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Enforced expression of miR-125b attenuates LPS-induced acute lung injury
Immunology Letters 162 (2014) 18–26
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Enforced expression of miR-125b attenuates LPS-induced acute lung injury Zhongliang Guo a,1 , Yutong Gu b,1 , Chunhong Wang c,1 , Jie Zhang a , Shan Shan c , Xia Gu c , Kailing Wang c , Yang Han d , Tao Ren c,∗ a
Department of VIP, East Hospital, Tongji University School of Medicine, Shanghai, China Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China c Department of Respiratory Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China d Department of Pathology, East Hospital, Tongji University School of Medicine, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 9 March 2014 Received in revised form 11 June 2014 Accepted 15 June 2014 Available online 6 July 2014 Keywords: Acute respiratory distress syndrome Acute lung injury miR-125b
a b s t r a c t The acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. Despite decades of research, few therapeutic strategies for clinical ARDS have emerged. Recent evidence implicated a potential role of miR125b in development of ALI. Here we evaluated the miR-125b-based strategy in treatment of ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. We found that up-regulation of miR-125b expression maintained the body weight and survival of ALI mice, and significantly reduced LPS-induced pulmonary inflammation as reflected by reductions in total cell and neutrophil counts, proinflammatory cytokines, as well as chemokines in BAL fluid. Further, enforced expression of miR-125b resulted in remarkable reversal of LPS-induced increases in lung permeability as assessed by reductions in total protein, albumin and IgM in BAL fluid, and ameliorated the histopathology changes of lung in LPS-induced ALI mice. Of interest, serum miR-125b expression was also decreased and inversely correlated with the disease severity in patients with ARDS. Our findings strongly demonstrated that enforced expression of miR-125b could effectively ameliorate the LPS-induced ALI, suggesting a potential application for miR-125b-based therapy to treat clinical ARDS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory lung disorders are characterized by increased leukocyte infiltration into lung tissues as the body’s immune response to infection or injury [1]. An important inflammatory lung disease of rapid onset is acute lung injury (ALI), which is a significant cause of morbidity and mortality in critically ill patients [1–5]. ALI is a diffuse, heterogeneous type of inflammatory lung disease clinically characterized by progressive hypoxemia, reduced lung compliance, and intense inflammation in the lung tissues [1,6]. The physiological hallmark of ALI is disruption of the alveolar–capillary membrane barrier, leading to development of noncardiogenic pulmonary edema, in which a proteinaceous exudate floods the alveolar spaces, impairs gas exchange, and precipitates respiratory failure [2,5–9]. ALI can result in persistent respiratory failure and
∗ Corresponding author. Tel.: +86 21 38804518x7217; fax: +86 21 58798999. E-mail address: [email protected] (T. Ren). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.imlet.2014.06.008 0165-2478/© 2014 Elsevier B.V. All rights reserved.
prolonged dependence on mechanical ventilation, increasing susceptibility to multiorgan dysfunction and mortality [9,10]. Despite extensive investigation aimed at early diagnostic and pathogenetic factors of ALI, current management is mainly supportive, as specific therapies have not been identified [5,9,11–15]. Therefore, new strategies are urgently required for achieving effective treatment of ALI, which might ultimately aid the clinical therapy for ARDS patients. MicroRNAs (miRNAs) have been shown to be centrally involved in the regulation of immune system development, differentiation of B and T cells, proliferation of monocytes and neutrophils, antibody production, release of inflammatory mediators and certain inflammatory lung diseases [1,16]. Recent study showed that miR125b, which could target the 3 -UTR of TNF-␣ and thus inhibit its expression, was down-regulated in macrophages in response to LPS stimulation in vitro, and was substantially decreased in spleens in C57BL/6 mice after injection with LPS [17]. Of interest, mice deficient in TNF-␣ or TNFR1 are resistant to LPS-induced shock whereas wild-type mice die within hours after LPS challenge, suggesting that the main cytokine responsible for this syndrome is TNF-␣ [17–19].
Z. Guo et al. / Immunology Letters 162 (2014) 18–26 Table 1 The clinical characters of ARDS patients. ARDS Demographics Male Female Age (years) Clinical parameters PaO2/FiO2 APACHE II SAPS II LIS Causes of ARDS Sepsis Lung contusion Shock
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according to the ethical guidelines of the Tongji University Laboratory Animal Care and Use Committee. Number
2.3. Murine model of LPS-induced ALI 11 5 57.3 ± 13.7 176.50 ± 13.76 17.13 ± 3.48 39.19 ± 8.51 2.94 ± 0.23 12 1 3
(1) Shown as patient numbers or mean ± standard deviation values. (2) APACHE II, Acute Physiology and Chronic Health Evaluation (APACHE) II. SAPS II, Simplified Acute Physiology Score (SAPS) II. LIS, Murray Lung Injury Score (LIS).
These findings promoted us to hypothesize that enforced expression of miR-125b might be able to ameliorate the LPS-induced ALI. To test our hypothesis, here we carefully evaluate the potential role of miR-125b-based strategy in treatment of ALI using the murine model of LPS-induced ALI. We found that up-regulation of miR-125b significantly reduced LPS-induced pulmonary inflammation and resulted in remarkable reversal of LPS-induced increases in lung permeability, accompanied by a significant reduction of histopathology changes of lung. Our findings strongly demonstrated that enforced expression of miR-125b could effectively ameliorate the LPS-induced acute lung injury, suggesting a potential role for miR-125b-based therapy to treat patients with ARDS. 2. Materials and methods 2.1. Patients The human study was approved by the Ethics Committee of Tongji University. Sixteen patients with ARDS were enrolled and all the peripheral blood samples were collected from human subjects after obtaining informed consent. ARDS was defined as the ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2/FiO2) <300 mm Hg, the presence of bilateral pulmonary infiltrates on a chest radiograph, and no clinical evidence of left atrial hypertension [20,21]. The clinical characteristics of ARDS patients were summarized in Table 1. Disease severity was assessed by the Acute Physiology and Chronic Health Evaluation (APACHE) II, Simplified Acute Physiology Score (SAPS) II, and the Murray Lung Injury Score (LIS), a composite variable that includes components of oxygenation, compliance, positive end expiratory pressure, and the appearance of the chest radiograph [22–24]. Arterial blood samples were drawn for blood gas analysis. Serological levels of TNF-␣ and IL-6 were determined using the human immunoassay kit (R&D Systems). Normal controls (n = 16) matched for gender and age (±2.1 years) were recruited from among the Institute personnel, and all were in excellent health at the time of the study. 2.2. Mice Female BALB/c mice at 6 weeks old were purchased from the Center of Experimental Animals of Tongji University. All mice were housed in the pathogen-free animal facilities of Tongji University School of Medicine. All animal experiments were performed
