Enhanced expression of coxsackievirus and adenovirus receptor in lipopolysaccharide-induced inflammatory macrophages is through TRIF-dependent innate immunity pathway

Life Sci. 2021 Jan 15:265:118832. doi: 10.1016/j.lfs.2020.118832. Epub 2020 Nov 28.

Abstract

Aims: Inflammatory macrophages have been proposed as a therapeutic target for joint disorders caused by inflammation. This study aimed to investigate the expression and regulation of coxsackievirus-adenovirus receptor (CAR) in lipopolysaccharide (LPS)-stimulated inflammatory macrophages whereby to evaluate the feasibility of virus-directed enzyme prodrug therapy (VDEPT).

Main methods: Macrophage cell lines (RAW264.7 and J774A.1) and primary macrophage cells derived from rat spleen were used to evaluate the expression of CAR protein or CAR mRNA. Specific inhibitors for TLR4 pathway were used to investigate the regulation of CAR expression. CAR expression in rat joints was documented by immunohistochemistry. Conditionally replicating adenovirus, CRAd-EGFP(PS1217L) or CRAd-NTR(PS1217H6), and non-replicating adenovirus CTL102 were used to transduce genes for enhanced green fluorescent protein (EGFP) or nitroreductase (NTR), respectively. The expression of EGFP, NTR, and the toxicity induced by CB1954 activation were evaluated.

Key findings: The in vitro experiments revealed that CAR upregulation was mediated through the TLR4/TRIF/IRF3 pathway in LPS-stimulated inflammatory macrophage RAW264.7 and J774A.1 cells. The inflammatory RAW264.7 cells upregulated CAR expression following LPS stimulation, leading to higher infectability, increased NTR expression, and enhanced sensitization to CB1954. In animal experiments, the induction of CAR expression was observed in the CD68-expressing primary macrophages and in the CD68-expressing macrophages within joints following LPS stimulation.

Significance: In conclusion, we report an enhanced CAR expression in inflammatory macrophages in vitro and in vivo through the immune response elicited by LPS. Thus, the TLR4/TRIF/IRF3 pathway of macrophages, when activated, could facilitate the therapeutic application of adenovirus-mediated VDEPT.

Keywords: Adenoviral vector; Coxsackievirus and adenovirus receptor (CAR); Innate immune response; Macrophages; Toll-like receptor 4 (TLR4).

MeSH terms

  • Adaptor Proteins, Vesicular Transport / metabolism*
  • Adenoviridae / genetics
  • Animals
  • Cell Line
  • Coxsackie and Adenovirus Receptor-Like Membrane Protein / genetics*
  • Genetic Vectors / administration & dosage
  • Immunity, Innate / immunology*
  • Inflammation / genetics
  • Inflammation / immunology
  • Inflammation / pathology*
  • Interferon Regulatory Factor-3 / metabolism
  • Lipopolysaccharides
  • Macrophages / immunology
  • Macrophages / pathology*
  • Male
  • Mice
  • RAW 264.7 Cells
  • Rats
  • Rats, Sprague-Dawley
  • Toll-Like Receptor 4 / metabolism

Substances

  • Adaptor Proteins, Vesicular Transport
  • Coxsackie and Adenovirus Receptor-Like Membrane Protein
  • Interferon Regulatory Factor-3
  • Irf3 protein, mouse
  • Lipopolysaccharides
  • TICAM-1 protein, mouse
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4