33 antibodies (AF3626; R&D systems) or goat isotype

In order to evaluate tissue inflammation, the upper lobe of the right lungs were fixed with 10% formalin (Boer lad, Chongqing, China) in PBS

The amount of extravascular lung water was calculated as an index of lung edema. The left lung was removed and the wet weight was measured and recorded. Then the lungs were incubated for 3–4 days at 60 °C to remove all moisture and then the lungs were re-weighed. The ratio of wet to dry weight was obtained by dividing the wet weight by the dry weight.

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S. Lin et al. / Clinical Immunology 173 (2016) 32–43

for 24 h, dehydrated in a graded ethanol series, embedded in paraffin, then paraffin sections were stained with hematoxylin and eosin (H&E), followed by microscopic assessments and documented by photographs. Then, the slides were histopathologically evaluated using a semi-quantitative scoring method in a double-blind manner. The score consisted of 0 (normal), 1 (mild), 2 (moderate), 3 (severe), or 4 (especially severe) points ranked according to four indicators (alveolar congestion, hemorrhage, edema, and neutrophil infiltration). Two pathology experts observed lung tissue slice under 10 × 40 high-

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power microscope. 10 different visual fields were randomly selected in lung slice for scoring. The average value was the final result. 2.9. Mouse serum cytokine measurements Blood was drawn from mice and coagulated for 1 h at room temperature, then the serum was obtained by centrifuging the blood samples for 10 min at 1000 g at 4 °C. The serum was then aliquoted and kept frozen at −80 °C until analysis. Serum IL-33 (R&D Systems, Inc.), TNF-α, IL-

Fig. 3. C57BL/6 mice were randomly divided into sham group and CLP group (n = 20 mice/group) and there were four time points in each group (n = 5 mice/time point). (a) Histology of lung tissues from representative mice 24 h after CLP was assessed by hematoxylin and eosin staining. Representative micrograph of lung tissue sections (400× magnification). (b) Lung injury score of CLP group and sham group. (c) The lung wet:dry weight ratios within 24 h after CLP. (d) The IL-6, TNF-α, and TGF-β1 levels in serum were evaluated by ELISA in mice within 24 h after CLP. (e) Lung IL-33 mRNA levels was determined by qRT-PCR in mice within 24 h after CLP. The relative levels of expression of the genes were expressed with the GAPDH housekeeping gene as an internal reference. (f) The serum IL-33 levels in serum were evaluated by ELISA in mice within 24 h after CLP. (f) Immunohistochemistry assessment and colorimetric detection with DAN (brown stain) was used to determine the expression of IL-33 in lung tissues at 24 h after CLP. The figure is presented at a magnification of 400×. ***P b 0.001 compared with 0 h. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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6, TGF-β1, IFN-γ, CXCL1, CXCL8, and CXCL10 (Hushang, Shanghai, China) levels were determined with ELISA kits according to the manufacturers' instructions. 2.10. RNA extraction and quantification Total RNA was extracted from lung tissues using TRIzol reagent (Takara, Dalian, China) according to the manufacturer's instructions. The RNA was then reversely transcribed to make cDNA using a Prime Script™ RT Reagent Kit (Takara). cDNA samples were amplified with SYBR Premix Ix Taq II (Takara) and run on a CFX-96™ Real-Time System (Bio-Rad, Hercules, California, USA). The primer sequences for IL-33 and GAPDH were as follows: IL-33, forward 5′-GCAAGACCAG GTGCTACTACGC-3′ and reverse 5′-GAGTAGTCCTTGTCGTTGGCATG-3′; and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), forward 5′-TTCACCACCATGGAGAAGGC-3′ and reverse 5′GGCATGGACTGTGGTCATGA-3′. The primers were purchased from Takara. The qPCR conditions were 30 s at 95 °C, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. As an internal control, each sample was measured in triplicate and normalized to that of GADPH. The 2−Δ ΔCt method was used to calculate the relative mRNA expression levels of target genes.

related to pneumonia, n = 13; pulmonary contusion, n = 6) or indirect lung injury (30 patients, related to major surgery, n = 7; multiple trauma, n = 8; diabetic ketoacidosis, n = 2; pregnancy-induced hypertension, n = 3; pancreatitis, n = 4; organic phosphorus poisoning, n = 1, transfusion, n = 5). The average APACHE II score was 17.6 ± 6.4. The average ventilator-free days (VFDs) and ICU-free days were 9.1 ± 6.7 and 11.8 ± 6.2 days, respectively.