The murine model of LPS-induced ALI was established as previous reported [5,9]. Briefly, female BALB/c mice (n = 6 per group) were anaesthetized and orally intubated with a sterile plastic catheter, and challenged with intratracheal instillation of 800 g of LPS (Escherichia coli 055:B5; Sigma) dissolved in 50 l of normal PBS. Naive mice (without LPS instillation) were injected with the same volume of pyrogen-free PBS to serve as controls. Mice were humanely killed at 3d after LPS challenge to collect tissues for analysis. 2.4. MiR-125b Quantitative RT-PCR analyses for miR-125b were performed using TaqMan micro-RNA assays (Applied Biosystems) as previously described [25]. All reagents, primers, and probes were obtained from Applied Biosystems. Retrovirus-mediated overexpression of miR-125b was generated based on pMX vector (Invitrogen) as previously described method [25]. Sequence encoding mutant miR-125b was cloned into the same vector and used as the control vector. Virus was produced and target cells were infected according to the user’s manual. To access the effect of miR125b-based strategy on the treatment of ALI, groups of mice were challenged with LPS plus the miR-125b expression vector (2 × 109 plaque-forming units per mouse). 2.5. Determination of total cells and neutrophils According to previously described [5,9], BAL was performed by instilling 0.9% NaCl containing 0.6 mmol/l ethylenediaminetetraacetic acids in two separate 0.5 ml aliquots. The fluid was recovered by gentle suction and placed on ice for immediate processing. An aliquot of the BAL fluid was processed immediately for total and differential cell counts. The remainder of the lavage fluid was centrifuged and the supernatant was removed aseptically and stored in individual aliquots at −70 ◦ C. Total cell counts in BAL fluid were determined using a haemocytometer. Number of neutrophils was calculated as the percentage of neutrophils multiplied by the total number of cells in the BAL fluid sample. All analyses were performed in a blinded fashion. 2.6. Measurement of proinflammatory cytokines, chemokines, albumin and IgM In line with previously described [5,9], BAL fluid collected was centrifuged at 800 g for 10 min, and supernatant was collected for analysis of total protein, albumin, IgM, and cytokine/chemokine levels. Proinflammatory cytokine levels including TNF-␣, IL-1 and IL-6 in BAL fluid were measured with murine cytokine-specific Quantikine ELISA kits (R&D Systems). Chemokine levels including Cxcl2, JE (the murine homolog of human CCL2) and KC (the murine homolog of human IL-8) in BAL fluid were measured using cytokine-specific bead kits (R&D Systems). Albumin and IgM levels in BAL fluid samples were measured using with a murine-specific albumin ELISA kit (ALPCO Diagnostics) and a murine specific IgM ELISA kit (Bethyl Laboratories), respectively. All the measurements were performed according to the manufacturer’s instructions. 2.7. Histopathology Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 m thick sections. Sections were stained
Z. Guo et al. / Immunology Letters 162 (2014) 18–26
BAL miR-125b expression
A
2.8. Statistical analysis Differences between the treated groups versus the injured group were assessed using a one-way ANOVA with statistic software (GraphPad Prism version 4.00). A value of p < 0.05 was considered statistically significant. 3. Results
B
3.1. Expression of miR-125b was down-regulated in BAL fluid of LPS-induced ALI mice To assess the potential role of miR-125b-based strategy in treatment of LPS-induced ALI, we determined the expression level of miR-125b in bronchoalveolar lavage (BAL) fluid of LPS-induced ALI mice at day 3 after LPS challenge. We found that the expression level of miR-125b in BAL fluid was significantly decreased on day 3 in mice challenged with LPS compared with the control groups (Fig. 1A, p < 0.05). Similarly, we revealed that the expression of miR125b in peripheral blood and splenocytes was also down-regulated on day 3 in LPS-induced ALI mice (Fig. 1B and C, p < 0.05). While genomic, biochemical and histology findings are striking at day 3 after LPS exposure, we found a remarkable reduction of miR-125b expression on day 3 of ALI mice (Supplementary Fig. 1). Combing these findings indicated that miR-125b might be involved in the development of ALI. 3.2. Enforced expression of miR-125b in LPS-induced ALI mice Given the decreased expression of miR-125b in LPS-induced ALI mice, we sought to detect whether enforced expression of miR125b could ameliorate the LPS-induced ALI. Thus, groups of mice were challenged with LPS plus the miR-125b expression vector, and then were determined for the expression level of miR-125b in bronchoalveolar lavage (BAL) fluid on day3. We found that the expression level of miR-125b in BAL fluid was significantly elevated by treatment with the miR-125b expression vector (Fig. 2A, p < 0.05). Further, the expression of miR-125b in peripheral blood and splenocytes was also up-regulated (Fig. 2B and C, p < 0.05). These findings demonstrated the enforced expression of miR-125b in LPS-induced ALI mice.
C
1.25 1.00 0.75
*
0.50 0.25 0.00
Serum miR-125b expression
with hematoxylin and eosin, and images were taken with a Nikon Eclipse E800 microscope (200×). For the lung injury score, images were evaluated by an investigator who was blinded to the identity of the slides as previously described [5,9]. The score for each animal was calculated by dividing the total score for the number of sections observed.
1.25
Splenic miR-125b expression
20
1.25
Control mice
ALI mice
1.00 0.75
*
0.50 0.25 0.00
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0.50 0.25 0.00
Control mice
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Fig. 1. MiR-125b was down-regulated in LPS-induced ALI mice. Groups of mice were challenged with LPS for 3 days. The level of miR-125b in BAL fluid (A), peripheral blood (B) and splenocytes (C) was determined in LPS-induced ALI mice or control mice. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
3.3. Enforced expression of miR-125b maintained the body weight and survival of LPS-induced ALI mice To access the effect of miR-125b-based strategy on the treatment of ALI, groups of mice were challenged with LPS plus the miR-125b expression vector. As shown in Fig. 3A, we revealed that up-regulation of miR-125b effectively abrogated the loss of body weight of LPS-induced ALI mice (p < 0.05). Furthermore, we found that the mortality was approximately 40% in LPS-induced ALI mice, while up-regulation of miR-125b in LPS-induced ALI mice effectively maintained their survival (Fig. 3B, p < 0.05). And we did not observe the death of miR-125b treated mice after day 3 during the later experiments (Supplementary Fig. 2). These findings suggested that enforced expression miR-125b was an effective strategy for treatment of ALI.
3.4. Enforced expression of miR-125b attenuated the LPS-induced pulmonary inflammation To investigate the possible mechanism underlying the protective effect of enforced expression of miR-125b on LPS-induced ALI, we detected the total cell and neutrophil counts in BAL fluid from mice treated with miR-125b expression vector. As shown in Fig. 4A, we found the up-regulation of miR-125b effectively decreased the total inflammatory cell count in the BAL fluid in LPS-induced ALI mice (p < 0.05). Further, up-regulation of miR-125b resulted in reduced the neutrophil counts in BAL fluid of LPS-induced ALI mice (Fig. 4B, p < 0.05). To further assess the anti-inflammatory effect of enforced expression of miR-125b, we further detected the
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Fig. 3. Up-regulation of miR-125b maintained the body weight and survival of ALI mice. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days, and then were assayed for their decreased percent of body weight (A) and survival (B). Three animal experiments and each time has six animals per group were performed. * p < 0.05.
