Fever-Range Hyperthermia Promotes Macrophage Polarization towards Regulatory Phenotype M2b

Abstract

Fever-range hyperthermia (FRH) is utilized in chronic disease treatment and serves as a model for fever's thermal component investigation. Macrophages, highly susceptible to heat, play a pivotal role in various functions determined by their polarization state. However, it is not well recognized whether this process can be modulated by FRH. To address this, we used two different macrophage cell lines that were treated with FRH. Next, to define macrophage phenotype, we examined their functional surface markers CD80 and CD163, intracellular markers such as inducible nitric oxide synthase (iNOS), arginase-1 (Arg-1), and the expression of interleukin-10 (IL-10) and tumor necrosis factor α (TNF-α). Additionally, in FRH-treated cells, we analyzed an expression of Toll-like receptor 4 (TLR-4) and its role in macrophage polarization. We also checked whether FRH can switch the polarization of macrophages in pro-inflammatory condition triggered by lipopolysaccharide (LPS). FRH induced M2-like polarization, evident in increased CD163, IL-10, and Arg-1 expression. Notably, elevated COX-2, TNF-α, and TLR-4 indicated potential pro-inflammatory properties, suggesting polarization towards the M2b phenotype. Additionally, FRH shifted lipopolysaccharide (LPS)-induced M1 polarization to an M2-like phenotype, reducing antimicrobial molecules (ROS and NO). In summary, FRH emerged as a modulator favoring M2-like macrophage polarization, even under pro-inflammatory conditions, showcasing its potential therapeutic relevance.

Keywords: fever; fever-range hyperthermia; inflammation; macrophage polarization; macrophages.

Figure 1

FRH-induced macrophage polarization. RAW264.7 cells (A–C) and J774A.1 cells (D–F) were cultured at 37 °C or 39 °C for 24 h. Shadowed bars indicate cells cultured at 39 °C. The expression of surface markers CD80 and CD163 was assessed by flow cytometry. Anti-CD163 antibodies were conjugated with APC, whereas anti-CD80 antibodies were conjugated with FITC. Bars represent the ratio of M1/M2 surface markers (B,E). Asterisks indicate the statistical significance (ns > 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

LPS-induced macrophage polarization changes under hyperthermic conditions. RAW264.7 cells (A–C) and J774A.1 cells (D–F) were treated with LPS and cultured at 37 °C or 39 °C for 24 h. Shadowed bars indicate cells cultured at 39 °C. The expression of surface markers CD80 and CD163 was assessed by flow cytometry. Anti-CD163 antibodies were conjugated with APC, whereas anti-CD80 antibodies were conjugated with FITC. Bars represent the ratio of M1/M2 surface markers (B,E). Asterisks indicate statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 3

mRNA expression of IL-10 (A,C) and TNF-α (B,D) in RAW264.7 cells (A,B) and J774A.1 cells (C,D) pre-treated with TAK-242 for 1 h at 37 °C and further treated with LPS at the concentration of 100 ng/mL and cultured at 37 °C and 39 °C for 4 h. Shadowed bars indicate cells cultured at 39 °C. mRNA expression was determined by quantitative real-time PCR. Data represent the mean and standard error of the mean (SEM) obtained from three independent experiments. Asterisks indicate the statistical significance (ns > 0.05; * p < 0.05; *** p < 0.001).

Figure 4

Western blot analysis of cyclooxygenase-2 (COX-2) and Toll-like receptor 4 (TLR-4) expression in RAW264.7 (A,B) and J774A.1 (C,D) cells simultaneously treated with LPS and FRH for 2 h. Actin was used as a protein loading control. Data represent the mean and standard error of the mean (SEM) obtained from three independent experiments. Asterisks indicate the statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Oxidative status of RAW264.7 (A,B) and J774A.1 (C,D) cells in response to simultaneous treatment with FRH and LPS for 24 h. The effect was measured as NO concentration (colorimetric) and the relative level of ROS (fluorescent) assessed by flow cytometry. Data represent the mean and standard error of the mean (SEM) obtained from three independent experiments. Asterisks indicate the statistical significance (ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

Macrophage polarization surface markers after TLR-4 inhibition. RAW264.7 cells (A–C) and J774.A cells (D–F) were pre-treated with 0.1 µM TAK-242 for 1 h at 37 °C, and further cultured at 37 °C and 39 °C for 24 h. Shadowed bars indicate cells cultured at 39 °C. The expression of surface markers CD80 and CD163 was assessed by flow cytometry. Anti-CD163 antibodies were conjugated with APC, whereas anti-CD80 antibodies were conjugated with FITC. Bars represent the ratio of M1/M2 surface markers (B,E). Asterisks indicate statistical significance (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 7

Western blot analysis of inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1) in RAW264.7 cells (A,B) and J774.A cells (C,D) pre-treated with TAK-242 for 1 h at 37 °C, and further cultured at 37 °C and 39 °C for 24 h. Actin was used as a protein loading control. Data represent the mean and standard error of the mean (SEM) obtained from three independent experiments. Asterisks indicate the statistical significance (ns p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001).