INTRODUCTION
Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are inflammatory diseases characterized by acute respiratory dysfunction resulting from pulmonary inflammation, noncardiogenic pulmonary edema, and disruption of the endothelium and epithelium (1 2
Toll-like receptors, acting as sensors for the presence of specific pathogen-associated molecular patterns, play a key role in host defense, innate immunity, and inflammation (3, 4 5 6
However, TLR activation is a double-edged sword. These receptors have important functions in host defense, but unchecked, excessive, or inappropriate TLR activation may lead to severe inflammation and inappropriate immunity-related tissue damage, such as ALI. To avoid detrimental and inappropriate inflammatory responses, the immune system requires negative regulators for TLR signaling including single Ig IL-1R-related molecule (SIGIRR) (7 8 9 8, 10 10 in vitro or in vivo administration of LPS (10, 11 11 11 In vitro , SIGIRR-deficient kidney and intestinal epithelial cells and splenocytes exhibit enhanced responsiveness to IL-1 and Toll ligands (11 9 In vivo , SIGIRR/Tir8-deficient mice are more susceptible to the systemic toxicity of LPS (9, 11 9 12, 13 10, 11, 14 15 16 15, 16
Taken together, these observations indicate that SIGIRR functions as an important modulator for the Toll-IL-1R system, including the LPS-TLR4 signaling pathway. Through negative regulation of TLR4 signaling, SIGIRR can suppress the inflammatory reaction to LPS and reduce tissue and organ damage. With this in mind, we hypothesized that enhancing SIGIRR expression with an adenoviral vector may be beneficial for LPS-induced lung injury. However, the precise and potential effect of SIGIRR on lung injury remains unclear, and currently, there is no direct evidence to prove the hypothesis mentioned above. Therefore, the present study was performed to investigate the role of enhanced expression of murine SIGIRR (mSIGIRR), a homolog of human SIGIRR, in LPS-induced ALI in mice.
MATERIALS AND METHODS
Cell culture
HEK293 cells were obtained from Microbix (Toronto, Ontario, Canada) and were grown in Dulbecco's modified Eagle medium (Hyclone, Logan, Utah), supplemented with 10% fetal bovine serum (Hyclone) and 2 mmol/L l-glutamine (Gibco/BRL, Grand Island, NY). Cells in the exponential growth phase were used in the experiments described in the following section.
Generation of adenoviral vectors
An adenovirus was generated that contained a single open reading frame encoding an mSIGIRR. First, cDNA coding for mSIGIRR was excised from pCMV-SPORT6-mSIGIRR (GeneCopoeia Inc, Guangzhou, China) and ligated into the adenoviral shuttle plasmid pDC316 (Microbix) to generate the plasmid pDC316-mSIGIRR. Recombinant viruses were then generated using HEK293 packaging cells cotransfected with pDC316-mSIGIRR and a plasmid containing cDNA for adenoviral proteins (pBHGlox) via the AdMax system (Microbix). Plaques were isolated after approximately 14 days and expanded in HEK293 cells. Viral stocks were purified using CsCl gradients. Meanwhile, a control vector, free of any transgenes, was constructed using the same method. The recombinant virus and control virus were named as Ad.mSIGIRR and Ad.V, respectively. Viral titer determination (plaque-forming units [PFUs]/mL) was performed by means of 50% tissue culture infectious dose (TCID50 ). The titers of Ad.mSIGIRR and Ad.V were 4 × 109 PFUs/mL and 1 × 109 PFUs/mL, respectively.