CCL2) and KC (the murine homolog of human IL-8), in BAL fluid in LPS-induced ALI mice (Fig. 4C and D, p < 0.05). 3.5. Enforced expression of miR-125b reduced the LPS-induced lung permeability
Fig. 2. Enforced expression of miR-125b in LPS-induced ALI mice. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. The level of miR-125b in BAL fluid (A), peripheral blood (B) and splenocytes (C) was determined. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
We next determined the concentrations of total protein, albumin, and IgM in BAL fluid to evaluate the integrity of the alveolar–capillary membrane barrier and assess pulmonary vascular leakage as a marker for ALI. As shown in Fig. 5A–C, we found that enforced expression of miR-125b effectively reduced the levels of total protein, albumin and IgM in BAL fluid in LPS-induced ALI mice (p < 0.05). 3.6. Enforced expression of miR-125b ameliorated the histopathology changes of lung in LPS-induced ALI mice
proinflammatory cytokines and chemokines in BAL fluid. We found that enforced expression of miR-125b substantially decreased the proinflammatory cytokines including TNF-␣, IL-1 and IL-6, as well as chemokines including Cxcl2, JE (the murine homolog of human
To evaluate the potential role of enforced miR-125b expression in the histopathology changes of lung in LPS-induced ALI mice, histological assessment of lung sections 3 days after the
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Z. Guo et al. / Immunology Letters 162 (2014) 18–26
Fig. 4. Up-regulation of miR-125b attenuated LPS-induced pulmonary inflammation. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. ((A) and (B)) Total cell (A) and neutrophil (B) counts in BAL fluid were detected to evaluate lung airspace inflammation. ((C) and (D)) The indicated proinflammatory cytokines and chemokines in BAL fluid were determined. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
administration of LPS with or without treatment was performed. We revealed that up-regulation of miR-125b significantly inhibited the marked inflammatory infiltrates, interalveolar septal thickening, and interstitial edema in LPS-induced ALI mice (Fig. 6A). Further, severity of lung injury was also scored using a semiquantitative histopathology score system [5,9,26], and we found that enforced expression of miR-125b could significantly reduce the lung injury score of LPS-induced ALI mice (Fig. 6B, p < 0.05). 3.7. Decreased levels of serum miR-125b correlated with clinical parameters in patients with ARDS To explore the clinical relevance of our above findings, we detected the serological level of miR-125b expression and analyzed its correlation with the pro-inflammatory cytokines and disease
activity in patients with ARDS. As shown in Fig. 7A, we found a decreased level of miR-125b expression in peripheral blood of patients with ARDS (p < 0.05). Further, we revealed that serological level of miR-125b was inversely correlated with the serological level of TNF-␣ and IL-6 in patients with ARDS (Fig. 7B and C, p < 0.05). Of important, we found that serum miR-125b expression was associated with the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2) (Fig. 7D, p < 0.05). We further revealed that the expression level of serum miR-125b was inversely correlated with disease activity as evidenced by the Acute Physiology and Chronic Health Evaluation (APACHE) II, Simplified Acute Physiology Score (SAPS) II, and the Murray Lung Injury Score (LIS) in patients with ARDS (Fig. 7E–G, p < 0.05). These resulted suggested that miR-125b was involved in pathogenesis of in patients with ARDS.
Z. Guo et al. / Immunology Letters 162 (2014) 18–26
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Fig. 6. Up-regulation of miR-125b ameliorated the histopathology changes of lung in LPS-induced ALI mice. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. (A) Representative images of hematoxylin and eosin stained lung sections from each experimental group. (B) Lung injury score was determined. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. * p < 0.05.
Fig. 5. Up-regulation of miR-125b reduced the LPS-induced lung permeability. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. The total protein (A), albumin (B) and IgM (C) in BAL fluid were determined at day 3. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
4. Discussion MiRNAs have emerged as key regulators of diverse biological processes, including the activation of the innate immune response [27,28]. Recently, accumulating evidence implicated a potential role of miRNAs in contributing to immune response in the context
of LPS exposure, sepsis, or ALI [29–32]. In present study, we found that the expression of miR-125b was substantially decreased in the BAL fluid in LPS-induced ALI mice compared with that in the control mice. Our data were, to some extent, consistent with previous study which showed that miR-125b were down-regulated in Raw 264.7 cells in response to LPS, and the same changes were observed when C57BL/6 mice were challenged with LPS [17]. These findings suggested that decreased expression of miR-125b might be involved in the pathogenesis of ALI. In this study, we revealed decreased miR-125b expressions in LPS induced ALI mice. Consistent with previous study [17], the LPS stimulation of macrophages, at least partly, might account for the reduced miR-125b in ALI mice. Of note, here we conducted a miR-125b-based strategy to treat ALI using the miR-125b expression vector. We showed that enforced expression of miR-125b indeed effectively reduced LPS-induced pulmonary inflammation and resulted in remarkable reversal of LPS-induced increases in lung permeability, accompanied by a significant reduction of histopathology changes of lung. In consistent, the active caspase 3 activity was also significantly decreased in miR-125b treated ALI mice (Supplementary Fig. 3). Our findings strongly demonstrated that enforced expression of miR-125b could effectively ameliorate the LPS-induced ALI, which could facilitate
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Z. Guo et al. / Immunology Letters 162 (2014) 18–26
B
*
1.2
120
Serum TNF- α (pg/ml)
Serum miR-125b expression
A
0.9 0.6 0.3 0.0
HC
r=-0.753 p<0.001
100 80 60 40 0.4
ALI
0.5
0.6
0.7
0.8
0.9
Serum miR-125b expression
D 220
Serum IL-6 (pg/ml)
50
r=-0.698 p=0.003
195
PaO2/FiO2
C
40
30
20 0.4
0.5
0.6
0.7
0.8
170 145 120 0.4
0.9
Serum miR-125b expression
E
0.7
0.8
0.9
r=-0.678 p=0.004
50
SAPS II
APACHE II
0.6
60
r=-0.646 p=0.007
20
15
10 0.4
0.5
Serum miR-125b expression
F
25
r=0.722 p=0.002
40 30
0.5
0.6
0.7
0.8
0.9
20 0.4
0.5
0.6
0.7
0.8
0.9
Serum miR-125b expression
Serum miR-125b expression
G 3.5
r=-0.663 p=0.005
LIS
3.0
2.5
2.0 0.4
0.5
0.6
0.7
0.8
0.9
Serum miR-125b expression Fig. 7. Serum miR-125b expression correlated with disease activity of ARDS patients. (A) The serological level of miR-125b was determined in ARDS patients and healthy controls. ((B) and (C)) The correlation between serological miR-125b and the serological TNF-␣ and IL-6 was analyzed. ((D)–(G)) The correlation between serum miR-125b expression and the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2) (D), the Acute Physiology and Chronic Health Evaluation (APACHE) II (E), Simplified Acute Physiology Score (SAPS) II (F), and the Murray Lung Injury Score (LIS, G) of patients with ARDS was determined respectively. Each dot represents the result from one patient. * p < 0.05.
