key: cord-0006177-544twg83 authors: Lee, Woonjeong; Kim, Insoo; Shin, Soyoung; Park, Kicheol; Yang, Keumjin; Eun, Jung woo; Sul, Haejoung; Jeong, Sikyoung title: Expression profiling of microRNAs in lipopolysaccharide-induced acute lung injury after hypothermia treatment date: 2016-10-07 journal: Mol Cell Toxicol DOI: 10.1007/s13273-016-0029-7 sha: a12f12436a3dfd4fc7207a16b8f64c7215929fad doc_id: 6177 cord_uid: 544twg83 We investigated the expression profiles of miRNAs in acute lung injury (ALI) rats after hypothermia treatment. ALI rats were induced with lipopolysaccharide (LPS) and maintained with hypothermia (HT) or normothermia (NT) for 6 hours. HT attenuated inflammatory cell infiltration in the lung and improved biochemical indicators of multi-organ dysfunction. Nineteen miRNAs were significantly differentially expressed in the HT group compared with the NT group. miR-142, miR-98, miR-541, miR-503, miR-653, miR- 223, miR-323 and miR-196b exhibited opposite patterns of expression between the two groups. These dysregulated miRNAs were mainly involved in the immune and inflammatory response on functional annotation analyses. This study shows that HT has lung protective effects and influences expression profiles of miRNAs in ALI. And dysregulated miRNAs after HT modulate the immune and inflammation in ALI. These results suggest that dysregulated miRNAs play a role in the mechanism of the lung protective effects of HT in ALI. esis, and metastasis. Therefore, altered miRNA expres sion levels are correlated with disease occurrence and progression 11, 12 . The expression profiles of miRNAs have been studied to identify the pathogenesis of various lung diseases, such as in a mouse model of ventilator-induced lung injury 13 , chronic obstructive pulmonary disease 14 , id iopathic pulmonary fibrosis 15 , and rat arDS 16 . Son koly et al. 17 reported that miRNAs are novel players in the regulation of immune function and inflamma tion, which is the first line of defense response. Tili's study 18 showed that upregulated miR-155 and down regulated miR-125b were responsible for the patho genesis of LPS-induced endotoxemia through the TNFα stimulation pathway. We put forward the hypothesizes that HT affects the expression profiles of miRNAs and the dysregulated miRNAs are correlated with the effects of HT in ALI. We confirmed the effects of HT in ALI by lung pathology studies, inflammatory cell counts in BAL fluid and multi-organ dysfunction studies and analysis of the differentially expressed miRNAs in rat ALI after HT treatment. The representative lung tissues from three different groups are shown in Figure 1 . Histopathological examination of lung tissues confirmed the presence of mod erate pulmonary injuries, congested alveolar capillar ies, hemorrhage, inflammatory cell infiltration, and some alveolar wall thickening in LPS (ali with nT treatment) comparing to lPS + HT (ali with HT treat ment) ( Figure 1B, 1C) . To quantify the effects of HT on lung injury, we estimated the LIS. Based on the scoring system, all 4 components for controls had the score 0. Consistent with the histopathological examination, LIS in NT rats (10.6±1.14) was significantly higher than that in the controls (P<0.001, Figure 1D ). In contrast, LIS in HT rats was lower than in NT rats (9.8±0.83 vs. 10.6± 1.14), but was not statistically significant. To investigate the protective effect of HT in LPS-in duced ALI, we measured the total cell number and the number of neutrophils in the BAL fluid. We found that HT decreased the number of total inflammatory cells and neutrophils in the BAL fluid compared with NT ( Figure 1E , 1F). Because the lethality of ALI is associated with multiorgan failure, we examined the dysfunction of major organs. Organ dysfunction was determined by measur In control rat lungs, the normal pulmonary struc tures, such as alveolar septa, alveolar lumen, and capillaries were well preserved. (B) In LPS-induced ALI, congested alveolar septa and inflammatory cell infiltration were observed. (C) In LPS-induced ALI rats with HT, lung injury was less prominent compared with the NT-treated rats. (D) Lung injury scores (LIS). Compared to the controls, the