Establishment of ALI mouse model and experimental design
This study was approved by the Animal Care and Use Committee of the Third Military Medical University. BALB/c mice (18-22 g) were purchased from the Laboratory Animal Center of Third Military Medical University. After acclimatization for 1 week in the animal housing facility, the mice were lightly anesthetized by ether inhalation and inoculated intranasally with 4 × 107 PFUs (in 50 μL) of Ad.mSIGIRR (group Ad.mSIGIRR) or Ad.V (group Ad.V). Forty-eight hours after intranasal administration of viruses, LPS (Escherichia coli 0127: B8; Sigma, St Louis, Mo) at 15 mg/kg (dissolved in saline) was injected into the peritoneal cavity of conscious mice to establish ALI mouse model. The mice were killed at 0, 2, 6, 12, 24, and 48 h after LPS challenge (n = 5 per time point in each group). Blood and lung samples were harvested aseptically for subsequent experiments. Blood samples were allowed to clot for 0.5 h on ice and then centrifuged. The sera were stored in small aliquots at −70°C until use for detection of TNF-α level. Lung samples were divided into two parts, one of which was snap-frozen and stored in liquid nitrogen for RNA extraction, protein isolation, and tissue homogenate. The other part was fixed immediately for histological evaluation and immunohistochemical analysis of SIGIRR. Additionally, to evaluate the effect of Ad.mSIGIRR on the outcome of LPS-induced ALI, another 30 mice were divided into two groups (15 mice/group) and given the same treatments as described above. Survivals were recorded for 7 days, and Kaplan-Meier curves were depicted.
Expression of SIGIRR
Determination of SIGIRR mRNA expression
The lung tissue was mechanically homogenized under RNase-free condition, dissociated with TRIzol Reagent (Invitrogen, Shanghai, China) on ice for 3 min, and then RNA was extracted according to the manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was finished in a two-step way using RNA PCR Kit (AMV) version 3.0 (TaKaRa, Dalian, China) according to the manufacturer's instructions. The primers for mSIGIRR were 5′-GGGAGGTGGAGATGAACG-3′ (sense) and 5′-GATAGGTCTGCGGGTGAG-3′ (antisense). The primers for murine glyceraldehyde-3-phosphate dehydrogenase (mGAPDH) were 5′-GTGCTGAGTATGTCGTGGAGTCT-3′ (sense) and 5′-GAGTGGGAGTTGCTGTTGAAGT-3 ′ (antisense). Murine GAPDH was selected as an internal standard. The single-stranded cDNA was amplified by PCR using 30 cycles. The PCR profile used for mSIGIRR amplification was as follows: 30 s of denaturation at 94°C, 30 s of annealing at 56°C, and 1 min of extension at 72°C. The profile for mGAPDH amplification was as follows: 30 s of denaturation at 94°C, 30 s of annealing at 65°C, and 1 min of extension at 72°C. Polymerase chain reaction products and molecular weight markers were subjected to electrophoresis on 2% agarose gels in Tris-acetate-EDTA buffers at 5 V/cm for 1 h and visualized by means of ethidium bromide staining. The gel was then photographed using AlphaImager 2200 system (Alpha Inotech, San Leandro, Calif), and the band optical density was quantified by Adobe Photoshop version 7.0.1 software (Adobe Systems, San Jose, Calif). The relative amount of mRNA levels of the mSIGIRR gene was calculated as the ratio to mGAPDH expression.
Western blot analysis of SIGIRR
The lung tissue was mechanically homogenized in ice-cold RIPA lysis buffer (Beyotime Chemical Co, Jiangsu, China), supplemented with protein inhibitor cocktail pill (Roche, Mannheim, Germany). At 10 min after incubation on ice, the homogenates were centrifuged at 12,000g for 5 min at 4°C. The supernatant containing the total protein from lung tissue was collected. After the concentration of the protein was measured by the BCA method (Beyotime Chemical Co), protein sample was analyzed by Western blotting. Briefly, equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% polyacrylamide gel). Then, the protein was transferred to a polyvinylidene difluoride membrane (Santa Cruz Biotechnology Inc, Santa Cruz, Calif) using the semidry transfer apparatus with the steady flow model (electric current 0.8 mA/cm2 ) over 2 h. The membrane was blocked in a 5% nonfat milk solution in Tris-buffered saline with 0.5% Tween 20. The membrane was allowed to react with primary antibody (Santa Cruz Biotechnology Inc) and secondary antibody-horseradish peroxidase (Beijing Golden Bridge Biotech, Beijing, China). Detection of specific proteins was done by enhanced chemiluminescence following the manufacturer's instructions (Beyotime Chemical Co). The membrane was then photographed using gel documentation and analysis systems (GBox-HR; Syngene, Frederick, Md), and band intensities were measured with Adobe Photoshop version 7.0.1 software (Adobe Systems) and normalized to the expression of mGAPDH for quantitative analysis.
