The Crucial Role of PPARγ-Egr-1-Pro-Inflammatory Mediators Axis in IgG Immune Complex-Induced Acute Lung Injury

Background The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR) γ plays crucial roles in diverse biological processes including cellular metabolism, differentiation, development, and immune response. However, during IgG immune complex (IgG-IC)-induced acute lung inflammation, its expression and function in the pulmonary tissue remains unknown. Objectives The study is designed to determine the effect of PPARγ on IgG-IC-triggered acute lung inflammation, and the underlying mechanisms, which might provide theoretical basis for therapy of acute lung inflammation. Setting Department of Pathogenic Biology and Immunology, Medical School of Southeast University Subjects Mice with down-regulated/up-regulated PPARγ activity or down-regulation of Early growth response protein 1 (Egr-1) expression, and the corresponding controls. Interventions Acute lung inflammation is induced in the mice by airway deposition of IgG-IC. Activation of PPARγ is achieved by using its agonist Rosiglitazone or adenoviral vectors that could mediate overexpression of PPARγ. PPARγ activity is suppressed by application of its antagonist GW9662 or shRNA. Egr-1 expression is down-regulated by using the gene specific shRNA. Measures and Main Results We find that during IgG-IC-induced acute lung inflammation, PPARγ expression at both RNA and protein levels is repressed, which is consistent with the results obtained from macrophages treated with IgG-IC. Furthermore, both in vivo and in vitro data show that PPARγ activation reduces IgG-IC-mediated pro-inflammatory mediators’ production, thereby alleviating lung injury. In terms of mechanism, we observe that the generation of Egr-1 elicited by IgG-IC is inhibited by PPARγ. As an important transcription factor, Egr-1 transcription is substantially increased by IgG-IC in both in vivo and in vitro studies, leading to augmented protein expression, thus amplifying IgG-IC-triggered expressions of inflammatory factors via association with their promoters. Conclusion During IgG-IC-stimulated acute lung inflammation, PPARγ activation can relieve the inflammatory response by suppressing the expression of its downstream target Egr-1 that directly binds to the promoter regions of several inflammation-associated genes. Therefore, regulation of PPARγ-Egr-1-pro-inflammatory mediators axis by PPARγ agonist Rosiglitazone may represent a novel strategy for blockade of acute lung injury.