ALI rats showed significantly increased LIS (P<0.001). In contrast, the HT rats showed decreased LIS compared with NT rats. (E, F) The total cell count and neutrophil count in the BAL fluid. Compared with the controls, the ALI rats showed significantly increased total cell and neutrophil counts (P<0.001). However, the HT group showed decreased total cell and neutrophil counts compared with the NT group. Cont: Saline-treated con trol rats; LPS: Normothermia-treated ALI rats after LPS challenge; LPS + HT: Hypothermia-treated ALI rats after LPS challenge. **P<0.001: significantly different from Cont. ing biochemical indicators in serum samples collected 6 hours after LPS challenge. LPS challenge signifi cantly increased the levels of each biomarker ( Figure 2 ). The concentrations of the liver enzymes released into the circulation upon injury, ALT and especially AST, were significantly lower in the HT group than the NT group. In kidney injury, Blood Urea Nitrogen (BUN) and creatinine were also significantly lower in the HT than in the nT group (P<0.05). The concen tration of lactate, an indicator of tissue hypoperfusion, was lower in the HT group than in the NT group, but the difference between the HT and the NT groups was not statistically significant. To identify the altered miRNAs in the lungs of the ALI rats, we performed miRNA profiling using an in-house printed microarray containing 690 rat miRNAs. In total, 126 miRNAs showed differences in expression levels between the LPS-induced ALI rats and the control rats. Out of the 126 miRNAs, 67 miRNAs were upregulat ed and 59 miRNAs were downregulated. Based on a P value of <0.05 and a fold change >1.5, 29 mirnas showed significant alteration after LPS challenge, and are presented in Table 1 To identify the effects of hypothermia in ALI, we analzyed the miRNAs inversely expressed in the HT group compared to the NT group, at a significant manner. We found that 8 miRNAs were inversely expressed between the two groups. Among them, 6 miRNAs (miR-142, miR-98, miR-541, miR-503, miR-653, and miR-223) were upregulated in the NT group and down regulated in the HT group. Two miRNAs (mir323 and miR-196b) were downregulated in the NT group and upregulated in the HT group (Table 1, Table 2 ). We hypothesized that the 8 miRNAs that are differentially expressed between the HT and the NT group may be involved in the pathways related to the effects of HT treatment. Since each miRNA potentially regulates a large number of targets, two miRNA target databases, the miranda and Targetscan (version 7.0), were used to improve the accuracy of target prediction. Gene ontology (GO) term and Kyoto Encyclopedia of Genes and Ge nomes (KEGG) pathway annotation analyses were then performed using the DAVID version 6.7. DAVID provides a tool for annotating biological meaning for input genes regulated by specific miRNAs and functional annotation clustering, and it uses an algorithm to explore relationships among the annotation terms via co-asso ciated genes 16. The 8 lists of predicted targets (upregulated and downregulated miRNAs) were separately submitted to the functional annotation tool. The similar annotation contents were clustered into annotation clusters due to their similar biological meaning. The results showed that dysregulated target genes could be categorized into several major categories. (1) immune re sponse : defense response, response to exter nal stimuli, (2) inflammation : inflammatory cell activation, (3) cell survival : regulation of apoptosis, (4) cellular growth : epidermal growth factor activation, (5) cell-cell inter action : cell-cell communication, and (6) cell migration and adhesion. The main biological process in which the differentially expressed 8 miRNAs in HT were involved was the immune response (cluster 1), inflammation (cluster 2) and cellular apoptosis (cluster 3) (Table 3) . The sorted gene lists of 8 miRNAs were overlapped with KEGG pathway database to identify signaling pathways regulated by those miRNAs. After removing redundant terms, we identified 12 annotated KEGG pathways for the differently expressed miRNAs ( Figure 3 ). Cytokine-cytokine receptor interaction, chemokine signaling