Immunohistochemical analysis of SIGIRR
The expression level of SIGIRR in the murine lung parenchyma was detected by means of immunohistochemical analysis. In brief, paraffin sections were dewaxed by passage through xylene and graded alcohols. Endogenous peroxidase activity was blocked with 0.6% hydrogen peroxidase in methanol, and nonspecific binding sites were blocked with normal rabbit serum. The sections were incubated in a humidified chamber at 4°C overnight with goat polyclonal anti-mouse SIGIRR (Santa Cruz Biotechnology Inc). The sections were further incubated for 30 min with rabbit anti-goat IgG and with streptavidin-conjugated horseradish peroxidase (Zymed Laboratories, San Diego, Calif). Samples were visualized after a few minutes of incubation with diaminobenzidine as the chromatogen, followed by hematoxylin counterstaining. The substitution of phosphate-buffered saline for the specific primary antibody was performed as the negative control.
Histological evaluation
Lungs were treated in a 4% paraformaldehyde phosphate buffer solution (pH 7.4) overnight, embedded, and sectioned at 6.0 μm, and tissue slides were stained with hematoxylin-eosin. Lung injury was scored by a blinded observer according to the following three criteria: (a ) alveolar and interstitial edema, (b ) alveolar hemorrhage, and (c ) infiltration or aggregation of neutrophils. Each criterion was graded according to a four-point scale: 0 = normal, 1 = mild change, 2 = moderate change, and 3 = severe change. The scores for criteria 1 through 3 were summed to represent the lung damage score (total score, 0-9) (17
Determination of TNF-α level
TNF-α levels in serum and lung tissue were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kits for mice. Lungs were homogenized in 0.15 g/mL ice-cold suspension buffer (phosphate-buffered saline with 0.05% Tween 20, pH 7.0; 2 μg/mL aprotinin; and 100 μg/mL phenylmethylsulfonyl fluoride) using a tissue homogenizer and were then sonicated on ice. The resultant homogenate was centrifuged (10,000g , 4°C), and the clear supernatant was transferred to sterile Eppendorf tubes and stored at −70°C. Supernatants (100 μL) from the blood or the lung tissue homogenate were added to ELISA plates precoated with anti-murine TNF-α, and ELISA was performed strictly following the protocols provided by the manufacturer (Dakewe Biotech Co, Shenzhen, China).
Determination of lung NO concentration
Lung tissue homogenates were prepared as described above. Then, the resultant homogenates were centrifuged (14,000g , 4°C) for 15 min, and the clear supernatants were transferred to sterile Eppendorf tubes. After detection of the protein content of the supernatants by Bradford method (Beyotime Chemical Co), the supernatants were used to assay NO concentration as described previously (18
Determination of lung myeloperoxidase activity
To measure tissue myeloperoxidase (MPO) activity, frozen lungs were thawed and extracted for MPO, following the homogenization and sonication procedure as described previously (19 2 O2 in the presence of o -dianisidine.
Determination of NF-κB activity
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay was performed by using a commercial kit (Roche). The nuclear extracts were prepared using a commercially available extraction kit (Active Motif, Carlsbad, Calif), according to the manufacturer's instructions. The extracts were stored at −70°C until use, and the protein content of the nuclear extracts was quantified by the BCA method (Beyotime Chemical Co). A total of 5 μg of the extracts was incubated with a digoxigenin-labeled NF-κB probe (Roche) and was then subjected to electrophoresis on a 6% nondenaturing polyacrylamide gel. Control samples were also incubated with an excess of unlabeled NF-κB probe to confirm specificity of binding. The gel contents were then transferred to a nylon membrane (Roche) and developed using a chemiluminescence system (Roche) and exposed to film (Hyperfilm; Amersham Biosciences, Buckinghamshire, UK).