thus amplifying IgG-IC-triggered expressions of inflammatory factors via association with their promoters.
Conclusion: During IgG-IC-stimulated acute lung inflammation, PPARg activation can relieve the inflammatory response by suppressing the expression of its downstream target Egr-1 that directly binds to the promoter regions of several inflammation-associated genes. Therefore, regulation of PPARg-Egr-1-pro-inflammatory mediators axis by PPARg agonist Rosiglitazone may represent a novel strategy for blockade of acute lung injury.
Keywords: acute lung injury, PPARg, Egr-1, pro-inflammatory mediators, inflammation BACKGROUND Excessive formation of IgG immune complexes in vivo has been demonstrated to be associated with a variety of diseases, such as systemic lupus erythematosus, rheumatoid arthritis, acute glomerulonephritis, mixed cryoglobulinemia and vasculitis (1)(2)(3)(4)(5)(6)(7). Moreover, the pulmonary diseases, such as acute lung injury (ALI) and its more severe form-acute respiratory distress syndrome (ARDS), are also reported to be related with deposition of immune complexes in the tissue (8,9). During IgG-IC induction of ALI/ARDS, the local macrophages are firstly activated through engagement of Fcg receptors with the ligands, resulting in the release of numerous cytokines and chemokines. Then, under the action of the inflammatory factors, alveolar epithelial cells are activated and neutrophils are recruited into the pulmonary tissue. Finally, acute lung injury is triggered by the severe inflammatory response. However, the molecular events responsible for productions of inflammation-associated mediators are poorly understood.
PPARg is a type II nuclear receptor, which is rich in adipose tissue. Actually, other types of cells and tissues also generate PPARg (10)(11)(12). A large number of studies focus on its roles in regulation of gene expressions associated with lipid uptake and adipogenesis, and find that PPARg is involved in a broad spectrum of lipid metabolism disorder-mediated diseases, such as obesity, diabetes and atherosclerosis (13)(14)(15). However, the importance of PPARg in immune regulations has also been recognized. For example, PPARg is required for IL-9 production in T H 2 cells, which might exert regulatory effects on acute allergic skin inflammatory response in human beings (16). Reddy et al. report that PPARg activation has protective effects against cigarette smoke-stimulated inflammatory responses during chronic obstructive pulmonary disease (17). Except for its effect on chronic pulmonary disease, the role of PPARg in acute lung injury has also been examined. There are data that ventilation-or LPS-induced acute lung injury is relieved by PPARg, but the conclusions are obtained by using PPARg antagonist or agonist (18)(19)(20). So, the nonspecific effects of the chemical reagents cannot be excluded. More importantly, the influence of PPARg on IgG-IC-induced acute lung injury and the corresponding mechanism have not been elucidated.
Egr-1 also named as NGFI-A (nerve growth factor-induced protein A) is a nuclear protein acting as a transcription factor. Its expression can be induced by a variety of stimulus including cytokines, neurotransmitters and growth factors (21). However, the effect of IgG-IC treatment on its expression remains enigmatic. Aberrant Egr-1 expression or activity has been detected in various human diseases, such as atherosclerosis, cancer, ischemic injury and TGF-b-dependent pro-fibrotic responses (21). Notably, the role of Egr-1 in inflammatory responses has also been extensively investigated. For example, cigarette smoke-induced chemokine release from primary human lung fibroblasts is dependent on Egr-1 (22). In addition, the secretion of inflammatory cytokines induced by Mycoplasma pneumonia infection in vitro and in vivo is also positively regulated by Egr-1 (23). However, its influence on acute lung inflammation and the underlying mechanism after intrapulmonary deposition of IgG-IC remain mysterious.
Given the importance of PPARg and Egr-1 in inflammatory responses, the present study intends to determine the roles of the two transcription factors in IgG-IC-induced acute lung injury, and the possible interaction between PPARg and Egr-1. We find, for the first time, that PPARg expression is downregulated, while Egr-1 expression is upregulated in IgG-IC-treated mouse lungs and macrophages. Furthermore, we prove that Egr-1 enhances IgG-IC-induced inflammatory response by directly binding to the promoter regions of the pro-inflammatory mediators, and PPARg suppresses inflammation by inhibiting production of Egr-1. Taken together, the data show that during IgG-ICinduced acute lung injury, PPARg may play an essential role in regulating inflammatory response by interfering with the expression of its downstream target Egr-1, and PPARg agonist Rosiglitazone may be used clinically for blockade of acute lung injury in the future.
ELISA kit for mouse albumin is purchased from Bethyl Laboratories. ELISA kits for TNF-a, MCP-1, MIP-1a and MIP-2 are purchased from R&D Systems. PPARg antagonist GW9662, PPARg agonist Rosiglitazone (ROSI) and puromycin are purchased from Cayman Chemical.

IgG-IC-Induced Acute Lung Injury and Analysis of Bronchoalveolar Lavage Fluids (BALFs)
Mice are anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital. Then rabbit anti-BSA antibodies are administrated into mouse lungs through the airways. Immediately after the operation, the mice are treated by intravenous injection of BSA dissolved in PBS. Four hours later, whole lungs or bronchoalveolar lavage fluids (BALFs) are collected.
BALFs are centrifuged at 3,000 rpm for 5 min at 4°C, and then cell-free supernatants are collected and subjected to analyze albumin, TNF-a, MCP-1, MIP-1a and MIP-2 levels by ELISA. Albumin contents derived from IgG-IC-treated mice are divided by those from the corresponding control mice to obtain values of lung permeability indexes. The remaining cell pellets are resuspended in HBSS containing 0.5% BSA. Total white blood cells are measured with hemocytometer. Cytospin centrifugation is used for preparation of samples for cell differentials. Samples are fixed and stained with Diff-Quik Stain (Solarbio ® LIFE SCIENCES, China), which is followed by analysis of percentage of neutrophils.