pathway had high scores and were likely to be controlled by the differentially expressed miRNAs in HT. The pathways for these miRNAs were associ ated with the immune response : Cytokine-cytokine receptor interaction, chemokine signaling pathway and JAK-STAT signaling pathway; the cell migration and adhesion (leukocyte trans endothelial migration and adherens junction). Individually, miR-142 was mainly associated with the immunity and the cell growth via JAK-STAT signaling pathway and cytokine-cytokine receptor interaction; and apoptosis via the MAPK sig naling pathway. miR-223 was also involved in cyto kine-cytokine receptor interaction, and miR-653 par ticipated in the cell adhesion and migration molecules through T cell receptor signaling pathway, tight junc tion, and leukocyte transendothelial migration. miR-98 was related to the hematopoietic cell lineage through NK cells, neutrophils, mast cells and platelets. To perform static topological analysis, a network of The DAVID informatics resources tool was used for the analysis. The sorted gene list was input to KEGG pathway analysis to re veal the potential biological functions and pathways, which were ranked by significance level. log (P-value) altered miRNAs after HT was established with their putative target molecules. After the predicted target genes associated with the HT-dysregulated miRNAs were imported into the network, the Cytoscape plugin NetworkAnalyzer was applied. As a result, we ob tained a network of 8 miRNAs and 769 target genes (Figure 4) , and found that miR-503-3p and miR-142 correlate with targets GZMB, C5orf51, and STAM, whereas miR-503-3p and miR-541-3p correlate with target SH3BGR, DCDC1, and LRRC19 (Table 4 ). There have been recent reports showing that induced mild hypothermia can improve clinical outcome in several severe clinical situations. In refractory ARDS patients'study, it was found that HT reduced metabolic rate and allowed successful ventilation with a very low tidal volume without an increase in PaCO2 and respira tory acidosis 25 . However, the therapeutic effect of HT is still under debate. Some studies have reported that HT can have beneficial effects in endotoxemic rats via modulation of the inflammatory response and attenua tion of lung injury 26, 27 . Kira et al. 28 also demonstrated that HT inhibits the adhesion, activation, and accumulation of neutrophils during the acute phase of ALI in rats and may have the potential to reduce ongoing inflam mation of ALI. In contrast, Torossian et al. 29 reported that HT increased mortality and impaired immune response in a septic rat model. Stewart et al. 30 also re ported that mild HT increased the levels of IL-6 and IL-10 in endotoxemic mice. In our current study, we studied the therapeutic effects of HT in LPS-induced ALI rat model compared with NT treatment. ALT/AST (hepatic dysfunction), BUN/Cr (renal dysfunction), and lactate (tissue hypoperfusion) have previously been investigated as predictive markers of organ dysfunc tion. Our results show that mild HT ameliorates liver and kidney injury and improves tissue perfusion. Upon histological examination, we observed that lung inju ries in the HT group were less prominent than in the NT group. These results led us to investigate the mech anisms that are involved in these therapeutic effects of HT in ali. Functional genomics approaches provide new prom ising insights into understanding genemechanism in teractions. Actually, the expression profiles of miRNAs have been studied for several diseases, including dif ferent types of malignancies, acute myocardial infarc tion, congestive heart failure, type1 diabetes mellitus, infectious diseases 23, 3133 . In present study, the differ ential expression profiles of miRNAs in ALI rats with HT compared to that in the nT group permitted us to find that up-regulated 6 miRNAs (mir142, mir98, miR-541, miR-503, miR-653, miR-223) in NT were inversely downregulated in the HT group. In contrast, 2 mirnas (miR-323, miR-196b) that were downregu lated in the NT were upregulated in the HT group. We searched for biological processes related with signifi cantly differently expressed 8 miRNAs in HT using with DAVID. We