A Synthetic Cannabinoid Uncouples Intracellular Inflammation from TNF Release During Infection

Aubrey Atkins
University of Florida

Co-Authors: Hailey A. Barker¹, Jorge J. Canas¹, Aubrey Atkins¹, Samantha M. Enslow¹, Dana R. Amin¹, Xin Wang¹, Mariola J. Ferraro¹
¹University of Florida

Background: Cannabinoid signaling is increasingly recognized as a regulator of immune function, but its effects during bacterial infection remain incompletely understood. WIN 55,212-2 is a synthetic cannabinoid receptor agonist with reported immunomodulatory activity; however, whether it suppresses or redirects inflammatory cytokine responses during intracellular bacterial infection is unclear.

Objectives: This study investigated how WIN 55,212-2 modulates macrophage inflammatory responses during Salmonella enterica serovar Typhimurium infection, with emphasis on TNFα production, intracellular cytokine accumulation, cytokine release, and cannabinoid receptor involvement.

Methods: RAW 264.7 macrophages and bone marrow-derived macrophages from wild-type, CB1R-deficient, and CB2R-deficient mice were treated with WIN 55,212-2 or vehicle and infected with Salmonella Typhimurium. Cytokine responses were assessed by ELISA, flow cytometry, Western blotting, RT-qPCR, and label-free quantitative proteomics. An oral Salmonella infection model was used to evaluate systemic inflammatory responses in vivo. Direct bacterial effects of WIN 55,212-2 were assessed using bacterial proteomics, growth curves, viability assays, and macrophage infection assays.

Results: WIN 55,212-2 broadly reshaped the macrophage proteome during Salmonella infection, altering pathways associated with metabolism, proteostasis, inflammatory signaling, and intracellular trafficking. Unexpectedly, WIN 55,212-2 increased intracellular abundance of inflammatory mediators, including TNFα, IL-6, and IFNγ, while markedly reducing extracellular TNFα accumulation. This divergent TNFα phenotype persisted in macrophages lacking either CB1R or CB2R alone. In vivo, WIN 55,212-2-treated mice showed reduced circulating TNFα during Salmonella infection, with altered splenic myeloid activation markers. Direct exposure of Salmonella to WIN 55,212-2 changed selected bacterial proteins but did not alter bacterial growth, viability, early macrophage infection, or macrophage TNFα induction.

Conclusions: WIN 55,212-2 does not simply suppress macrophage inflammation during bacterial infection. Instead, it uncouples intracellular cytokine accumulation from extracellular TNFα release, revealing a distinct mode of cannabinoid-associated immunomodulation during Salmonella infection.