3.2. ARDS patient IL-33 levels We first compared the serum IL-33 levels in ARDS patients and healthy controls. The serum IL-33 levels in patients with ARDS were significantly higher than controls (p b 0.001, Fig. 1a). In addition, we showed that IL-33 concentrations were clearly higher in the serum from patients with pulmonary ARDS than patients with extrapulmonary ARDS (p b 0.01, Fig. 1b). Based on our ROC analysis, a cut-off level of serum IL-33 for the diagnosis of ARDS was set at 3.24 pg/ml. The specificity, sensitivity, negative predictive value and positive predictive value were 79%, 86% 90% and 72%, respectively.

3.3. Relationship between serum IL-33 and other cytokines in ARDS 2.11. Immunohistochemistry Immunohistochemistry was performed on paraformaldehyde-fixed and paraffin-embedded lung tissues. Five-micrometre sequential sections were mounted on poly-L-lysine-coated glass slides, deparaffinized in xylene, and rehydrated in graded alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide (H2O2) for 10 min. The slides were incubated in 10 mM citrate buffer (pH 6.0) for 20 min in a microwave oven for immunogenic retrieval. Then, tissue sections were blocked for non-specific binding with diluted normal goat serum in PBS at room temperature at 20 min, and the sections were incubated overnight at 4 °C in a humidified chamber with the appropriate goat anti-mouse IL-33 polyclonal antibody (15 μg/ml dilution; R&D Systems, Inc.). The next day, sections were rinsed and incubated with a biotin-conjugated rabbit anti-goat IgG antibody (Zhongshan, Beijing, China) for 20 min at 37 °C. Finally, the sections were incubated with streptavidin-peroxidase (Zhongshan) for 20 min at 37 °C, stained with DAB(Zhongshan), and counterstained with hematoxylin. Specificity control slides were analogously stained using non-immune goat IgG (1:500 dilution; TDY Biotech, Beijing, China) as a primary antibody. 2.12. Statistical analyses SPSS 19.0 was used for all statistical analyses. Values are shown as the mean ± SEM. Differences between groups were analysed by nonparametric Mann–Whitney test or Student's t-test. Statistical analysis was performed using the one-way analysis of variance (ANOVA) followed by LSD multiple comparison test or Dunnett for comparing multiple groups. To test for a correlation between two parameters, a non-parametric Spearman's rank correlation coefficient was used. Statistical significance was set at p b 0.05.

Based on correlation analyses between IL-33 and cytokines in patients with ARDS, the concentration of IFN-γ (r = 0.715, P b 0.01, Fig. 2a) and IL-2 (r = 0.680, P b 0.05, Fig. 2b) were significantly correlated with serum IL-33. The increases in serum IL-33 were associated with increases in serum TNF-α, but did not reach statistical significance (P N 0.09 for TNF-α, Fig. 2c). The concentration of IL-6, IL-1β, CXCL1, CXCL8, CXCL10, IL-4, IL-5 or IL-13 did not correlated with the level of IL-33 (P N 0.05, Fig. 2d-k).

3.4. Changes in histopathology, IL-33 production, and serum cytokine levels in a murine model of CLP-induced lung inflammation/injury To further study IL-33 expression in the lungs, we first established a murine model of extrapulmonary ARDS. In the CLP model, there was widespread alveolar wall thickness caused by edema, severe hemorrhage in the alveoli, alveolar collapse and prominent inflammatory cell infiltration, according to lungs stained with hematoxylin-eosin (Fig. 3a). Lung injury score of CLP group was significantly higher than sham group (Fig. 3b). Within 24 h after CLP, the lung wet:dry weight ratios were progressively increased (Fig. 3c). We also measured the concentrations of serum TNF-α, TGF-β1, and IL-6, which increased significantly 6 h after CLP (Fig. 3d). The levels of IL-33 were measured with qRT-PCR, an ELISA assay, and immunohistochemistry staining to determine whether IL-33 is involved in CLP-induced lung inflammation/injury. The results showed that IL-33 mRNA levels in the lungs and IL-33 protein in the serum were not significantly different after CLP (Fig. 3e, f). Furthermore, the immunohistochemistry staining results also showed that IL-33 expression in lung tissues was not significantly different compared with the sham group (Fig. 3g).

