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. 2018 Jan;59(1):102-112.
doi: 10.1194/jlr.M080028. Epub 2017 Nov 27.

Genomic and lipidomic analyses differentiate the compensatory roles of two COX isoforms during systemic inflammation in mice (V体育ios版)

Xinzhi Li  1 , Liudmila L Mazaleuskaya  2 , Laurel L Ballantyne  1 , Hu Meng  2 , Garret A FitzGerald  2 , Colin D Funk  3
Affiliations

Genomic and lipidomic analyses differentiate the compensatory roles of two COX isoforms during systemic inflammation in mice

Xinzhi Li et al. J Lipid Res. 2018 Jan.

V体育ios版 - Abstract

Both cyclooxygenase (COX)-1 and COX-2, encoded by Ptgs1 and Ptgs2, function coordinately during inflammation. But the relative contributions and compensations of COX-1 and COX-2 to inflammatory responses remain unanswered. We used three engineered mouse lines where the Ptgs1 and Ptgs2 genes substitute for one another to discriminate the distinct roles and interchangeability of COX isoforms during systemic inflammation. In macrophages, kidneys, and lungs, "flipped" Ptgs genes generate a "reversed" COX expression pattern, where the knock-in COX-2 is expressed constitutively and the knock-in COX-1 is lipopolysaccharide inducible. A panel of eicosanoids detected in serum and kidney demonstrates that prostaglandin (PG) biosynthesis requires native COX-1 and cannot be rescued by the knock-in COX-2. Our data further reveal preferential compensation of COX isoforms for prostanoid production in macrophages and throughout the body, as reflected by urinary PG metabolites. NanoString analysis indicates that inflammatory networks can be maintained by isoform substitution in inflamed macrophages. However, COX-1>COX-2 macrophages show reduced activation of inflammatory signaling pathways, indicating that COX-1 may be replaced by COX-2 within this complex milieu, but not vice versa. Collectively, each COX isoform plays a distinct role subject to subcellular environment and tissue/cell-specific conditions, leading to subtle compensatory differences during systemic inflammation VSports手机版. .

Keywords: animal model; cyclooxygenase; eicosanoid; lipopolysaccharide; macrophage; prostaglandin, gene targeting V体育安卓版. .

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Figures

Fig. 1.
Fig. 1.
COX expression in the kidney, stomach, and lung of Ptgs gene-exchanged mice. Western blot analysis of COX-1 and COX-2 expression in tissues of mice challenged either with LPS for 6 h or with an equivalent volume of PBS alone. Images are representative of two separate experiments.
Fig. 2.
Fig. 2.
Effects of Ptgs gene exchange on AA metabolism signaling pathways in macrophages from mice with systemic inflammation. A: Western blot analysis of COX isoforms in peritoneal macrophages harvested from mice either challenged in vivo with LPS (2 mg/kg, ip) or an equivalent volume of PBS for 6 h. Images are representative of two separate experiments. B: RNA counts from NanoString analysis of Ptgs1 and Ptgs2 expression in macrophages. Note that NanoString Ptgs1 probe (exon 11) and Ptgs2 probe (exon 5) can detect both native Ptgs and knock-in Ptgs. Data are presented as mean ± SEM, n = 6. C: Quantitative PCR for prostanoid synthases (mPGES-1, PGIS, AKR1B3, TBXAS1, and H-PGDS) in macrophages. Data are expressed as relative mRNA expression to 18S rRNA (mean ± SEM, n = 3). *P < 0.05 versus WT. D: RNA counts from NanoString analysis of Alox15, Alox5, Cysltr1, Ltb4r1, Pla2g4a, Ptger2, and Tbxa2r expression in macrophages. Data are presented as mean ± SEM, n = 6.
Fig. 3.
Fig. 3.
Prostanoid profiles in peritoneal lavage from mice challenged with LPS in vivo. Peritoneal lavage was harvested after 6 h of LPS peritoneal administration, and then subjected to LC-MS/MS assays. Data are presented as mean ± SEM, n = 6–8.
Fig. 4.
Fig. 4.
Prostanoid profiles in mice challenged with LPS in vivo. Serum or plasma were obtained after 6 h of LPS peritoneal administration, and then subjected to LC-MS/MS assays. BLD, below the limit of detection. Data are presented as mean ± SEM, n = 6–8. *P < 0.05 and **P < 0.01.
Fig. 5.
Fig. 5.
AA and downstream eicosanoid profiles of mice challenged with LPS in vivo. Serum or plasma were obtained after 6 h of LPS peritoneal administration, and then subjected to LC-MS/MS assays. BLD, below the limit of detection. Data are presented as mean ± SEM, n = 6–8. *P < 0.05 and **P < 0.01.
Fig. 6.
Fig. 6.
Total urinary prostanoid biosynthesis in mice. Urinary prostanoid metabolites were measured in urine samples collected over 3 h from mice challenged with LPS intraperitoneally. Data are presented as mean ± SEM, n = 6–11.
Fig. 7.
Fig. 7.
Prostanoid profiles in the renal medulla. Dissected inner renal medulla was homogenized and then subjected to LC-MS/MS assays. Data are presented as mean ± SEM, n = 6–8.
Fig. 8.
Fig. 8.
Differential gene expression and inflammatory signaling pathway of COX isoforms in macrophages. Peritoneal macrophages were harvested from mice either challenged in vivo with LPS (2 mg/kg, ip) or an equivalent volume of PBS (Control) for 6 h. A: Venn diagram showing the number of differentially expressed (fold change >2 vs. Control) genes induced by LPS in WT (132), in COX-2>COX-1 (144), in COX-1>COX-2 (128), and in Reversa (140) mice. Of the 248 genes represented in the array, 113 (69%) changed more than 2-fold in all four LPS-challenged genotypes of mice. B: Heat map of the results of hierarchical clustering analysis for the 113 genes. Each row represents a transcript and the column represents grouped and normalized signals for different treatments and different genotypes of mice (n = 6). Red and blue represent up- and down-regulation, respectively. C: Comparison of NanoString count analysis and qPCR-determined relative expression for Ccl2, Ccl3, Il1b, and Il6. Arrows indicate near absence of gene expression in Control mice detected under these conditions. D: nSolver 3.0-generated pathway score analysis for cytokine, chemokine, and associated inflammatory responses. Data are presented as mean ± SEM, n = 6 except for the qPCR data, n = 4. *P < 0.05 versus WT. E: Evaluation of ear inflammatory responses. Data are expressed as ear weight increase of an 8 mm diameter biopsy after AA treatment (left ear) compared with vehicle treatment (right ear). *P < 0.05 versus WT (n = 8).
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