Construction of Expression Vectors and Promoter-Driven Reporters
Adenoviral vectors promoting ectopic expression of PPARg are constructed by following the instruction provided by BD Biosciences. Briefly, PPARg-encoding sequences (Accession Number NM_001127330) are amplified from mouse lung cDNA library, and the DNA sequences are ligated with the shuttle vector-pShuttle2, to form the recombinational plasmids -pShuttle2-PPARg. Enzyme-digested pShuttle2-PPARg is extracted and linked with the adenoviral vector. Then the recombinational adenovirus vectors are linearized by the restriction enzyme PacI, which is followed by transfection of the linear DNA into HEK293 cells to make the infectious adenovirus (Ad-PPARg). The obtained Ad-PPARg is stored at -80°C, and tittered by utilizing the titer kit from BD Biosciences before use.
Control plasmid is constructed by ligating the amplified EGFP encoding fragment from pIRES-EGFP (Clontech) with pcDNA3.1-Myc-His (Invitrogen). Egr-1-encoding sequences (Accession Number NM_007913) are cloned from mouse lung cDNA library by PCR. The recombinational plasmids expressing Egr-1 are then formed by ligating the amplified DNA sequences with pcDNA3.1-Myc-His.
We firstly generate double-stranded oligos containing the PPARg or Egr-1 interference sequence, and then link them to the vector-miRZip ™ shRNA Expression Lentivector (System Biosciences). The transfer plasmids together with two other plasmids-pMD2.G and psPAX2 (Addgene plasmids 12259 and 12260) are co-transfected into HEK293 cells by using Lipofectamine 2000 (Invitrogen) to make mature viral particles that are titered by measuring GFP positivity after transduction.
The full length of TNF-a (Accession Number NC_000083)/ MCP-1 (Accession Number NC_000077)/MIP-1a (Accession Number) promoter region is amplified by using mouse genomic DNA as the template. Then TNF-a Luc, MCP-1 Luc and MIP-1a Luc are constructed by ligating their corresponding promoter fragments with pGL4-basic vector (Promega). Mutations are introduced to the Egr-1 binding sites in TNF-a, MCP-1 and MIP-1a promoter regions by using the Mut Express II Fast Mutagenesis Kit V2 (Vazyme, China). pRL-TK plasmids used as internal references are obtained from Promega.

Airway Injection of Recombinational Viral Vectors
Ad-GFP (BD Biosciences) or Ad-PPARg is intratracheally administrated into mice. Three days later, the infected mice are used for the subsequent experiments. Mice receiving airway administration of shRNA NC or Egr-1 shRNA are used 5 days after infection.

Construction of Stable Cell Line
RAW264.7 cells are infected by control or PPARg/Egr-1 shRNA lentiviral particles at a MOI of 30. Seventy-two hours later, the cells are treated with 3 mg/ml puromycin. The survival cells are selected, and the knockdown efficiency is confirmed by qPCR.

Western Blot Analysis
Total cellular or tissue proteins are extracted by using radioimmune precipitation assay buffer from Beyotime Biotechnology (China). Then 30 mg of proteins are run on a 12% SDS-polyacrylamide gel and examined by using the following antibodies: goat anti-PPARg (Santa Cruz, catalog number sc-1984X), mouse anti-Egr-1 (Santa Cruz, catalog number sc-515830), and mouse anti-GAPDH (Proteintech, catalog number 60004-1-Ig).

Measurement of Pulmonary Myeloperoxidase (MPO) Activity
Four hours after airway deposition of IgG-IC, mouse lungs are harvested and homogenized in the buffer containing the following components: 0.5% hexadecyltrimethylammonium bromide, 50 mM potassium phosphate buffer and 5 mM EDTA. Then the homogenates are subjected to sonication. Cell-free supernatants are mixed with the MPO analysis buffer consisted of 5 mg/ml H 2 O 2 , 100 mM potassium, and 167 mg/ml odianisidine dihydrochloride. MPO contents are determined by measuring the change in optical density (at 450 nm) per min using the 96-well plate reader.