3. Results 3.1. Baseline characteristics of the study population

3.5. IL-33 production was increased in a murine model of LPS-induced lung inflamma- tion/injury

There were 77 subjects (49 patients with ARDS and 28 healthy controls) who participated in the study, according to the inclusion and exclusion criteria. Table 1 shows the baseline characteristics of the subjects. The mean age of the ARDS patients was 51 ± 13 years and 19 of the patients were females. All patients met the ARDS PaO2:FiO2 criteria, with a mean PaO2:FiO2 ratio of 142.5 ± 49.7 mmHg. The causes of ARDS in the study participants was direct lung injury (19 patients,

A murine model of pulmonary ARDS was established with intranasal instillation of LPS. An ELISA and qRT-PCR were used to measure the expression of IL-33 protein and mRNA, respectively. The results showed that IL-33 mRNA levels in the lung were substantially increased 6 h after LPS administration, and recovered to normal levels 24 h after LPS administration (Fig. 4a), and IL-33 protein in serum was significantly increased 24 h after LPS administration (Fig. 4b).

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Fig. 4. C57BL/6 mice were randomly divided into LPS group and control group (n = 20 mice/group) and there were four time points in each group (n = 5 mice/time point). IL-33 levels were elevated in LPS-induced ARDS. (a) qRT-PCR was used to determine lung IL-33 mRNA levels in mice at 6 h after LPS administration. Relative levels of expression of the genes were expressed with the GAPDH housekeeping gene as an internal reference. (b) The serum IL-33 levels were determined by ELISA in mice at 24 h after LPS administration. **P b 0.01, ***P b 0.001 when compared between groups denoted by horizontal lines.

3.6. Effect of IL-33 neutralizing antibody treatment on LPS-induced lung inflammation/injury We analyzed whether IL-33 neutralizing antibodies can correct LPSinduced lung inflammation/injury. Mice received an intraperitoneal injection of an equal dose of control IgG 30 min prior to LPS administration or a single dose of IL-33 neutralizing antibodies. We further analyzed inflammatory cell infiltration and the effects of anti-IL-33 treatment with histologic examination of lung sections stained with H&E. We found that LPS administration induced widespread alveolar wall thickness related to edema alveolar collapse, severe hemorrhage, and apparent inflammatory cell infiltration. Treatment with anti-IL-33 antibodies severely curbed the inflammation/injury of lung tissues. There was no effect with control antibody treatment on inflammation/ injury lung tissues (Fig. 5a). Lung injury score of LPS group was significantly higher than control group, and the score was significantly decreased after treatment with anti-IL-33 antibody (Fig. 5b). At the same time, the pulmonary edema significantly improved after treatment with anti-IL-33 antibody (Fig. 5c). We also examined the IL-33 protein levels in lung tissues after LPS administration and treatment with antiIL-33 antibody by immunohistochemistry staining. The results showed that the IL-33 detected by immunohistochemistry was significantly increased in the lung tissues after LPS administration compared with control mice. IL-33 protein levels in lung tissues were significantly decreased following treatment with anti-IL-33 antibodies (Fig. 5d). In addition, IL-33 neutralizing antibody pre-treatment efficiently reduced the production of TNF-α, IL-6, IFN-γ, CXCL1, CXCL8, and CXCL10 (P b 0.05 in all cases, Fig. 5e).

4. Discussion Many studies have shown that IL-33 exerts a pro-inflammatory effect in inflammatory diseases. In ulcerative colitis (UC) and experimental colitis, IL-33 is significantly up-regulated and induces GATA-3, a master regulator gene of Th2 differentiation, expression in mucosal T cells, then the differentiated T helper cells produce a large amount of Th2 cytokines and aggravate inflammation of colitis [28]. In rheumatoid arthritis (RA), there is excessive IL-33 expression, enhanced TNF-α-induced synthesis of the pro-inflammatory molecules, IL-6 and IL-8, and matrix metalloproteinase-3 in rheumatoid arthritis synovial fibroblasts (RA-SFs) [12]. IL-33 has also been closely associated with the development of atopic dermatitis (AD) [29]. Recent studies have demonstrated that IL-33 plays a crucial pro-inflammatory role in several pulmonary inflammatory diseases, such as allergic asthma, chronic obstructive