Enzyme-linked immunosorbent assay
The nuclear extracts were prepared as described above. Nuclear factor κB p65 DNA binding activity was assessed on isolated nuclear extracts by ELISA using the TransAM NF-κB transcription factor assay kit according to the manufacturer's protocol (Active Motif).
Statistical analysis
Data in text and figures are expressed as the mean ± SEM. ANOVA was used to compare experimental groups to control values. Comparisons between multiple groups were done using Student-Newman-Keuls test. Kaplan-Meier curves were analyzed using the log-rank test. Statistical significance was determined as P < 0.05.
RESULTS
SIGIRR gene and protein expression in lung
To determine whether expression of SIGIRR was enhanced in group Ad.mSIGIRR, we tested SIGIRR mRNA and protein expression levels in lungs using RT-PCR, Western blot, and immunohistochemistry. Single Ig IL-1 receptor-related molecule was constitutively expressed (mRNA and protein) in normal murine lungs, which was consistent with previous reports (8, 10 P > 0.05 vs. normal mice; Fig. 1 ). After LPS stimulation, SIGIRR protein and mRNA levels were downregulated and maintained at a very low level until 12 h after LPS administration (P < 0.05 vs. its own 0-h time point). Compared with those in group Ad.V, SIGIRR mRNA and protein expression were enhanced in group Ad.mSIGIRR at each time point, although decreases were noted at 2 and 6 h after LPS administration (P < 0.05; Fig. 1 ). The result of immunohistochemical analysis was similar to that of Western blot, and protein expression of SIGIRR was mainly detected in the pulmonary epithelial cells (Fig. 1C ).
Fig. 1: SIGIRR gene and protein expression in lung . A, SIGIRR mRNA levels (normalized to GAPDH) in lungs of normal mice and two groups (group Ad.Vand Ad.mSIGIRR) at 0, 2, 6, 12, 24, and 48 h after LPS injection by RT-PCR. Upper panel-representative image. N indicates normal mice. Lower panel-quantitative data. B, Western blot of SIGIRR protein levels (normalized to GAPDH) in lungs of normal mice and two groups of mice at 0, 2, 6, 12, 24,and48 h after LPS injection. Upper panel-representative image. N indicates normal mice. Lower panel-quantitative data. All data are expressed as mean±SEM (n= 5); *P < 0.05 vs. group Ad.V at each time point, +P < 0.05 vs. its own 0 h time point, and #P < 0.05 vs. its own 0 h time point. C, RepresentativeSIGIRR immunostained lung sections of normal mice and two groups of mice at 0, 12, or 48 h after LPS challenge. Negative control (a, b), normal mice (c), (0h; Ad.mSIGIRR [d]), (12 h; Ad.mSIGIRR [e]), (48 h; Ad.mSIGIRR [f]), (0 h; Ad.V [g]), (12 h; Ad.V [h]), (48 h; Ad.V [i]). Brown represents positive staining.
SIGIRR ameliorated lung histological changes
The histopathology of lung tissues from mice pretreated with Ad.V or Ad.mSIGIRR was examined as described above at 0, 2, 6, 12, 24, and 48 h after LPS challenge. Compared with normal mice lung tissue, no significant histological changes were observed either in Ad.V- or Ad.mSIGIRR-pretreated mice before LPS challenge (Fig. 2A [a-c]), demonstrating that there was no effect of adenovirus on lung injury. After LPS challenge, typical histological features of ALI were observed in group Ad.V, inclusive of diffuse alveolar damage, infiltration of numerous neutrophils, alveolar hemorrhage, and interstitium edema (Fig. 2A [d-f]). However, in mice given Ad.mSIGIRR, the pathological changes in lung tissue were attenuated (Fig. 2A [g-i]). Compared with those in group Ad.V, the lung damage scores were significantly reduced in the Ad.mSIGIRR-pretreated group at each time point (Fig. 2B ).
