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

Abstract

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.

Keywords: Egr-1; PPARγ; acute lung injury; inflammation; pro-inflammatory mediators.

Figure 1

Ectopic expression of peroxisome proliferator-activated receptor (PPAR)γ in the lung inhibits IgG immune complex (IgG-IC)-induced acute lung injury. (A) Different doses of adenovirus are injected into lungs via airways. Seventy-two hours later, lungs are harvested and total proteins are extracted. Then Western blot assays are conducted by using antibodies recognizing PPARγ and GAPDH, respectively. Mice are treated by airway administration of 1 × 10⁸ PFU of Ad-GFP or Ad-PPARγ. Three days later, acute lung injury is induced by treating the mice with IgG-IC. Four hours later, bronchoalveolar lavage (BAL) fluids and whole lungs are collected to analyze lung permeability indexes (B), measurement of pulmonary myeloperoxidase (MPO) contents (C), total white blood cells (D), and neutrophils counts (E) in BAL fluids, and levels of TNF-α (F), MCP-1 (G), MIP-1α (H), and MIP-2 (I) in BAL fluids, respectively. Data are expressed as means ± S. E. M. N=3 for α-BSA-treated mice, N=5 for Ad-GFP+IgG-IC group, and N=6 for Ad-PPARγ+IgG-IC group. *, ** and *** indicate statistically significant difference—p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 2

Peroxisome proliferator-activated receptor (PPAR)γ decreases IgG immune complex (IgG-IC)-stimulated inflammatory response in macrophages. RAW264.7 cells are treated with 100 μg/ml of IgG-IC for different time points. Then RNAs and proteins are extracted, and qPCR (A) and Western blot (B) are performed to verify PPARγ generation at mRNA and protein levels, respectively. RAW264.7 cells are pre-treated with DMSO or 10 μM Rosiglitazon–ROSI. One hour later, the cells are further treated with IgG-IC. Then total cellular RNAs are isolated 4 h later, and cell-free supernatants are harvested 8 h later. TNF-α (C, F), MCP-1 (D, G), and MIP-1α (E, H) expressions are examined at both RNA and protein levels. (I) RAW264.7 cells are infected by control or PPARγ shRNA lentiviral particles at a MOI of 30. Seventy-two hours later, the cells are treated with 3 μg/ml of puromycin. The survival cells are selected, and the knockdown efficiency is confirmed by qPCR. RAW264.7 cells expressing PPARγ shRNA are treated with DMSO or 10 μM ROSI for 1 h. Then the cells are treated with IgG-IC for 4 h, which is followed by measurement of TNF-α (J), MCP-1 (K), and MIP-1α (L) expressions. Data are expressed as means ± S. E. M. (N=3 for qPCR, and N=6 for ELISA). *, ** and *** indicate statistically significant difference—p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 3

Early growth response protein 1 (Egr-1) production is negatively regulated by peroxisome proliferator-activated receptor (PPAR)γ in macrophages. RAW264.7 cells are challenged by IgG immune complex (IgG-IC) for distinct time points. Then RNAs and proteins are extracted, and qPCR (A) and Western blot (B) are conducted to analyze Egr-1 expression. (C) RAW264.7 cells are pre-treated with DMSO or 10 μM Rosiglitazone (ROSI) for 1 h. Then the cells are incubated with IgG-IC for 3 h, and Egr-1 expression is examined. (D) RAW264.7 cells expressing control or PPARγ shRNA are treated with IgG-IC for 3 h, which is followed by measurement of Egr-1 generation. Data are expressed as means ± S. E. M. (N=3). ** and *** indicate statistically significant difference—p < 0.01, and p < 0.001, respectively.

Figure 4

Early growth response protein 1 (Egr-1) positively regulates IgG immune complex (IgG-IC)-triggered expressions of TNF-α, MCP-1, and MIP-1α in macrophages by binding to their promoter regions. (A) RAW264.7 cells are infected by control or Egr-1 shRNA lentiviral particles at a MOI of 30. Seventy-two hours later, the cells are treated with 3 μg/ml of puromycin. The survival cells are selected, and the knockdown efficiency is confirmed by qPCR. RAW264.7 cells expressing control or Egr-1 shRNA are treated with IgG-IC. Then RNAs and cell-free supernatants are harvested 4 and 8 h later, respectively. qPCR and ELISA are performed to analyze TNF-α (B, E), MCP-1 (C, F), and MIP-1α (D, G) expressions. (H) Egr-1-overexpressing plasmids are constructed, and confirmed in HEK293 cells by Western blot. (I–N) HEK293 cells are transfected with the indicated plasmids. Twenty-four hours later, the cells are lysed, and the lysates are subjected to luciferase assays. The detailed information about the luciferase-expressing constructs is indicated in the figures. The data are calculated by dividing the value of the indicated promoter-driven firefly luciferase by the corresponding value of thymidine kinase promoter-driven renilla luciferase, and then set to 1 in control cells. Data are expressed as means ± S. E. M. (N=3 for qPCR and luciferase assays, and N=6 for ELISA). *, ** and *** indicate statistically significant difference—p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 5

IgG immune complex (IgG-IC)-mediated pulmonary early growth response protein 1 (Egr-1) expression is inhibited by ectopic production of peroxisome proliferator-activated receptor (PPAR)γ. Four hours after pulmonary deposition of IgG-IC, lungs are harvested. The tissue RNAs and proteins are isolated separately. Then the Egr-1 expression is examined by qPCR (A) and Western blot (B), respectively. (C) Mice are treated with airway injection of Ad-GFP or Ad-PPARγ at a dose of 1 × 10⁸ PFU. Seventy-two hours later, the mice are intratracheally challenged by IgG-IC for 4 h. Then the lungs are harvested, and proteins are extracted for Western blot analysis by using antibodies against Egr-1 and GAPDH, respectively. Data are expressed as means ± S. E. M. (For qPCR, N=3 for α-BSA treated mice, and N=5 for IgG-IC-treated mice; N=3 for Western blot). ** indicates statistically significant difference—p < 0.01.

Figure 6

Downregulating early growth response protein 1 (Egr-1) expression attenuates IgG immune complex (IgG-IC)-mediated acute lung injury. (A) Lentiviruses (3 × 10⁷ TU—transduction unit) expressing control or Egr-1 shRNA are injected into mouse lungs through airways. Five days later, the lungs are harvested, and RNAs are extracted, which is followed by verification of Egr-1 expression by qPCR. Mice infected by control or Egr-1 shRNA lentiviral particles are intratracheally treated by IgG-IC for 4 h. Then bronchoalveolar lavage (BAL) fluids and whole lungs are harvested. Lung permeability index (B), and measurement of pulmonary myeloperoxidase (MPO) activities (C) in whole lungs are measured. In addition, influxes of total leukocytes (D) and neutrophils (E) into alveolar spaces are counted. Also, productions of TNF-α (F), MCP-1 (G), MIP-1α (H), and MIP-2 (I) in BAL fluids are assayed. Data are expressed as means ± S. E. M. (N=3 for qPCR; For ELISA, N=3 for α-BSA-treated mice, N=5 for shRNA NC+IgG-IC group, and N=6 for Egr-1 shRNA+IgG-IC group). *, ** and *** indicate statistically significant difference—p < 0.05, p < 0.01, and p < 0.001, respectively.