pulmonary diseases (COPD), lung fibrosis, and pneumonia [10,11,30, 31]. ARDS is also a kind of pulmonary inflammatory diseases, studies have confirmed that the expression of IL-33 is significantly increased in the mice model of intratracheal administration of LPS- induced acute lung injury and the rat model of ventilator-induced acute lung injury [21–23]. Schmitz et al. demonstrated that the administration of IL33 induced an infiltration of eosinophils and mononuclear cells around pulmonary vessels [8]. Furthermore, IL-33 can be released from damaged lung endothelial and epithelial cells [7] or dendritic cells, macrophages, and mast cells [8,32] through autocrine and paracrine pathways, then activate mast cells, eosinophils, basophils, or lung endothelial and epithelial cells to produce a variety of cytokines and chemokines [33–37]. All of the inflammatory cells can accumulate during lung injury and participate in inflammatory reactions. We therefore hypothesized that IL-33 plays a crucial role in pulmonary inflammation that triggers or progresses to ARDS by promoting the inflammatory cells to produce a variety of cytokines and chemokines. Puzzlingly, there was a study demonstrated that the expression of IL-33 is not increased in the serum and BALF of ARDS patients. Why this study result is inconsistent with the results of animal studies? In the animal studies, they all established the direct lung injury model to study the role of IL-33 in ARDS. At present, there was no research to study the role of IL-33 in the indirect lung injury model. However, in the clinical study performed by Mato et al., pulmonary ARDS and extrapulmonary ARDS patients have not been separately studied. Different etiologies of ARDS may have different underlying mechanisms, IL-33 may only play a role in pulmonary ARDS, but don't play a role in the extrapulmonary ARDS. Therefore, the result of clinical study is inconsistent with the results of animal studies. In our study, we studied the role of IL-33 in pulmonary ARDS and extrapulmonary ARDS, respectively. The results from this study showed that serum IL33 levels in ARDS patients were higher than in healthy controls and IL-33 is positively correlated with some pro-inflammatory cytokines in serum. Our study showed that IL-33 was increased in a murine model of LPS-induced direct lung injury. Treatment with IL-33 neutralizing antibody significantly reduced pulmonary inflammation and injury. Together, this data clearly indicates that IL-33 plays a pro-inflammatory role in the development and progression of ARDS. ARDS is involved in direct and indirect lung injury. Serum IL-33 levels in pulmonary and extrapulmonary ARDS patients were measured and the results showed that serum IL-33 concentrations were significantly higher in pulmonary ARDS patients compared to extrapulmonary ARDS patients. The difference in serum IL-33 levels in patients with extrapulmonary ARDS compared to healthy controls was not significant. To further study the role of the IL-33 in pulmonary and extrapulmonary ARDS, we established two

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Fig. 5. C57BL/6 mice were randomly divided into LPS group, Control group, LPS + Anti-IL-33 group and LPS + IgG group (n = 20 mice/group) and there were four time points in each group (n = 5 mice/time point). Pre-treatment with IL-33 neutralizing antibody attenuated LPS-induced ARDS. (a) Lung samples from each experimental group were subjected to histologic examination after hematoxylin and eosin staining (400×). Mice that were exposed to intratracheal LPS alone or to LPS + IgG for 24 h led to profound neutrophil infiltration and alveolar hemorrhage compared with control mice. These conditions were dramatically decreased in mice pre-treated with IL-33 neutralizing antibody prior to LPS challenge. (b) Lung injury score of LPS group, Control group, LPS + Anti-IL-33 group and LPS + IgG group. (c) The lung wet:dry weight ratios from each experimental group 24 h after LPS administration. (d) Immunohistochemistry analysis and colorimetric detection with DAB (brown stain) was used to determine IL-33 expression levels in lung tissues at 24 h after LPS administration. The figure is presented at a magnification of 400×. (e) Serum levels of cytokines and chemokines, including TNF-α, IL-6, IFN-γ, CXCL1, CXCL8, and CXCL10, 6 h after LPS administration. *P b 0.05, **P b 0.01, and ***P b 0.001 when compared between groups denoted by horizontal lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

models of ARDS in mice. Our study showed that both the LPS-induced direct and CLP-induced indirect lung injury models had characteristic lung injury, such as widespread alveolar wall thickness, severe hemorrhage in the alveoli, edema, alveolar collapse, and prominent inflammatory cell infiltration; however, we only detected increased IL-33 levels in the serum of mice in the LPS-induced direct lung injury model. In the CLP-induced indirect lung injury model, IL-33 levels did not differ from the sham control group. Thus, IL-33 may only play a pro-