Fig. 2: Histological evaluation of ALI. A, Histological evaluation of ALI . A, Hematoxylin-eosin-stained histological lung section of mice. Lung section of normal mice (a). Lung section of Ad.V-treated mice before LPS injection (b). Lung section of Ad.mSIGIRR-treated mice before LPS injection (c). The second and third rows of this figure are representative lung sections at 6, 12, or 48 h after LPS challenge. (6 h; Ad.V [d]), (12 h; Ad.V [e]), (48 h; Ad.V [f]), (6 h; Ad.mSIGIRR [g]), (12 h; Ad.mSIGIRR [h]), (48 h; Ad.mSIGIRR [i]). B, Lung damage score of the two groups after LPS injection. Data are expressed as mean ± SEM (n = 5). *P < 0.05 vs. 0 h time point and #P < 0.05 vs. group Ad.V at each time point.
SIGIRR decreased TNF-α level in serum and lung
The proinflammatory cytokine TNF-α level was detected by ELISA. The basal cytokine levels in lung and serum showed no difference between the two groups. Exposure to LPS increased the TNF-α level in serum and lung (P < 0.05 vs. their respective 0-h time points). In group Ad.mSIGIRR, this increase was significantly reduced except for the 48-h time point in lung. In fact, the TNF-α level of two groups in lung had returned to their respective basal levels at 48 h (P > 0.05 vs. 0-h time point) (Fig. 3B ). Meanwhile, the TNF-α level of group Ad.mSIGIRR in serum also returned close to its baseline (P > 0.05 vs. 0-h time point) (Fig. 3A ).
Fig. 3: Enhanced expression of SIGIRR decreased LPS-mediated TNF-α release in serum (A) and lung (B) . Data are expressed as mean ± SEM (n = 5). *P < 0.05 vs. 0 h time point and #P < 0.05 vs. group Ad.V at each time point.
SIGIRR prevented NO production in lung
In unstimulated mice, the concentrations of NO were low. In response to LPS, there was a marked increase in the levels of NO (P < 0.05 vs. 0-h time point). Ad.mSIGIRR pretreatment significantly reduced LPS-mediated NO production in lung (P < 0.05 vs. group Ad.V at 2-, 6-, 12-, and 24-h time points). At 48 h after LPS treatment, the concentration of NO in the Ad.mSIGIRR group approximated the basal level, and there was no significant difference between the two (P > 0.05) (Fig. 4 ).
Fig. 4: Enhanced expression of SIGIRR prevented LPS-mediated NOproduction in lung . Data are expressed as mean ± SEM (n = 5). *P < 0.05vs.0 h time point and #P < 0.05 vs. group Ad.V at each time point.
SIGIRR suppressed pulmonary MPO activity
Before LPS challenge, lung tissue MPO levels in the two groups were very low (0.65 ± 0.07 U/g wet lung tissue and 0.66 ± 0.10 U/g wet lung tissue, respectively). The MPO activity in the lung parenchyma increased sharply after LPS injection and persisted during the entire experiment (P < 0.05 vs. 0-h time point). Compared with that in group Ad.V, the increased MPO activity in group Ad.mSIGIRR was significantly reduced at each time point after LPS challenge (P < 0.05) (Fig. 5 ).
Fig. 5: Enhanced expression of SIGIRR prevented LPS-stimulated MPO activity in lung . Data are expressed as mean ± SEM (n = 5). *P < 0.05 vs. 0 h time point and #P < 0.05 vs. group Ad.V at each time point.
SIGIRR reduced the levels of activated NF-κB activation in lung
We used two methods to detect the activity of NF-κB in LPS-induced ALI. All LPS-injected mice exhibited a marked increase in the amount of translocated, active p65 levels in lung nuclear extracts, compared with mice not given LPS (P < 0.05 vs. 0-h time point). However, compared with the group Ad.V, pretreatment of mice with Ad.mSIGIRR significantly attenuated the LPS-induced increase in NF-κB activation and translocation (P < 0.05 vs. group Ad.V at each time point). At the end point of the experiment, the NF-κB activation was extinguished in group Ad.mSIGIRR (Fig. 6 ).
