The formyl peptide receptor 2/3 (Fpr2/3) agonist, Compound 43 (Cpd43), attenuates and promotes the release of pro-inflammatory, and anti-inflammatory cytokines, respectively

E. S. Wickstead, S. J. Getting, S. McArthur. Science & Technology, University of Westminster, London, UNITED KINGDOM.

Introduction

Inflammation contributes to many central nervous system (CNS) disorders (1). Microglia are the first line of immune defence in the brain and spinal cord, but can undergo inappropriate and persistent activation, entering a chronic inflammatory state and leading to extensive neuronal damage (2). Dampening or reversing this state may protect neurons from chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated and a resolving phenotype induced in the immortalised murine microglial cell line, BV-2. Cells activated with LPS were then treated with the novel compound Cpd43 (N-(4-Chlorophenyl)-N-[2,3-dihydro-1-methyl-5-(1-methylethyl)-3-oxo-2-phenyl-1H-pyrazol-4-yl]-urea), a synthetic agonist of a known pro-resolution receptor, Fpr2/3, and levels of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored.

Methods

Cells were seeded onto either 96-well or 12-well plates at a density of 1x105 cells/cm2, and following adhesion were serum starved in Dulbecco's modified Eagle's medium (DMEM) for 24h before treatment. Cells were pre-exposed to LPS (50ng/ml) for 1 h before being treated with Cpd43 (0, 10 or 100nM). Both LPS and Cpd43 were dissolved in DMEM. Samples were collected at 24h and 48h for cytokine analysis. NO release was determined with the use of the Griess reagent. TNFα and IL-10 production was assessed using commercial ELISA kits according to manufacturer's instructions. Data are given as mean±SEM. Statistical analysis was performed using two-way ANOVA with post hoc analysis using Tukey's HSD test. #P < 0.05 vs. Untreated; *P < 0.05 vs. LPS Only.

Results

LPS increased TNFα and NO release at 24h, when compared to untreated cells (Table 1), but did not significantly affect IL-10 at 48h. Cpd43 (10 and 100nM) limited the rise in TNFα and NO, with 100nM Cpd43 stimulating an increase in IL-10 release at 48h compared to untreated cells.

Conclusions

In summary, LPS administration increases BV-2 release of TNFα and NO at 24h. This was attenuated by Cpd43, with 100nM showing reductions of approximately 25% for both. Further, at 48h, Cpd43 (100nM) significantly increases the production of the anti-inflammatory cytokine, IL-10, to levels approximately 4 times greater than untreated cells. These data suggest that Cpd43 may not just be anti-inflammatory, but may have the ability to be immunomodulatory.

References

(1) Streit WJ et al. (2004). J Neuroinflammation 1: 14.

(2) Cunningham C (2013). Glia 61: 71-90.