also found that the dysregulated miRNAs in the HT rats were involved in the immunity and the inflammation pathways, as determined by the KEGG pathway analysis. Previous studies have shown that miRNAs may have a role in the immune response and inflammation in sepsis, or ARDS. One such example is that upregulated miR-155 and downregulated miR-125b is one of the pathogenesis in LPS-induced endo toxemic rats, and acts through tumor necrosis factor (TNF)-α stimulation 18 . Dysregulation of miR-146, mir155, mir181b, mir21, and mir301a were re lated to NF-κB activation, which is an essential medi ator in the immune system 34 . miR-146 inhibited Tolllike receptor and cytokine signaling through downreg ulation of IL-1 receptor-associated kinase 1 and TNF receptor-associated factor 6 (TRAF 6) 35 . In this study, dysregulated miR-142 and miR-223 are involved in the inflammatory response by modulating interleukin 2/3 (IL 2/3), interleukin 6 signal tTransducer (IL6ST), IL-7, TNF-α/TNFR (TNF receptor) interaction, and CCL4 (chemokine ligand 4) in JAK-STAT signaling pathway and cytokine-cytokine receptor interaction. The interaction of the inflammatory mediators, TNF-α/ TNFR is very important due to its position at the apex of the proinflammatory cytokine cascade and its dom inance in the pathogenesis of various diseases. More over, TNF-α is one of the main cytokines involved in the response to lPS 36 . Thus, we hypothesize that the alteration of the expression of many genes involved in immune response and inflammation contributes to the therapeutic effects of HT in ALI. In this study, analysis of the differentially expressed miRNAs in HT by KEGG pathway analysis showed that they may be involved in the regulation of apoptosis. Apoptosis of epithelial and endothelial cells is known for the major metabolic pathway that activated in the lung of ARDS patients 37 . lee et al. 38 also reported that the levels of apoptosis mediators were increased in the BAL fluid of ARDS patients, and, a delayed apoptosis of intra-alveolar neutrophils and increased apoptosis of alveolar epithelium increased the severity of lung injury. Some studies showed that miRNAs are related with the regulation of apoptosis. Upregulated miR-26a increased apoptosis in hypoxic rat cardiomyocytes via the caspase-3 pathway 39 and miR let-7 regulated apoptosis in tumors 40 . In this study, the upregulated miR-142 expression profile in NT was downregulated after HT treatment. We confirmed that miR-142 is participated in the regulation of apoptosis through ASK2 (apoptosis signal-regulating kinase), MLK3 (mixed lineage protein kinase 3), and TAB2 (TAK1 binding protein 2) in MAPK signaling pathway in Gene ontol ogy and functional annotation analysis. There have been a few studies about miR-223 that is differently expressed in different situations 4143 . Serum miR-223 expression levels are significantly reduced in septic patients compared with healthy controls 41 , but lung miR-223 levels are increased in influenza virus infected mice 42 . Another study suggested that the protective effect of 5, 14-HEDGE was related to decreased serum miR-223 level through down-regulation of MyD88/TAK1/IKKb/IkB-a/NF-kB pathway in the rat model of septic shock 36 . In our study, lung miR-223 expression levels were increased at 6 h after LPS chal lenge but decreased in HT-treated rat ALI. The dis crepancy among previous studies as well as our results could be attributed to factors such as differences in between humans and animals, type of infection, type of sample, and time points for measurement of miRNA expression levels. In a review of miR-223, miR-223 was described to affect multiple targets simultaneously for key process es. Such processes include hematopoietic cell differ entiation, particularly towards the granulocyte lineage (where miR-223 is abundant) and the myeloid lineage (where miR-223 expression decreases). NF-kB is an important inflammatory mechanism that is dampened by miR-223 43 . anca et al. 44 concluded that miR-223 regulate leukocyte chemotaxis by directly targeting the chemoattractants CXCL2, CCL3, and IL-6 in myeloid cells in TB 44 . Sinilarly, downregulated miR-223 in HT was involved in the