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our further understanding of the pathogenesis of ARDS, and provide a clue for developing new strategy for treatment of ARDS patients. Inflammatory responses are initiated by monocytes that upon recognition of pathogens differentiate into macrophages, which then become activated to produce cytokines and chemokines, which help recruit effector cells to the site of infection and induce endothelial cell activation to increase vascular permeability [27,33]. Within the inflammatory mediators, TNF-␣/TNFR interaction has received great attention due to its position at the apex of the proinflammatory cytokine cascade and its dominance in the pathogenesis of various disease processes [9,34–37]. TNF-␣ is one of the main cytokines involved in the response to LPS, and miR125b was shown to be able to target TNF-␣ transcripts at their 3 -UTR, suggesting that relative high levels of miR-125b in the absence of LPS may be needed to help to ensure that the LPS pathway remains turned off in the absence of microbial infection [17]. Here we extended previous study by demonstrating that enforced expression of miR-125b could inhibit the inflammatory responses induced by LPS pathway. We showed that up-regulation of miR125b resulted in decreased expression of TNF-␣ in LPS-induced ALI mice. Low TNF-a levels in BAL fluid were also observed on day 5 of miR-125b treated ALI mice (Supplementary Fig. 4). Further, we validated the in vivo relevance of our findings in patients with ARDS, and revealed that serum miR-125b level was inversely correlated with the serum level of inflammatory cytokines and the disease severity. Here we did not explore the target cells of miR-125b in ALI mice. Given the important roles of macrophages in secreting TNF-␣ and development of ALI, we speculated that microphages might be the potential targets of miR-125b. However, the precise mechanisms involved in protective effect of miR-125b on ALI development undoubtedly deserved successive studies. In summary, here we demonstrated that enforced expression of miR-125b could ameliorate the development of ALI using the LPS-induced model. Our findings could enlarge our understanding of the effect of miR-125b on development of ALI, and emphasize the potential of miR-125b-based therapeutic strategy for possible implications for treatment of ARDS patients. Acknowledgements This work was supported by National Natural Science Foundation of China (81372347 and 81370174), Shanghai Committee of Science and Technology (134119a4900), Shanghai Pudong New Area Academic Leader in Health System (PWRd2010-01), and Basic Research Program supported by Shanghai Committee of Science and Technology (11JC1410900). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imlet. 2014.06.008. References [1] Zhou T, Garcia JG, Zhang W. Integrating microRNAs into a system biology approach to acute lung injury. Transl Res 2011;157:180–90. [2] Herridge MS, Tansey CM, Matté A, Tomlinson G, Diaz-Granados N, Cooper A, et al. Functional disability 5 years after acute respiratory distress syndrome. N Engl J Med 2011;364:1293–304. [3] Mendez JL, Hubmayr RD. New insights into the pathology of acute respiratory failure. Curr Opin Crit Care 2005;11:29–36. [4] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. [5] Mei SH, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPSinduced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med 2007;4(9):e269.
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[6] Tsushima K, King LS, Aggarwal NR, De Gorordo A, D’Alessio FR, Kubo K. Acute lung injury review. Intern Med 2009;48:621–30. [7] Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit Care Med 2001;163:1376–83. [8] Guidot DM, Folkesson HG, Jain L, Sznajder JI, Pittet JF, Matthay MA. Integrating acute lung injury and regulation of alveolar fluid clearance. Am J Physiol Lung Cell Mol Physiol 2006;291:L301–6. [9] Guo Z, Li Q, Han Y, Liang Y, Xu Z, Ren T. Prevention of LPS-induced acute lung injury in mice by progranulin. Mediators Inflamm 2012;2012: 540794. [10] D’Alessio FR, Tsushima K, Aggarwal NR, West EE, Willett MH, Britos MF, et al. CD4 + CD25 + Foxp3 + Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. J Clin Invest 2009;119: 2898–913. [11] Crimi E, Slutsky AS. Inflammation and the acute respiratory distress syndrome. Best Pract Res Clin Anaesthesiol 2004;18:477–92. [12] Meade MO, Cook DJ, Guyatt GH, Slutsky AS, Arabi YM, Cooper DJ, et al. Ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acute lung injury and acute respiratory distress syndrome: a randomized controlled trial. JAMA 2008;299: 637–45. [13] Esper AM, Martin GS. Evolution of treatments for patients with acute lung injury. Expert Opin Investig Drugs 2005;14:633–45. [14] Mehta D, Bhattacharya J, Matthay MA, Malik AB. Integrated control of lung fluid balance. Am J Physiol Lung Cell Mol Physiol 2004;287:L1081–90. [15] Gattinoni L, Caironi P, Cressoni M, Chiumello D, Ranieri VM, Quintel M, et al. Lung recruitment in patients with the acute respiratory distress syndrome. N Engl J Med 2006;354:1775–86. [16] Lindsay MA. MicroRNAs and the immune response. Trends Immunol 2008;29:343–51. [17] Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol 2007;179:5082–9. [18] Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med 1996;184:1397–411. [19] Pfeffer K, Matsuyama T, Kündig TM, Wakeham A, Kishihara K, Shahinian A, et al. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 1993;73: 457–67. [20] Raghavendran K, Napolitano LM. Definition of ALI/ARDS. Crit Care Clin 2011;27:429–37. [21] Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, et al. The American–European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994;149:818–24. [22] Hua F, Wang X, Zhu L. Terlipressin decreases vascular endothelial growth factor expression and improves oxygenation in patients with acute respiratory distress syndrome and shock. J Emerg Med 2013;44:434–9. [23] Perkins GD, McAuley DF, Thickett DR, Gao F. The beta-agonist lung injury trial (BALTI): a randomized placebo-controlled clinical trial. Am J Respir Crit Care Med 2006;173:281–7. [24] Murray JF, Matthay MA, Luce JM, Flick MR. An expanded definition of the adult respiratory distress syndrome. Am Rev Respir Dis 1988;138: 720–3. [25] Li Q, Li X, Guo Z, Xu F, Xia J, Liu Z, et al. MicroRNA-574-5p was pivotal for TLR9 signaling enhanced tumor progression via down-regulating checkpoint suppressor 1 in human lung cancer. PLoS One 2012;7:e48278. [26] Dorr AD, Wilson MR, Wakabayashi K, Waite AC, Patel BV, van Rooijen N, et al. Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies. J Appl Physiol 2011;111:177–84. [27] Worm J, Stenvang J, Petri A, Frederiksen KS, Obad S, Elmén J, et al. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF. Nucleic Acids Res 2009;37:5784–92. [28] Dai R, Ahmed SA. MicroRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases. Transl Res 2011;157: 163–79. [29] Moschos SA, Williams AE, Perry MM, Birrell MA, Belvisi MG, Lindsay MA. Expression profiling in vivo demonstrates rapid changes in lung microRNA levels following lipopolysaccharide-induced inflammation but not in the antiinflammatory action of glucocorticoids. BMC Genomics 2007;8:240. [30] Nahid MA, Pauley KM, Satoh M, Chan EK. miR-146a is critical for endotoxin-induced tolerance: implication in innate immunity. J Biol Chem 2009;284:34590–9. [31] Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Nat Acad Sci USA 2009;106:15819–24. [32] Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, et al. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 2002;110:1703–16.
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[33] Huang H, Park CK, Ryu JY, Chang EJ, Lee Y, Kang SS, et al. Expression profiling of lipopolysaccharide target genes in RAW264.7 cells by oligonucleotide microarray analyses. Arch Pharm Res 2006;29:890–7. [34] Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003;3:745–56. [35] Mackay F, Kalled SL. TNF ligands and receptors in autoimmunity: an update. Curr Opin Immunol 2002;14:783–90.
[36] Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 2003;66: 1403–8. [37] Li T, Luo N, Du L, Zhou J, Zhang J, Gong L, et al. Tumor necrosis factor-␣ plays an initiating role in extracorporeal circulation-induced acute lung injury. Lung 2013;191:207–14.
