Caffeine’s Effect on Macrophage Pro-inflammatory Cytokine Expressions

Background: Caffeine is used primarily for the treatment of apnea of prematurity and has been shown to decrease the rate of bronchopulmonary dysplasia. One of the proposed mechanisms of action of caffeine is that it is a non-specific adenosine receptor antagonist, a pivotal player in regulating macrophage response to inflammation. Macrophages play an important role in healthy innate immune responses to infection, which has been shown to be essential to BPD development in preterm infants. Our study is to determine at what concentration(s) of caffeine will attenuate proinflammatory responses in E coli lipopolysaccharide (LPS)-exposed macrophages. The aim of this study was to test the hypothesis that there is an optimal concentration of caffeine that will inhibit pro-inflammatory cytokine expression in an LPS exposed undifferentiated human monocyte cell model when caffeine is given as both treatment and prophylaxis.

Methods: THP-1 human monocytes were grown in RMPI-1640 medium in a humidified incubator. There were four experimental groups: saline control, caffeine controls, LPS exposed (10ug/mL), and caffeine as treatment/prophylaxis to LPS-exposed monocytes. Caffeine concentrations of 25, 50, 100, and 150uM were studied (clinically correlating with serum concentrations of 5, 10, 20, and 30ug/mL, respectively). Cells were harvested after 2.5 hours. RT-PCR was used to measure pro-inflammatory (TNFα, IL-8, NF-kB) and anti-inflammatory (PPARy) gene expressions. ΔCt values were calculated relative to PPIA (housekeeping gene). Data analyzed using nonparametric T-tests on ΔΔCt values and one-way ANOVA with posthoc Tukey test on ΔCt values. Data shown in the figures are represented by fold change relative to the LPS exposed group, 2^(-ΔΔCt). Significance assigned as p < 0.05.

Results: When the THP-1s were exposed to LPS there was an increase in pro-inflammatory gene expression of TNFα and NF-kB compared to saline controls (p < 0.05). THP-1 cells treated with caffeine alone at 25, 50, 100, 150uM did not alter endogenous TNFα and NF-kB gene expressions. When caffeine was given as treatment to LPS-exposed THP-1s, only caffeine of 100uM showed a significant decrease in TNFα gene expression (p < 0.05, figure 1) and caffeine of 50 and 100uM caused a significant decreased in NF-kB gene expression respectively (p < 0.05, figure 2). When caffeine was given as prophylaxis, there was a significant decrease in both TNFα and NF-kB gene expressions for all concentrations of caffeine (figure 3 and 4).

Conclusion: In this in vitro LPS-stimulated human monocyte model, caffeine concentrations of 50 and 100uM (equivalent to serum levels of 10 and 20ug/mL) had a significant anti-inflammatory effect. This effect is dose-dependent and specific to TNFα and NF-kB when given as treatment. However, prophylactic use of caffeine had a protective effect for monocytes for any of the studied caffeine concentrations. These findings may indicate a benefit of caffeine in BPD pathology beyond neuro-respiratory control.