Luciferase Assays
Cells are cultured in 12-well plates. Twelve hours later, the cells are transfected with total of 0.5 mg plasmids using Fugene ® 6 Transfection Reagent (Promega) according to the manufacturer's guideline. Twenty-four hours after transfection, cells are lysed and the lysates are subjected to measurement of luciferase activities with the Dual-Luciferase Reporter Assay System purchased from Promega.

Statistical Assay
All data are represented as the mean ± S. E. M. Significance is indicated when p value is less than 0.05. Data sets are analyzed using Student's t test or one-way ANOVA, with individual group means being compared with the Student-Newman-Keuls multiple comparison test.

PPARg Negatively Regulates IgG-IC-Induced Acute Lung Injury
The roles of PPARg in pulmonary diseases have been extensively explored. However, during IgG-IC-stimulated acute lung injury, its expression and function have not been determined. So, we first examine if PPARg production is changed in the mouse lung after intratracheal deposition of IgG-IC. As shown in Figure  S1A, IgG-IC treatment leads to a 36% decrease in PPARg production at mRNA level, which is consistent with the Western blot assay ( Figure S1B).
We then investigate the role of PPARg in IgG-IC-initiated acute lung injury which is reflected by lung permeability, and MPO activity-an indicator of neutrophil accumulation. Firstly, we overexpress PPARg in the mouse pulmonary tissue by using adenovirus as the vector (24), and find that Ad-PPARg greatly increases PPARg expression even at a dose of 1 × 10 8 PFU (plaque forming unit) ( Figure 1A). Importantly, mice receive intratracheal treatment of 1 × 10 8 PFU of adenoviral vectors do not show any signs of acute lung injury ( Figure S2). Therefore, in the following studies, 1 × 10 8 PFU of Ad-PPARg is applied to the animal experiments. As shown in Figure 1B, when compared with the control group, lung permeability index is increased by more than 2.5 folds after IgG-IC stimulation, which is almost reduced to basal level by ectopic expression of PPARg. As expected, the increase in the pulmonary MPO activity caused by IgG-IC treatment is also significantly inhibited by PPARg overexpression ( Figure 1C).
Recruitment of white blood cells, especially neutrophils, into alveolar spaces plays a critical role in acute lung injury. We observe that influxes of total leukocytes and neutrophils into alveolar compartments are greatly induced by IgG-IC treatment ( Figures 1D, E). However, 4 h after initiation of IgG-IC-induced acute lung injury, the numbers of total white blood cells and neutrophils recovered from BAL fluids of Ad-PPARg mice are obviously reduced as compared with the control (Figures 1D, E).
To further elucidate the important role of PPARg in IgG-ICinduced acute lung injury, chemical reagent GW9662-an antagonist of PPARg, is used to block PPARg activation. As shown in Figure S3, IgG-IC-induced elevation of acute lung injury-related indicators including lung permeability, MPO activity, alveolar recruitment of leukocytes, especially neutrophils, and expressions of pro-inflammatory mediators are further exacerbated in mice receiving GW9662 treatment. However, acute lung injury induced by IgG-IC is alleviated by PPARg agonist-ROSI ( Figure S4)