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Enforced expression of miR-125b attenuates LPS-induced acute lung injury Zhongliang Guo a,1 , Yutong Gu b,1 , Chunhong Wang c,1 , Jie Zhang a , Shan Shan c , Xia Gu c , Kailing Wang c , Yang Han d , Tao Ren c,∗ a
Department of VIP, East Hospital, Tongji University School of Medicine, Shanghai, China Department of Pulmonary Medicine, Zhongshan Hospital, Fudan University, Shanghai, China c Department of Respiratory Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China d Department of Pathology, East Hospital, Tongji University School of Medicine, Shanghai, China b
a r t i c l e
i n f o
Article history: Received 9 March 2014 Received in revised form 11 June 2014 Accepted 15 June 2014 Available online 6 July 2014 Keywords: Acute respiratory distress syndrome Acute lung injury miR-125b
a b s t r a c t The acute respiratory distress syndrome (ARDS), a severe form of acute lung injury (ALI) in humans, is a leading cause of morbidity and mortality in critically ill patients. Despite decades of research, few therapeutic strategies for clinical ARDS have emerged. Recent evidence implicated a potential role of miR125b in development of ALI. Here we evaluated the miR-125b-based strategy in treatment of ARDS using the murine model of lipopolysaccharide (LPS)-induced ALI. We found that up-regulation of miR-125b expression maintained the body weight and survival of ALI mice, and significantly reduced LPS-induced pulmonary inflammation as reflected by reductions in total cell and neutrophil counts, proinflammatory cytokines, as well as chemokines in BAL fluid. Further, enforced expression of miR-125b resulted in remarkable reversal of LPS-induced increases in lung permeability as assessed by reductions in total protein, albumin and IgM in BAL fluid, and ameliorated the histopathology changes of lung in LPS-induced ALI mice. Of interest, serum miR-125b expression was also decreased and inversely correlated with the disease severity in patients with ARDS. Our findings strongly demonstrated that enforced expression of miR-125b could effectively ameliorate the LPS-induced ALI, suggesting a potential application for miR-125b-based therapy to treat clinical ARDS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Inflammatory lung disorders are characterized by increased leukocyte infiltration into lung tissues as the body’s immune response to infection or injury [1]. An important inflammatory lung disease of rapid onset is acute lung injury (ALI), which is a significant cause of morbidity and mortality in critically ill patients [1–5]. ALI is a diffuse, heterogeneous type of inflammatory lung disease clinically characterized by progressive hypoxemia, reduced lung compliance, and intense inflammation in the lung tissues [1,6]. The physiological hallmark of ALI is disruption of the alveolar–capillary membrane barrier, leading to development of noncardiogenic pulmonary edema, in which a proteinaceous exudate floods the alveolar spaces, impairs gas exchange, and precipitates respiratory failure [2,5–9]. ALI can result in persistent respiratory failure and
∗ Corresponding author. Tel.: +86 21 38804518x7217; fax: +86 21 58798999. E-mail address: [email protected] (T. Ren). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.imlet.2014.06.008 0165-2478/© 2014 Elsevier B.V. All rights reserved.
prolonged dependence on mechanical ventilation, increasing susceptibility to multiorgan dysfunction and mortality [9,10]. Despite extensive investigation aimed at early diagnostic and pathogenetic factors of ALI, current management is mainly supportive, as specific therapies have not been identified [5,9,11–15]. Therefore, new strategies are urgently required for achieving effective treatment of ALI, which might ultimately aid the clinical therapy for ARDS patients. MicroRNAs (miRNAs) have been shown to be centrally involved in the regulation of immune system development, differentiation of B and T cells, proliferation of monocytes and neutrophils, antibody production, release of inflammatory mediators and certain inflammatory lung diseases [1,16]. Recent study showed that miR125b, which could target the 3 -UTR of TNF-␣ and thus inhibit its expression, was down-regulated in macrophages in response to LPS stimulation in vitro, and was substantially decreased in spleens in C57BL/6 mice after injection with LPS [17]. Of interest, mice deficient in TNF-␣ or TNFR1 are resistant to LPS-induced shock whereas wild-type mice die within hours after LPS challenge, suggesting that the main cytokine responsible for this syndrome is TNF-␣ [17–19].
Z. Guo et al. / Immunology Letters 162 (2014) 18–26 Table 1 The clinical characters of ARDS patients. ARDS Demographics Male Female Age (years) Clinical parameters PaO2/FiO2 APACHE II SAPS II LIS Causes of ARDS Sepsis Lung contusion Shock
19
according to the ethical guidelines of the Tongji University Laboratory Animal Care and Use Committee. Number
2.3. Murine model of LPS-induced ALI 11 5 57.3 ± 13.7 176.50 ± 13.76 17.13 ± 3.48 39.19 ± 8.51 2.94 ± 0.23 12 1 3
(1) Shown as patient numbers or mean ± standard deviation values. (2) APACHE II, Acute Physiology and Chronic Health Evaluation (APACHE) II. SAPS II, Simplified Acute Physiology Score (SAPS) II. LIS, Murray Lung Injury Score (LIS).
These findings promoted us to hypothesize that enforced expression of miR-125b might be able to ameliorate the LPS-induced ALI. To test our hypothesis, here we carefully evaluate the potential role of miR-125b-based strategy in treatment of ALI using the murine model of LPS-induced ALI. We found that up-regulation of miR-125b significantly reduced LPS-induced pulmonary inflammation and resulted in remarkable reversal of LPS-induced increases in lung permeability, accompanied by a significant reduction of histopathology changes of lung. Our findings strongly demonstrated that enforced expression of miR-125b could effectively ameliorate the LPS-induced acute lung injury, suggesting a potential role for miR-125b-based therapy to treat patients with ARDS. 2. Materials and methods 2.1. Patients The human study was approved by the Ethics Committee of Tongji University. Sixteen patients with ARDS were enrolled and all the peripheral blood samples were collected from human subjects after obtaining informed consent. ARDS was defined as the ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2/FiO2) <300 mm Hg, the presence of bilateral pulmonary infiltrates on a chest radiograph, and no clinical evidence of left atrial hypertension [20,21]. The clinical characteristics of ARDS patients were summarized in Table 1. Disease severity was assessed by the Acute Physiology and Chronic Health Evaluation (APACHE) II, Simplified Acute Physiology Score (SAPS) II, and the Murray Lung Injury Score (LIS), a composite variable that includes components of oxygenation, compliance, positive end expiratory pressure, and the appearance of the chest radiograph [22–24]. Arterial blood samples were drawn for blood gas analysis. Serological levels of TNF-␣ and IL-6 were determined using the human immunoassay kit (R&D Systems). Normal controls (n = 16) matched for gender and age (±2.1 years) were recruited from among the Institute personnel, and all were in excellent health at the time of the study. 2.2. Mice Female BALB/c mice at 6 weeks old were purchased from the Center of Experimental Animals of Tongji University. All mice were housed in the pathogen-free animal facilities of Tongji University School of Medicine. All animal experiments were performed
