Modulation of Mrna Expression in Peripheral Tissue in a Rodent E. Coli Sepsis Model

The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates pathophysiology in many pro‐inflammatory conditions, such as irritable bowel diseases, atherosclerosis and acute lung injury. Sepsis, a highly morbid condition, results from a dysregulated host response to infection. Despite extensive study the transcriptional regulation of cytokines and the ECS during sepsis remains to be established. We hypothesized that cannabinoid receptor (Cnr1 & 2) mRNA expression increases with systemic infection and these increases coincide with increases in pro‐inflammatory cytokines. We implanted a fibrin clot that contained either 25×106 or 0 E. coli in the peritoneal cavity of adult, male rats (n=8). Rats were euthanized at 3, 6, 12 or 24 h following the implant; lung, liver and serum were harvested. Total RNA was extracted using Trizol reagent, and cDNA was synthesized using reverse transcription PCR. Quantitative real‐time PCR was performed in duplicate for each sample using TaqMan Gene® Expression Assays specific for 18S ribosomal RNA (18S), Cnr1 & 2, interleukin (IL) IL‐4 & IL‐6 and tumor necrosis factor – alpha (TNF‐α). Each target gene was normalized to 18 S content, and relative quantitation of gene expression levels were performed using the comparative CT method (2−ΔΔCT method) with the 0 E. coli fibrin clot at each time point as the comparative control. The time course of cytokine expression depended on the cytokine and the tissue compartment. In liver, IL‐6 peaked at 3h (1086 ± 59% vs 0.36 ± 0.1%, p<0.01). In serum, IL‐4 peaked at 6h (168 ± 34% vs 86 ± 5%). In lung, both IL‐6 and TNF‐α expression levels peaked at 3h (IL‐6: 755 ± 33% vs 8 ± 0.5%, p<0.01; TNF‐a: 496 ± 230% vs 8 ± 0.2%); whereas, IL‐4 levels peaked at 24h (291 ± 31% vs 3 ± 2%, p<0.05). Interestingly, at 24h Cnr2 mRNA increased concomitantly with IL‐4 in the lung (360.0 ± 10% vs 30 ± 5%, p<0.01), whereas the expression levels of Cnr1 mRNA did not increase above sham surgical levels during the time course of this study. These preliminary data and analyses support our hypothesis that induction of ECS, as characterized by the increased transcription of Cnr2 mRNA, is associated with upregulation of mRNA of pro‐inflammatory cytokines.Support or Funding InformationNIH 5T32HL007913‐18, NIH U01 EB021960, VA Research Service I01BX004197This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.