Inflammation Is Relieved by PPARg in IgG-IC-Treated Macrophages
During IgG-IC-induced acute lung injury, macrophage is one of the most important positive regulators of the severity of tissue damage. Therefore, the role of PPARg in IgG-IC-induced inflammatory response in macrophages is analyzed. Firstly, we investigate whether PPARg expression in macrophages is affected by IgG-IC at mRNA level. As shown in Figure 2A, PPARg expression in macrophages is decreased in a time-dependent manner within 6 h after onset of IgG-IC treatment. Even after 24 h of IgG-IC stimulation, PPARg maintains at low level ( Figure 2A). Furthermore, the above qPCR results are consistent with the data obtained from Western blot analysis ( Figure 2B).
We then determine the effect of PPARg on IgG-IC-stimulated pro-inflammatory mediators' generation by using PPARg agonist Rosiglitasone (ROSI). We find that expressions of TNF-a, MCP-1 and MIP-1a at mRNA level are increased by 22-, 10-, and seven-folds, respectively in macrophages treated with IgG-IC ( Figures 2C-E). ROSI treatment causes IgG-IC-mediated productions of TNF-a, MCP-1, and MIP-1a to decrease by 36%, 34%, and 32%, respectively ( Figures 2C-E). We next verify the influence of ROSI on IgG-IC-initiated inflammatory reaction at protein level, and observe that the data obtained from ELISA are consistent with the qPCR results ( Figures 2F-H).
To exclude the off-target effect of ROSI, we silence the expression of PPARg in macrophages. As shown in Figure 2I, macrophages treated with PPARg shRNA show a 73% decrease in PPARg production when compared with the control cells. Of note, IgG-IC-mediated productions of TNF-a, MCP-1 and MIP-1a are no longer reduced by ROSI in macrophages expressing PPARg specific shRNA ( Figures 2J-L), indicating that ROSI negatively regulates IgG-IC-induced expressions of cytokine and chemokine via activating PPARg.

Egr-1 Expression Is Reduced by PPARg in Macrophages
To probe the mechanism by which PPARg decreases IgG-ICinduced inflammatory response, we tend to find out a bridge molecule linking PPARg with pro-inflammatory mediators. Time course experiment shows that Egr-1 mRNA level peaks after 6 h of IgG-IC stimulation, and then declines progressively ( Figure 3A), which is consistent with Western blot results ( Figure 3B). More importantly, we find that in the presence of IgG-IC, Egr-1 expression in ROSI-treated macrophages is only 37% of the control ( Figure 3C). On the contrary, IgG-IC-stimulated Egr-1 expression is increased by approximately twofolds with downregulation of PPARg production ( Figure  3D), suggesting that Egr-1 might be the possible molecule connecting PPARg with pro-inflammatory mediators.

Egr-1 Amplifies IgG-IC-Induced Expressions of Pro-Inflammatory Mediators in Macrophages by Binding to Their Promoter Regions
Egr-1 expression is controlled by PPARg, but its effect on IgG-IC-triggered pro-inflammatory mediators' expressions remains elusive. Therefore, we first downregulate Egr-1 expression in macrophages by using lentivirus expressing Egr-1 specific shRNA ( Figure 4A). In the presence of IgG-IC, TNF-a, MCP-1, and MIP-1a mRNA levels are increased by more than 20, 300, and 50 times, respectively ( Figures 4B-D). Egr-1 shRNA treatment reduces the expressions of the above inflammatory mediators to 41%, 1%, and 74% of their respective controls ( Figures 4B-D). Furthermore, the suppressive role of Egr-1 shRNA in inflammation is also confirmed at protein level in macrophages challenged by IgG-IC ( Figures 4E-G).  To reveal the mechanism involved in Egr-1 regulation of proinflammatory mediators' generation, we firstly construct Egr-1 overexpression plasmid ( Figure 4H). Then we investigate if productions of pro-inflammatory mediators are influenced by ectopic expression of Egr-1. As shown in Figures 4I-K, TNF-a, MCP-1, and MIP-1a promoter-driven luciferase activities are increased by Egr-1 to 242%, 277%, and 126% of their respective controls. According to software prediction (JASPAR), there are potential Egr-1 binding sites in the promoter regions of the above pro-inflammatory mediators. Thus, we further introduce mutations to the Egr-1 sites of the above inflammatory mediators' promoters, and find that Egr-1 site mutation causes TNF-a, MCP-1 and MIP-1a promoter-driven luciferase activities to decrease by 47%, 67%, and 47%, respectively ( Figures 4L-N).