The murine model of LPS-induced ALI was established as previous reported [5,9]. Briefly, female BALB/c mice (n = 6 per group) were anaesthetized and orally intubated with a sterile plastic catheter, and challenged with intratracheal instillation of 800 g of LPS (Escherichia coli 055:B5; Sigma) dissolved in 50 l of normal PBS. Naive mice (without LPS instillation) were injected with the same volume of pyrogen-free PBS to serve as controls. Mice were humanely killed at 3d after LPS challenge to collect tissues for analysis. 2.4. MiR-125b Quantitative RT-PCR analyses for miR-125b were performed using TaqMan micro-RNA assays (Applied Biosystems) as previously described [25]. All reagents, primers, and probes were obtained from Applied Biosystems. Retrovirus-mediated overexpression of miR-125b was generated based on pMX vector (Invitrogen) as previously described method [25]. Sequence encoding mutant miR-125b was cloned into the same vector and used as the control vector. Virus was produced and target cells were infected according to the user’s manual. To access the effect of miR125b-based strategy on the treatment of ALI, groups of mice were challenged with LPS plus the miR-125b expression vector (2 × 109 plaque-forming units per mouse). 2.5. Determination of total cells and neutrophils According to previously described [5,9], BAL was performed by instilling 0.9% NaCl containing 0.6 mmol/l ethylenediaminetetraacetic acids in two separate 0.5 ml aliquots. The fluid was recovered by gentle suction and placed on ice for immediate processing. An aliquot of the BAL fluid was processed immediately for total and differential cell counts. The remainder of the lavage fluid was centrifuged and the supernatant was removed aseptically and stored in individual aliquots at −70 ◦ C. Total cell counts in BAL fluid were determined using a haemocytometer. Number of neutrophils was calculated as the percentage of neutrophils multiplied by the total number of cells in the BAL fluid sample. All analyses were performed in a blinded fashion. 2.6. Measurement of proinflammatory cytokines, chemokines, albumin and IgM In line with previously described [5,9], BAL fluid collected was centrifuged at 800 g for 10 min, and supernatant was collected for analysis of total protein, albumin, IgM, and cytokine/chemokine levels. Proinflammatory cytokine levels including TNF-␣, IL-1 and IL-6 in BAL fluid were measured with murine cytokine-specific Quantikine ELISA kits (R&D Systems). Chemokine levels including Cxcl2, JE (the murine homolog of human CCL2) and KC (the murine homolog of human IL-8) in BAL fluid were measured using cytokine-specific bead kits (R&D Systems). Albumin and IgM levels in BAL fluid samples were measured using with a murine-specific albumin ELISA kit (ALPCO Diagnostics) and a murine specific IgM ELISA kit (Bethyl Laboratories), respectively. All the measurements were performed according to the manufacturer’s instructions. 2.7. Histopathology Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 m thick sections. Sections were stained
Z. Guo et al. / Immunology Letters 162 (2014) 18–26
BAL miR-125b expression
A
2.8. Statistical analysis Differences between the treated groups versus the injured group were assessed using a one-way ANOVA with statistic software (GraphPad Prism version 4.00). A value of p < 0.05 was considered statistically significant. 3. Results
B
3.1. Expression of miR-125b was down-regulated in BAL fluid of LPS-induced ALI mice To assess the potential role of miR-125b-based strategy in treatment of LPS-induced ALI, we determined the expression level of miR-125b in bronchoalveolar lavage (BAL) fluid of LPS-induced ALI mice at day 3 after LPS challenge. We found that the expression level of miR-125b in BAL fluid was significantly decreased on day 3 in mice challenged with LPS compared with the control groups (Fig. 1A, p < 0.05). Similarly, we revealed that the expression of miR125b in peripheral blood and splenocytes was also down-regulated on day 3 in LPS-induced ALI mice (Fig. 1B and C, p < 0.05). While genomic, biochemical and histology findings are striking at day 3 after LPS exposure, we found a remarkable reduction of miR-125b expression on day 3 of ALI mice (Supplementary Fig. 1). Combing these findings indicated that miR-125b might be involved in the development of ALI. 3.2. Enforced expression of miR-125b in LPS-induced ALI mice Given the decreased expression of miR-125b in LPS-induced ALI mice, we sought to detect whether enforced expression of miR125b could ameliorate the LPS-induced ALI. Thus, groups of mice were challenged with LPS plus the miR-125b expression vector, and then were determined for the expression level of miR-125b in bronchoalveolar lavage (BAL) fluid on day3. We found that the expression level of miR-125b in BAL fluid was significantly elevated by treatment with the miR-125b expression vector (Fig. 2A, p < 0.05). Further, the expression of miR-125b in peripheral blood and splenocytes was also up-regulated (Fig. 2B and C, p < 0.05). These findings demonstrated the enforced expression of miR-125b in LPS-induced ALI mice.
C
1.25 1.00 0.75
*
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Serum miR-125b expression
with hematoxylin and eosin, and images were taken with a Nikon Eclipse E800 microscope (200×). For the lung injury score, images were evaluated by an investigator who was blinded to the identity of the slides as previously described [5,9]. The score for each animal was calculated by dividing the total score for the number of sections observed.
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Splenic miR-125b expression
20
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Fig. 1. MiR-125b was down-regulated in LPS-induced ALI mice. Groups of mice were challenged with LPS for 3 days. The level of miR-125b in BAL fluid (A), peripheral blood (B) and splenocytes (C) was determined in LPS-induced ALI mice or control mice. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
3.3. Enforced expression of miR-125b maintained the body weight and survival of LPS-induced ALI mice To access the effect of miR-125b-based strategy on the treatment of ALI, groups of mice were challenged with LPS plus the miR-125b expression vector. As shown in Fig. 3A, we revealed that up-regulation of miR-125b effectively abrogated the loss of body weight of LPS-induced ALI mice (p < 0.05). Furthermore, we found that the mortality was approximately 40% in LPS-induced ALI mice, while up-regulation of miR-125b in LPS-induced ALI mice effectively maintained their survival (Fig. 3B, p < 0.05). And we did not observe the death of miR-125b treated mice after day 3 during the later experiments (Supplementary Fig. 2). These findings suggested that enforced expression miR-125b was an effective strategy for treatment of ALI.
3.4. Enforced expression of miR-125b attenuated the LPS-induced pulmonary inflammation To investigate the possible mechanism underlying the protective effect of enforced expression of miR-125b on LPS-induced ALI, we detected the total cell and neutrophil counts in BAL fluid from mice treated with miR-125b expression vector. As shown in Fig. 4A, we found the up-regulation of miR-125b effectively decreased the total inflammatory cell count in the BAL fluid in LPS-induced ALI mice (p < 0.05). Further, up-regulation of miR-125b resulted in reduced the neutrophil counts in BAL fluid of LPS-induced ALI mice (Fig. 4B, p < 0.05). To further assess the anti-inflammatory effect of enforced expression of miR-125b, we further detected the
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Fig. 3. Up-regulation of miR-125b maintained the body weight and survival of ALI mice. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days, and then were assayed for their decreased percent of body weight (A) and survival (B). Three animal experiments and each time has six animals per group were performed. * p < 0.05.
