Effects of Fasting on THP1 Macrophage Metabolism and Inflammatory Profile

Abstract

Fasting can affect the body's inflammatory response, and this has been linked to potential health benefits, including improvements for people with rheumatic diseases. In this work, we evaluated, in vitro, how changes in nutrient availability alter the inflammatory response of macrophages. Macrophage-differentiated THP1 cells were cultured, deprived of FCS or subjected to cycles of FCS deprivation and restoration to mimic intermittent fasting. Changes in the macrophage phenotype, the cells' response to inflammatory stimuli and the level of mitochondrial alteration were assessed. The results indicate that while periods of serum starvation are associated with a decrease in IL1β and TNFα expression, consistent with an anti-inflammatory response, intermittent serum starvation cycles promote a pro-inflammatory phenotype. Rapid changes in reducing capacity and mitochondrial response were also observed. Of note, while some changes, such as the production of oxygen free radicals, were reversed with refeeding, others, such as a decrease in reducing capacity, were maintained and even increased. This study shows that different fasting protocols can have diverging effects and highlights that time-limited nutrient changes can significantly affect macrophage functions in cell cultures. These findings help elucidate some of the mechanisms by which specific fasting dietary interventions could help control inflammatory diseases.

Keywords: fasting; inflammation; intermittent fasting; macrophages; rheumatic diseases.

Figure 1

SD modulates the macrophages’ profiles and response to LPS stimulation. (A) mRNA expression of IL-1β in macrophages cultured for 3 h at different FCS concentrations and the effect of LPS (100 ng/mL) treatment, as measured by qPCR (n = 4). (B) Expression of IL-1β (n = 3), TNFα (n = 4), MRC1 and ARG1 (n = 3) in macrophages under different conditions and ratios: IL-1β/MR and TNFα/ARG1 (n = 3). (C) Gene expression of IL-1β in macrophages stimulated with LPS (100 ng/mL) with different periods of nutrient availability (n = 4). Data are expressed as mean ± SEM. * p < 0.05 vs. control (10% FCS) group, + p < 0.05 vs. SD group. ANOVA with Tukey’s post-test was used to obtain p-values. ISD1C, intermittent SD, 1 cycle; ISD3C, intermittent SD, 3 cycles.

Figure 2

SD effects on NF-κB activation. Immunofluorescence of p65 subunit of NF-κB in macrophages under the FCS conditions described in Figure 1. In control and SD groups, p65 subunit of NFkB remains in cytoplasmatic localization. Nuclear translocation was mainly observed in ISD groups (n = 3).

Figure 3

SD affects the metabolic activity of macrophages independently of cell viability. (A) MTS assay (n = 4); (B) total cell number; and (C) live cell number of macrophages undergoing different SD and refeeding time periods (n = 3). (D) Macrophages grown under different SD and refeeding time periods; pictures were taken with a microscope at the end of the treatments. Scale bars represent 200 μm (n = 3). (E) Kinetics of MTS of macrophages undergoing different SD and refeeding cycles; measurements were performed every 5 min for an hour after each change of media. Data are expressed as mean ± SEM. * p < 0.05 vs. control group (n = 3). ANOVA with Tukey’s post-test was used to obtain p-values. SD, serum deprivation; R, refeeding.

Figure 4

SD and ISD alter cell metabolism caused by regulating mitochondrial dynamics. NADH and NAD⁺ concentrations and NAD⁺/NADH ratio (n = 2), phosphorylated AMPK (n = 2), TMRE (n = 3) and ROS (n = 2) under the different SD protocols. Data are expressed as mean ± SEM. * p < 0.05 vs. control group; + p < 0.05 vs. fasting group. ISD1C, intermittent SD, 1 cycle; ISD3C, intermittent SD, 3 cycles; NAD, nicotinamide adenine dinucleotide; AMPK, 5′ adenosine monophosphate-activated protein kinase; TMRE, tetramethylrhodamine, ethyl ester; ROS, reactive oxygen species.