During IgG-IC-Induced Acute Lung Injury, Egr-1 Expression Is Reduced by PPARg
To clarify the effect of PPARg on Egr-1 expression in vivo, we first examine the production of Egr-1 during IgG-IC-induced acute lung injury. As shown in Figure 5A, Egr-1 mRNA level in the lung tissue is increased to 13.7-folds of the control group. Furthermore, IgG-IC also stimulates an obvious increase in Egr-1 protein expression when compared with the control treatment ( Figure 5B). More importantly, during IgG-IC-induced acute lung injury, we find that mice receiving airway administration of Ad-PPARg show a great reduction in Egr-1 generation when compared with their littermates treated by Ad-GFP ( Figure 5C).

Egr-1 Aggravates IgG-IC-Induced Acute Lung Injury Through Amplifying Expressions of Pro-Inflammatory Mediators
Next, we analyze the role of Egr-1 in IgG-IC-induced acute lung injury by downregulating its expression with the lentiviral vector (25) ( Figure 6A). Of note, lentiviral vector itself could not stimulate acute lung injury ( Figure S5). We find that after airway deposition of IgG-IC, mice treated with Egr-1 shRNA display reduced tissue damage signs including lung permeability and MPO activity as compared with the control mice receiving shRNA NC treatment ( Figures 6B, C). Additionally, IgG-ICinduced recruitment of white blood cells including neutrophils into the alveolar compartments is significantly relieved by downregulating Egr-1 expression in the lungs (Figures 6D, E).
Productions of pro-inflammatory mediators promote acute lung injury. So we examine the effect of Egr-1 on expressions of cytokine and chemokine. As shown in Figures 6F-I, IgG-IC stimulation dramatically elevates TNF-a, MCP-1, MIP-1a, and MIP-2 expressions in BAL fluids, which are significantly decreased with downregulation of Egr-1 generation in the lung tissue.