CCL2) and KC (the murine homolog of human IL-8), in BAL fluid in LPS-induced ALI mice (Fig. 4C and D, p < 0.05). 3.5. Enforced expression of miR-125b reduced the LPS-induced lung permeability
Fig. 2. Enforced expression of miR-125b in LPS-induced ALI mice. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. The level of miR-125b in BAL fluid (A), peripheral blood (B) and splenocytes (C) was determined. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
We next determined the concentrations of total protein, albumin, and IgM in BAL fluid to evaluate the integrity of the alveolar–capillary membrane barrier and assess pulmonary vascular leakage as a marker for ALI. As shown in Fig. 5A–C, we found that enforced expression of miR-125b effectively reduced the levels of total protein, albumin and IgM in BAL fluid in LPS-induced ALI mice (p < 0.05). 3.6. Enforced expression of miR-125b ameliorated the histopathology changes of lung in LPS-induced ALI mice
proinflammatory cytokines and chemokines in BAL fluid. We found that enforced expression of miR-125b substantially decreased the proinflammatory cytokines including TNF-␣, IL-1 and IL-6, as well as chemokines including Cxcl2, JE (the murine homolog of human
To evaluate the potential role of enforced miR-125b expression in the histopathology changes of lung in LPS-induced ALI mice, histological assessment of lung sections 3 days after the
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Fig. 4. Up-regulation of miR-125b attenuated LPS-induced pulmonary inflammation. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. ((A) and (B)) Total cell (A) and neutrophil (B) counts in BAL fluid were detected to evaluate lung airspace inflammation. ((C) and (D)) The indicated proinflammatory cytokines and chemokines in BAL fluid were determined. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
administration of LPS with or without treatment was performed. We revealed that up-regulation of miR-125b significantly inhibited the marked inflammatory infiltrates, interalveolar septal thickening, and interstitial edema in LPS-induced ALI mice (Fig. 6A). Further, severity of lung injury was also scored using a semiquantitative histopathology score system [5,9,26], and we found that enforced expression of miR-125b could significantly reduce the lung injury score of LPS-induced ALI mice (Fig. 6B, p < 0.05). 3.7. Decreased levels of serum miR-125b correlated with clinical parameters in patients with ARDS To explore the clinical relevance of our above findings, we detected the serological level of miR-125b expression and analyzed its correlation with the pro-inflammatory cytokines and disease
activity in patients with ARDS. As shown in Fig. 7A, we found a decreased level of miR-125b expression in peripheral blood of patients with ARDS (p < 0.05). Further, we revealed that serological level of miR-125b was inversely correlated with the serological level of TNF-␣ and IL-6 in patients with ARDS (Fig. 7B and C, p < 0.05). Of important, we found that serum miR-125b expression was associated with the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2) (Fig. 7D, p < 0.05). We further revealed that the expression level of serum miR-125b was inversely correlated with disease activity as evidenced by the Acute Physiology and Chronic Health Evaluation (APACHE) II, Simplified Acute Physiology Score (SAPS) II, and the Murray Lung Injury Score (LIS) in patients with ARDS (Fig. 7E–G, p < 0.05). These resulted suggested that miR-125b was involved in pathogenesis of in patients with ARDS.
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Fig. 6. Up-regulation of miR-125b ameliorated the histopathology changes of lung in LPS-induced ALI mice. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. (A) Representative images of hematoxylin and eosin stained lung sections from each experimental group. (B) Lung injury score was determined. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. * p < 0.05.
Fig. 5. Up-regulation of miR-125b reduced the LPS-induced lung permeability. Groups of mice were challenged with LPS plus miR-125b expression vector or control vector for 3 days. The total protein (A), albumin (B) and IgM (C) in BAL fluid were determined at day 3. Data are represented as mean ± standard deviation of one experiment consisting of three replicates. Experiments were performed in triplicate. * p < 0.05.
4. Discussion MiRNAs have emerged as key regulators of diverse biological processes, including the activation of the innate immune response [27,28]. Recently, accumulating evidence implicated a potential role of miRNAs in contributing to immune response in the context
of LPS exposure, sepsis, or ALI [29–32]. In present study, we found that the expression of miR-125b was substantially decreased in the BAL fluid in LPS-induced ALI mice compared with that in the control mice. Our data were, to some extent, consistent with previous study which showed that miR-125b were down-regulated in Raw 264.7 cells in response to LPS, and the same changes were observed when C57BL/6 mice were challenged with LPS [17]. These findings suggested that decreased expression of miR-125b might be involved in the pathogenesis of ALI. In this study, we revealed decreased miR-125b expressions in LPS induced ALI mice. Consistent with previous study [17], the LPS stimulation of macrophages, at least partly, might account for the reduced miR-125b in ALI mice. Of note, here we conducted a miR-125b-based strategy to treat ALI using the miR-125b expression vector. We showed that enforced expression of miR-125b indeed effectively reduced LPS-induced pulmonary inflammation and resulted in remarkable reversal of LPS-induced increases in lung permeability, accompanied by a significant reduction of histopathology changes of lung. In consistent, the active caspase 3 activity was also significantly decreased in miR-125b treated ALI mice (Supplementary Fig. 3). Our findings strongly demonstrated that enforced expression of miR-125b could effectively ameliorate the LPS-induced ALI, which could facilitate
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B
*
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120
Serum TNF- α (pg/ml)
Serum miR-125b expression
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r=-0.753 p<0.001
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D 220
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30
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60
r=-0.646 p=0.007
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20 0.4
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G 3.5
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0.7
0.8
0.9
Serum miR-125b expression Fig. 7. Serum miR-125b expression correlated with disease activity of ARDS patients. (A) The serological level of miR-125b was determined in ARDS patients and healthy controls. ((B) and (C)) The correlation between serological miR-125b and the serological TNF-␣ and IL-6 was analyzed. ((D)–(G)) The correlation between serum miR-125b expression and the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (PaO2/FiO2) (D), the Acute Physiology and Chronic Health Evaluation (APACHE) II (E), Simplified Acute Physiology Score (SAPS) II (F), and the Murray Lung Injury Score (LIS, G) of patients with ARDS was determined respectively. Each dot represents the result from one patient. * p < 0.05.
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our further understanding of the pathogenesis of ARDS, and provide a clue for developing new strategy for treatment of ARDS patients. Inflammatory responses are initiated by monocytes that upon recognition of pathogens differentiate into macrophages, which then become activated to produce cytokines and chemokines, which help recruit effector cells to the site of infection and induce endothelial cell activation to increase vascular permeability [27,33]. Within the inflammatory mediators, TNF-␣/TNFR interaction has received great attention due to its position at the apex of the proinflammatory cytokine cascade and its dominance in the pathogenesis of various disease processes [9,34–37]. TNF-␣ is one of the main cytokines involved in the response to LPS, and miR125b was shown to be able to target TNF-␣ transcripts at their 3 -UTR, suggesting that relative high levels of miR-125b in the absence of LPS may be needed to help to ensure that the LPS pathway remains turned off in the absence of microbial infection [17]. Here we extended previous study by demonstrating that enforced expression of miR-125b could inhibit the inflammatory responses induced by LPS pathway. We showed that up-regulation of miR125b resulted in decreased expression of TNF-␣ in LPS-induced ALI mice. Low TNF-a levels in BAL fluid were also observed on day 5 of miR-125b treated ALI mice (Supplementary Fig. 4). Further, we validated the in vivo relevance of our findings in patients with ARDS, and revealed that serum miR-125b level was inversely correlated with the serum level of inflammatory cytokines and the disease severity. Here we did not explore the target cells of miR-125b in ALI mice. Given the important roles of macrophages in secreting TNF-␣ and development of ALI, we speculated that microphages might be the potential targets of miR-125b. However, the precise mechanisms involved in protective effect of miR-125b on ALI development undoubtedly deserved successive studies. In summary, here we demonstrated that enforced expression of miR-125b could ameliorate the development of ALI using the LPS-induced model. Our findings could enlarge our understanding of the effect of miR-125b on development of ALI, and emphasize the potential of miR-125b-based therapeutic strategy for possible implications for treatment of ARDS patients. Acknowledgements This work was supported by National Natural Science Foundation of China (81372347 and 81370174), Shanghai Committee of Science and Technology (134119a4900), Shanghai Pudong New Area Academic Leader in Health System (PWRd2010-01), and Basic Research Program supported by Shanghai Committee of Science and Technology (11JC1410900). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.imlet. 2014.06.008. References [1] Zhou T, Garcia JG, Zhang W. Integrating microRNAs into a system biology approach to acute lung injury. Transl Res 2011;157:180–90. [2] Herridge MS, Tansey CM, Matté A, Tomlinson G, Diaz-Granados N, Cooper A, et al. Functional disability 5 years after acute respiratory distress syndrome. N Engl J Med 2011;364:1293–304. [3] Mendez JL, Hubmayr RD. New insights into the pathology of acute respiratory failure. Curr Opin Crit Care 2005;11:29–36. [4] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005;353:1685–93. [5] Mei SH, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPSinduced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin 1. PLoS Med 2007;4(9):e269.