cytokine-cytokine receptor inter action via CCL4, CCL3, and CCL8, and, the hemato poiesis via il13. Despite a few studies on miRNAs expression pro filing in ARDS or sepsis, there have been no previous attempts to identify the expression profiles in rat ALI after HT treatment. In conclusion, this study showed that mild hypothermia restored indicators of multiorgan dysfunction and attenuated lung injury. More over, we systematically analyzed the expression of miRNAs in ALI after HT treatment compared with those after NT. The expression levels of lung miR-98, miR-142, miR-223, miR-503, and miR-541, miR-653, miR-323 and miR-196b were significantly altered in HT treatment comparing to NT. These differently ex pressed 8 miRNAs may play certain roles in the ther apeutic effects of HT in ALI by targeting genes that regulate the immunity, inflammation, and apoptosis. This is the first report of miRNAs expression profile in hypothermia treated rat ALI. Further studies should be encouraged because mild hypothermia may provide an approach to the modulation of ongoing inflammatory response in ali. Male Sprague Dawley rats (180200 g) purchased from Samtaco Bio Korea (Cheonan, South Korea) were used for this study. The rats were housed under virtually iden tical conditions for at least 3 days for adaptation. They had free access to standard food and filtered water, were under a 12 : 12 h light-dark cycle, and were kept at a room temperature maintained at 23-26°C. They were grouped as two or three animals per cage before any procedure and singly after the procedures. All pro cedures were performed by the same investigator in order to minimize variability. To induce ALI, bacterial lipopolysaccharide (lPS, serotype 055:B5, 20 mg/kg, Sigma Chemical, St Louis, MO), which is the outer membrane of Escherichia Coli, diluted in saline was used. Bacterial LPS is an effective trigger of the inflammatory response during infection with gram-negative bacilli 19 . ali rats were intraperito neally infiltrated with LPS (5 mg/kg) at time 0, control rats received the same amount of saline intraperito neally. At 16 h after LPS or saline challenge, the rats were anesthetized with an intraperitoneal injection of 1% ketamine (80 mg/kg) and xylazine (5 mg/kg). Rats were placed in a supine position at a 60° angle. Rats were endotracheally intubated with a sterile polyethylene catheter (PE-190 BD, NJ, USA). LPS (10 mg/kg, 100 μL) or saline (100 μL) was infiltrated via the endo tracheal catheter slowly, and 4 mL of air was infused to expand LPS diffusely into both the lungs. All rats were randomly assigned to three groups: salinetreated controls (Cont, n = 5), lPSinduced ali with normothermia (lPS, n = 5), and lPSinduced ali with Hypothermia (lPS + HT, n = 5). Body temperature was monitored using a rectal probe (Physitemp, Physitemp Instrument Inc, NJ). After intra-tracheal infiltration of LPS, rectal temperature was maintained at 32-34°C for 6 h by external cooling with ice cube and spray in LPS + HT group. The target temperature was reached with in 10 minutes. The LPS group kept in normothermia, 37±0.5°C, was placed on a heated pad (FHC, Bow doinham, ME, USA) and a blanket for 6 h. anesthesia was maintained with an intermittent infusion of 1% ketamine (80 mg/kg) and xylazine (5 mg/kg) for 6 h. Sepsis-related lung injury rat model induced by LPS study showed that at 6 hours after LPS administration, LPS developed the severe state, thrombocytopenia, elevated lactate levels, hypoxemia, and liver and renal injury. Hypoxemia at 6 hours was severe and slightly improved at 24 hours. So we collected the BAL and the sample at 6 hours after LPS infiltration 45 . The rats were sacrificed at 6 h after the NT or HT treatments. Blood was collected through cardiac punc ture and centrifuged at 3,000 rpm for 10 mins to obtain plasma. Plasma samples were frozen at -70°C before measurements were made using a specific kinetic enzymatic analyzer, IDEXX VetTest ® Chemistry Analyzer (IDEXX Laboratories, Inc., ME). The trachea was incised, and a 14-G tube was placed into the trachea. Left (Lt.) main bronchus was ligated with 3-0 silk. After 5 mL of PBS was flushed back and forth three times