DISCUSSION
Due to lacking of specific therapy methods in clinic, the mortality rates of ALI/ARDS in the United States can be as high as 60%, despite the recent development of protective lung ventilation strategies (26,27). Thus, we should further investigate the molecular mechanisms of ALI/ARDS to promote the development of specific therapies. In the present study, we utilize IgG-IC-induced acute lung injury model to reveal the underlying mechanism of acute lung injury. We have proved that IgG-IC treatment causes an obviously acute inflammatory response, characterized by the infiltration of neutrophils into the pulmonary tissue, and the elevated expressions of cytokine and chemokine, which are similar to clinical symptoms. Furthermore, we find that PPARg-Egr-1-proinflammatory mediators axis is involved in IgG-IC-triggered pulmonary damage, and intervention of the axis might be a useful strategy for treatment of patients suffering from ALI/ARDS.
After intrapulmonary deposition of IgG-IC, local macrophages expressing high levels of Fcg receptors, but not alveolar epithelial cells first perceive the stimulation. Then early response cytokines including TNF-a and IL-1b are released from the macrophages, which are crucial for stimulating lung endothelial cells to express adhesion molecules (28,29). Moreover, the early response cytokines promote chemokine secretion in the lung tissue (30)(31)(32). Finally, pulmonary injury and massive influx of neutrophils into the lung tissue are induced by the joint action of a variety of inflammationrelated mediators including cytokines, chemokines and adhesion molecules. Actually, all the above processes are controlled by the network composed of various transcription factors. There are reports that the DNA binding activities of NF-kB and AP-1 are increased during IgG-IC-induced acute lung injury (31,33). Both transcription factors can directly bind to a broad spectrum of proinflammatory mediators' promoter regions, and increase their productions. In addition, our findings demonstrate that both C/ EBPb and C/EBPd, which belong to C/EBP family members, can increase IgG-IC-induced inflammation via association with the C/ EBP elements in the promoter regions of inflammatory genes (34,35). In the present study, we find that PPARg expression is significantly downregulated following IgG-IC treatment. However, as an important transcription factor, the role of PPARg in IgG-ICinitiated inflammation has not been determined.
In the current paper, we find that PPARg inhibits IgG-ICinduced TNF-a, MCP-1 and MIP-1a productions in vitro and in vivo. Previous studies have proved that PPARg activation alleviates postoperative ileus-related gene expression through suppression of Egr-1 production (36). Also, Rosiglitazone-mediated PPARg activation inhibits Egr-1 generation, leading to suppression of bleomycin-induced scleroderma and profibrotic responses (37). Thus, during IgG-IC-induced inflammatory responses, Egr-1 might be the potential bridge molecule linking PPARg with proinflammatory mediators' productions. We find that Egr-1 expressions in macrophages and lungs are greatly upregulated by IgG-IC treatment. In addition, we observe that activation of PPARg decreases Egr-1 expression, while lowing PPARg level increases its generation, indicating that Egr-1 is negatively regulated by PPARg. Furthermore, we demonstrate that during IgG-IC-induced acute inflammatory response, expressions of pro-inflammatory mediators are positively regulated by Egr-1. Thus, during IgG-IC-induced acute lung injury, PPARg can negatively regulate IgG-IC-induced inflammation through an indirect way, which is similar to the method used by STAT3 (38). Of note, during IgG-IC-mediated acute lung injury, whether there are other PPARg-regulated transcription factors that directly control inflammation responses remains an open question.
Egr-1, a critical transcription factor, belongs to the EGR family of Cys2His2-type zinc finger proteins. It is composed of transactivation and inhibitory domains, as well as three DNAbinding zinc finger domains which interact with GC-rich regions in the promoters of target genes (39). Its roles in lung diseases, especially in chronic lung diseases have been widely reported. Lee et al. find that Egr-1-mediated cell apoptosis is crucial for TGF-belicited lung fibrosis (40), indicating that Egr-1 plays a detrimental role in the disease. In contrast, Kramer et al. report that the deficiency of Egr-1 obviously exacerbates TGF-a-induced pulmonary fibrosis, which is independent of the tissue inflammatory response (41), which manifests the protective effect of Egr-1 in the disease. The above contradictory phenomena might be explained by the distinct stimulus used in the model. Therefore, the role of Egr-1 is complicated, and should be discussed in specific contexts. In the present paper, we observe that Egr-1 accelerates inflammation induced by IgG-IC through association with the promoter regions of pro-inflammatory mediators including TNFa, MCP-1 and MIP-1a. Our previous data have demonstrated that the activities of both p38 MAPK and ERK1/2 are elicited, and are essential for cytokine/chemokine expressions during IgG-ICinduced inflammation (34). Others find that p38 MAPK activation is required for Egr-1-dependent expressions of proinflammatory mediators (42). Moreover, there are data that the IL-33-ERK/JNK/p38/Egr-1/TSLP (thymic stromal lymphopoietin) axis is involved in allergic skin Th2 inflammation (43). Thus, we speculate that there may be crosstalk between MAPK signaling pathway and PPARg-Egr-1 axis during inflammatory reactions triggered by IgG-IC. However, this remains an open question, which should be supported by more studies. Additionally, the role of the inhibitory domain of Egr-1 in IgG-IC-induced inflammation should also be examined in the future.
On the basis of our present data, we conclude that PPARg protects against the development of IgG-IC-induced acute lung injury through the reduction of inflammatory responses. This protection afforded by PPARg features reduced cytokine/ chemokine expressions and accumulation of neutrophils in the lung tissue. Furthermore, the anti-inflammatory effects of PPARg after intrapulmonary deposition of IgG-IC are mediated by inhibiting generation of Egr-1, which can bind to the promoter regions of pro-inflammatory mediators including TNF-a, MCP-1, and MIP-1a. Therefore, PPARg activation might represent a promising therapeutic option for IgG-IC-induced acute lung injury. Though PPARg agonist Rosiglitazone has been used for treatment of type 2 diabetes in clinic, whether the protective effects of PPARg detected in the animal model could be extrapolated to the clinical application is subject to the future clinical trials.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Animal Experimental Ethical Committee of Southeast University.