inflammatory role in pulmonary ARDS, possibly because different etiologies of ARDS have different underlying mechanisms. Intranasal instillation of LPS created an experimental model of direct lung injury, and led to lung tissue damage without causing multiple organ failure or systemic inflammation, which is different from the CLP model [26]. In addition, several studies have demonstrated that IL-33 mRNA and protein expression levels were profoundly increased in response to a LPS challenge [37,38], then IL-33 potentiated LPS-induced IL-6 and TNF-α

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Fig. 5 (continued).

production by macrophages through up-regulation of the myeloid differentiation factor 88 (MyD88) adaptor molecule and the LPS receptor (MD2/CD14/TLR4). Pro-inflammatory cytokine production by macrophages was not induced solely by IL-33. Such effects of IL-33 on LPS-mediated activation were abolished by treatment with neutralization of IL33 activity. This, perhaps, is the another reason for that IL-33 levels didn't increase in CLP model. Our study also showed that IL-33 expression was significantly increased as early as 6 h after LPS administration in the lungs, suggesting that IL-33 production may be an initial event in the inflammatory process of pulmonary ARDS and might become an indicator of pulmonary ARDS. Therefore, inhibition of IL-33 in the early stages of disease may be a useful therapeutic method to treat pulmonary ARDS. Our results showed that treatment with IL-33 neutralizing antibody can reduce lung edema and inflammatory cell recruitment to the airways and decrease the levels of serum inflammatory, including IL-6, TNF-α, IFN-γ, CXCL1, CXCL8, and CXCL10, in a murine model of LPS-induced direct lung injury. Thus, blockade of IL-33 can effectively control the excessive

inflammatory reaction during pulmonary ARDS. Indeed, this is the first study to show that anti-IL-33 antibody treatment can prevent the development of pulmonary ARDS in a murine model. Therefore, the blockade of IL-33 is a potential therapeutic strategy for pulmonary ARDS. Based on the ROC analysis, we know the specificity, sensitivity, positive predictive value, and negative predictive value to diagnose ARDS by IL-33 were 79%, 86%, 72%, and 90%, respectively. IL-33 is likely a new biomarker for ARDS. IL-33 limitations as a biomarker should be considered. Only 7 of the 14 ARDS patients had higher serum IL-33 levels than the maximum serum IL-33 levels of healthy controls. Thus, the serum IL-33 level may not differentiate ARDS patients from healthy controls. This situation may be associated with the different etiology of ARDS. In the present study, we showed that serum IL-33 levels were significantly higher in patients with pulmonary ARDS than patients with extrapulmonary ARDS. In animal experiments, IL-33 levels in the serum and lungs were only increased in the LPS-induced direct lung injury murine model. In the CLP-induced indirect lung injury mouse model, IL-33 levels were not different from the sham control group;

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S. Lin et al. / Clinical Immunology 173 (2016) 32–43

Fig. 5 (continued).

the small sample size of this study may be a major reason. Thus, samples collected a number of times in a large population of both pulmonary and extrapulmonary ARDS patients are necessary to further determine the usefulness of IL-33 as a diagnostic and prognostic biomarker for ARDS.

None of the authors has any potential financial conflict of interest related to this manuscript.

5. Conclusions

Acknowledgements

In conclusion, IL-33 levels were increased in serum of patients with ARDS, and positively correlated with some inflammatory cytokines in the serum. In animal models, we demonstrated that IL-33 is increased only in the LPS-induced direct lung injury murine model and treatment with IL-33 neutralizing antibodies lowered pulmonary inflammation and injury significantly, which suggested that IL-33 could be a potential therapeutic target in pulmonary ARDS.

We thank the laboratory of Lipid & Glucose Metabolism at The First Affiliated Hospital of Chongqing Medical University to provide laboratory facilities. Thanks for Jingxian Wu and Yanlin Chen from Chongqing Medical University to help us evaluate the histological changes using a semi-quantitative scoring method. This study was supported by National Natural Science Foundation grants of China (No: 81570069, to FX) and Basic science and cutting-edge technology research projects of

Disclosures

S. Lin et al. / Clinical Immunology 173 (2016) 32–43

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