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[6] Tsushima K, King LS, Aggarwal NR, De Gorordo A, D’Alessio FR, Kubo K. Acute lung injury review. Intern Med 2009;48:621–30. [7] Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit Care Med 2001;163:1376–83. [8] Guidot DM, Folkesson HG, Jain L, Sznajder JI, Pittet JF, Matthay MA. Integrating acute lung injury and regulation of alveolar fluid clearance. Am J Physiol Lung Cell Mol Physiol 2006;291:L301–6. [9] Guo Z, Li Q, Han Y, Liang Y, Xu Z, Ren T. Prevention of LPS-induced acute lung injury in mice by progranulin. Mediators Inflamm 2012;2012: 540794. [10] D’Alessio FR, Tsushima K, Aggarwal NR, West EE, Willett MH, Britos MF, et al. CD4 + CD25 + Foxp3 + Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury. J Clin Invest 2009;119: 2898–913. [11] Crimi E, Slutsky AS. Inflammation and the acute respiratory distress syndrome. Best Pract Res Clin Anaesthesiol 2004;18:477–92. [12] Meade MO, Cook DJ, Guyatt GH, Slutsky AS, Arabi YM, Cooper DJ, et al. Ventilation strategy using low tidal volumes, recruitment maneuvers, and high positive end-expiratory pressure for acute lung injury and acute respiratory distress syndrome: a randomized controlled trial. JAMA 2008;299: 637–45. [13] Esper AM, Martin GS. Evolution of treatments for patients with acute lung injury. Expert Opin Investig Drugs 2005;14:633–45. [14] Mehta D, Bhattacharya J, Matthay MA, Malik AB. Integrated control of lung fluid balance. Am J Physiol Lung Cell Mol Physiol 2004;287:L1081–90. [15] Gattinoni L, Caironi P, Cressoni M, Chiumello D, Ranieri VM, Quintel M, et al. Lung recruitment in patients with the acute respiratory distress syndrome. N Engl J Med 2006;354:1775–86. [16] Lindsay MA. MicroRNAs and the immune response. Trends Immunol 2008;29:343–51. [17] Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, et al. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol 2007;179:5082–9. [18] Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med 1996;184:1397–411. [19] Pfeffer K, Matsuyama T, Kündig TM, Wakeham A, Kishihara K, Shahinian A, et al. Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to L. monocytogenes infection. Cell 1993;73: 457–67. [20] Raghavendran K, Napolitano LM. Definition of ALI/ARDS. Crit Care Clin 2011;27:429–37. [21] Bernard GR, Artigas A, Brigham KL, Carlet J, Falke K, Hudson L, et al. The American–European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination. Am J Respir Crit Care Med 1994;149:818–24. [22] Hua F, Wang X, Zhu L. Terlipressin decreases vascular endothelial growth factor expression and improves oxygenation in patients with acute respiratory distress syndrome and shock. J Emerg Med 2013;44:434–9. [23] Perkins GD, McAuley DF, Thickett DR, Gao F. The beta-agonist lung injury trial (BALTI): a randomized placebo-controlled clinical trial. Am J Respir Crit Care Med 2006;173:281–7. [24] Murray JF, Matthay MA, Luce JM, Flick MR. An expanded definition of the adult respiratory distress syndrome. Am Rev Respir Dis 1988;138: 720–3. [25] Li Q, Li X, Guo Z, Xu F, Xia J, Liu Z, et al. MicroRNA-574-5p was pivotal for TLR9 signaling enhanced tumor progression via down-regulating checkpoint suppressor 1 in human lung cancer. PLoS One 2012;7:e48278. [26] Dorr AD, Wilson MR, Wakabayashi K, Waite AC, Patel BV, van Rooijen N, et al. Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies. J Appl Physiol 2011;111:177–84. [27] Worm J, Stenvang J, Petri A, Frederiksen KS, Obad S, Elmén J, et al. Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF. Nucleic Acids Res 2009;37:5784–92. [28] Dai R, Ahmed SA. MicroRNA, a new paradigm for understanding immunoregulation, inflammation, and autoimmune diseases. Transl Res 2011;157: 163–79. [29] Moschos SA, Williams AE, Perry MM, Birrell MA, Belvisi MG, Lindsay MA. Expression profiling in vivo demonstrates rapid changes in lung microRNA levels following lipopolysaccharide-induced inflammation but not in the antiinflammatory action of glucocorticoids. BMC Genomics 2007;8:240. [30] Nahid MA, Pauley KM, Satoh M, Chan EK. miR-146a is critical for endotoxin-induced tolerance: implication in innate immunity. J Biol Chem 2009;284:34590–9. [31] Liu G, Friggeri A, Yang Y, Park YJ, Tsuruta Y, Abraham E. miR-147, a microRNA that is induced upon Toll-like receptor stimulation, regulates murine macrophage inflammatory responses. Proc Nat Acad Sci USA 2009;106:15819–24. [32] Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, et al. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 2002;110:1703–16.
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[33] Huang H, Park CK, Ryu JY, Chang EJ, Lee Y, Kang SS, et al. Expression profiling of lipopolysaccharide target genes in RAW264.7 cells by oligonucleotide microarray analyses. Arch Pharm Res 2006;29:890–7. [34] Aggarwal BB. Signalling pathways of the TNF superfamily: a double-edged sword. Nat Rev Immunol 2003;3:745–56. [35] Mackay F, Kalled SL. TNF ligands and receptors in autoimmunity: an update. Curr Opin Immunol 2002;14:783–90.
[36] Gaur U, Aggarwal BB. Regulation of proliferation, survival and apoptosis by members of the TNF superfamily. Biochem Pharmacol 2003;66: 1403–8. [37] Li T, Luo N, Du L, Zhou J, Zhang J, Gong L, et al. Tumor necrosis factor-␣ plays an initiating role in extracorporeal circulation-induced acute lung injury. Lung 2013;191:207–14.