Fig. 6: Enhanced expression of SIGIRR inhibited LPS-induced NF-κB activation . A, Electrophoretic mobility shift assay for NF-κB activity at 0, 6, 12, and48 h after LPS injection. Upper panel of A is a representative photograph of five; cold indicates cold competitions with unlabeled oligonucleotide. Lower panelrepresents densitometries analysis of the bands. B, Enzyme-linked immunosorbent assay for NF-κB activation at 0, 2, 6, 12, 24, and 48 h after LPS injection. All data are expressed as means ± SEM (n = 5). *P < 0.05 vs. 0 h time point and #P < 0.05 vs. group Ad.V at each time point.
SIGIRR improved 7-day survival rate
To determine the effect of enhanced expression of SIGIRR on the outcome of LPS-induced ALI, the survival rate between group Ad.V and Ad.mSIGIRR was compared by using the log-rank test. Mice receiving Ad.mSIGIRR had an overall survival advantage of 73%, whereas the control animals had an overall survival of 33% over 7 days (P < 0.05) (Fig. 7 ).
Fig. 7: Survival rate of mice after LPS challenge over 7 days . Mice were pretreated with Ad.V (n = 15) or Ad.mSIGIRR (n = 15) 48 h before LPS injection. Survivals were recorded for 7 days, and the survival curves were compared by the log-rank test. *P < 0.05 vs. group Ad.V.
DISCUSSION
The present study shows that the enhanced expression of SIGIRR attenuated the extent of lung injury, decreased the production of TNF-α and NO, inhibited the neutrophil infiltration and NF-κB activation, and improved the survival rate in an LPS-induced ALI mouse model. To our knowledge, this is the first study to demonstrate amelioration of LPS-induced ALI in mice by the administration of the recombinant adenoviral vector that carries the mSIGIRR gene therapy.
SIGIRR is a novel member of TIR superfamily. Our results confirmed the findings of previous studies, which had demonstrated the pattern of SIGIRR expression (10, 11, 15, 16, 20 20 adenoviral vector successfully. The decrease in SIGIRR expression levels in the lungs of group Ad.mSIGIRR mice may also be due to consumption of SIGIRR in the process of ameliorating inflammation.
Enhancement of SIGIRR expression to resist the consumption at the early stage may timely inhibit excessive inflammation response and improve the prognosis of inflammatory diseases. Thus, we determined the lung pathological changes, the severity of inflammation, and the survival rate in two groups and found that enhanced expression of SIGIRR significantly decreased the levels of TNF-α and NO, inhibited the activation of NF-κB, attenuated lung pathological changes and neutrophil infiltration, and improved the outcome of ALI. These findings are similar to previous studies in vitro (10, 11, 14-16 10 in vitro (16
We were not able to determine blood-gas indices, as the mouse has low total blood volume, and all blood was used for measurement of serum TNF-α level. Nonetheless, damage to vascular barrier and respiratory membrane and deterioration of gas exchange are known components of ARDS (1, 21
In addition, we used an LPS model of lung injury to investigate the protective effect of SIGIRR. Although the clinical relevance of this model is controversial (22, 23 24 25-28
In conclusion, we have demonstrated that transient overexpression of SIGIRR in the lungs, mediated by an adenoviral vector , could suppress the inflammatory reaction, attenuate lung pathological impairments, and decrease mortality in an LPS-induced murine model. Future research will focus on the emerging issues from this study and on the detailed molecular mechanisms of SIGIRR protection against lung injury.
ACKNOWLEDGMENTS
The authors thank Mr Zhou Qin (Institute of Combined Injuries, Third Military Medical University) and Ms Chen Huaping (Institute of Respiratory Diseases, Xinqiao Hospital, Third Military Medical University) for their excellent technical assistance.
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