through the 14-G tube placed in trachea, BAL fluid was collected from the Right (Rt.) lung. The BAL fluid was centrifuged (3,000 rpm, 4℃, 10 mins), the pellet was diluted with 500 μL PBS, and the total cells were counted using LUNA automated cell counter (logos Biosystems, VA) according to the manufacturer's in structions. Of the 500 μL diluted pellet, 200 μL was cytospinned, prepared on a slide, and then Wright-Giemsa-stained. Differential cell counts were performed by counting 100 cells under a microscope (Olympus, Tokyo, Japan) in four representative slide sections, and the number of neutrophils was calculated as the per centage of neutrophils multiplied by the total number of cells in the BAL fluid. Lt. upper lobe was fixed with 10% formalin for hematoxylin and eosin (HE) stain, and Lt. lower lobe was stored at -70°C for miRNA study. After 24 h, the lung tissue was dehydrated and embedded in paraffin. Sections were stained with HE for evaluation of the severity of lung injury. Each lung section was blindly assigned by a clinical pathologist to obtain the lung injury score (LIS). LIS comprising of 4 components (alveolar capillary congestion, hemorrhage, inflammatory cells infiltrating the airspace or interstitium, and thickness of the alveolar wall) scored on a 5-point scale (0 = min imal damage, 1 = mild damage, 2 = moderate damage, 3 = severe damage, 4 = maximal damage) each and summed 20 . Total RNA was extracted from the rat lung tissue using the Tri reagenT (MRC, OH) according to the man ufacturer's instructions. Following homogenization, 1 mL of solution was transferred to a 1.5 mL Eppendorf tube and centrifuged at 12,000 rpm for 10 min at 4°C to remove insoluble material. The supernatant contain ing RNA was collected, mixed with 0.2 mL of chloro form, and centrifuged at 12,000 rpm for 15 mins at 4°C. After RNA in the aqueous phase was transferred into a new tube, the RNA was precipitated by mixing 0.5 mL of isopropyl alcohol and recovered by centrifuging the tube at 12,000 rpm for 10 mins at 4°C. The RNA pellet was washed briefly in 1 mL of 75% ethanol and centrifuged at 7,500 rpm for 5 mins at 4°C. Finally, the total RNA pellet was dissolved in Nuclease-free water, and its quality and quantity was assessed by an Agilent Bioanalyzer 2100. Rat microRNA expression was analyzed using miR CURY LNA TM microRNA Array (7 th genhas, mmu & rno array; Exiqon, Vedbaek, Denmark), covering 690 well-characterized rat microRNA among 3,100 capture probes for rat miRNAs. In this procedure, 5′phosphates from 800 ng of total RNA were removed by treating with Calf Intestinal Alkaline Phosphatase (CIP) fol lowed by labeling with Hy3 green fluorescent dye. Labeled samples were subsequently hybridized by loading onto a microarray slide using Hybridization Chamber Kit part # G2534A (Agilent Technologies, Santa Clara, CA, USA) and Hybridization Gasket Slide Kit part # G2534-60003 (Agilent Technologies). Hybridization was performed over 16 h at 56°C followed by washing the microarray slide as recommended by the manufacturer. Processed microarray slides were then scanned with Agilent G2565CA Microarray Scanner System (Agilent Technologies). Scanned images were imported by Agilent Feature Extraction software ver sion 10.7.3.1 (Agilent Technologies), and fluorescence intensities of each image were quantified using the mod ified Exiqon protocol and corresponding GAL files. miRNA target prediction was performed by the miRanda and Targetscan, version 7.0. To identify the functions of the differentially expressed miRNAs, the lists of predicted targets of dysregulated miRNAs were sepa rately submitted to the functional annotation tool pro vided by the Database for Annotation, Visualization, and Integrated Discovery (DAVID), version 6.7 21, 22 . The predicted targets were annotated by the Kyoto En cyclopedia of Genes and Genomes (KEGG) pathway analysis. A pathway was considered to be significant only if it passed the count threshold of three genes per annotation term and presented EASE score, with Ben jamini-Hochberg correction set to <0.05 23, 24 . The results are expressed as the mean±standard devi ation. 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