Efficacy of caerulomycin A in modulating macrophage polarization and cytokine response in a murine model of lipopolysaccharide-induced sepsis

Abstract

Background: Sepsis is characterized by an excessive immune response. Modulation of the immune response, particularly macrophage polarization, may provide therapeutic benefit. The effects of Caerulomycin A (caeA), a known STAT1 phosphorylation inhibitor, on macrophage polarization and inflammatory markers were explored using a lipopolysaccharide (LPS)-induced sepsis mouse model.

Methods: A sepsis model was established in C57BL/6 mice induced by intraperitoneal injection of LPS, and the survival rate of mice was observed after treatment with different doses of caeA to determine the optimal therapeutic dose. For in-vitro assays using the RAW264.7 macrophage cell line, the concentration of caeA that was non-toxic to cell survival was screened using the MTT assay, followed by the analyses by qRT-PCR, ELISA, Western blot and flow cytometry for M1/M2 type macrophage markers (CD86, NOS2, CD206, ARG1) and inflammatory factors (IL-1β, IL-6, TNF-α, IL-4, and IL-10) expression. In addition, the phosphorylation levels of STAT1 and STAT6 in the JAK-STAT signaling pathway were detected.

Results: The results of in-vivo experiments showed that caeA treatment (20 mg/kg) significantly increased the survival of LPS-induced septic mice and decreased the expression of M1-type macrophage markers (CD86 and NOS2) and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) while increasing the expression of M2-type markers (CD206 and ARG1) and anti-inflammatory cytokines (IL-4 and IL-10) expression. In in-vitro experiments, 20 μM caeA effectively inhibited LPS-induced polarization of M1-type macrophages without affecting the activity of RAW264.7 cells, and caeA significantly inhibited the phosphorylation of STAT1 yet enhanced the phosphorylation level of STAT6, as detected by Western blot.

Conclusions: CaeA effectively modulates macrophage polarization and attenuates the inflammatory response in septic mice, possibly by affecting the JAK-STAT signaling pathway. These findings support further exploration of the potential of caeA as a therapeutic agent for sepsis.

Keywords: Caerulomycin A; Cytokine modulation; Immunomodulation; Macrophage polarization; Sepsis therapy.

Fig. 1

Inflammatory indicators and expression levels of anti-inflammatory factors in Sepsis. A Survival curves of sepsis mice and normal mice. B Relative CD86 mRNA expression levels in sepsis and normal mice. C Relative mRNA expression levels of NOS2 in sepsis mice and normal mice. D The level of IL-1β in sepsis mice and normal mice. E The levels of IL-6 in sepsis mice and normal mice. F The levels of TNF-α in sepsis mice and normal mice. G The level of IL-4 protein in sepsis mice and normal mice. H The level of IL-10 protein in sepsis mice and normal mice. Con refers to C57BL/6 mice untreated for use as controls, and Sepsis group refers to mice induced into a sepsis model by intraperitoneal injection of 10 mg/kg LPS. All data of three independent trials were expressed as mean ± standard deviation

Fig. 2

Macrophages undergo M1 polarization in a mouse model of sepsis. A Cell types in the peripheral blood of sepsis mice. B Expression level of CD86 mRNA in macrophages. C Expression level of NOS2 mRNA in macrophages. D Expression level of CD206 mRNA in macrophages. E Expression level of ARG1 mRNA in macrophages. Con refers to C57BL/6 mice untreated for use as controls, and Sepsis group refers to mice induced into a sepsis model by intraperitoneal injection of 10 mg/kg LPS. All data of three independent trials were expressed as mean ± standard deviation

Fig. 3

In-vitro cellular assay confirming that macrophages in sepsis tend to be M1 polarized. A Flowchart of LPS-stimulated mouse monocyte macrophage leukemia cell line RAW264.7. B CD86 mRNA expression level in RAW264.7 cells. C Expression level of CD206 mRNA in RAW264.7 cells. D The level of IL-1β in RAW264.7 cells. E The level of TNF-α in RAW264.7 cells. F Expression level of IL-4 protein in RAW264.7 cells. G The level of IL-10 in RAW264.7 cells. H Expression level of JAK-STAT signaling pathway proteins in RAW264.7 cells by WB. I Phosphorylation level of STAT1 in RAW264.7 cells. J Phosphorylation level of STAT6 in RAW264.7 cells. Con refers to untreated RAW264.7 cells to be used as a control group, and LPS group refers to RAW264.7 cells treated in 10 ng/mL of LPS for 6 h. All data of three independent trials were expressed as mean ± standard deviation

Fig. 4

caeA can inhibit macrophage activity. A CCK-8 assay confirms that caeA can inhibit macrophage activity. B Optimal therapeutic concentration of caeA was determined

Fig. 5

caeA inhibits pro-inflammatory cytokine expression in sepsis. A Experimental flowchart of cells in control, LPS-stimulated, and LPS + caeA-treated groups. B Expression levels of CD86 mRNA in the three treatment groups.C Expression levels of CD206 mRNA in the three treatment groups. D The levels of IL-1β in the three treatment groups. E The level of TNF-α in the three treatment groups. F The level of IL-4 in the three treatment groups. G The level of IL-10 in the three treatment groups. Con refers to untreated RAW264.7 cells to be used as a control group, LPS group refers to RAW264.7 cells treated in 10 ng/mL of LPS for 6 h, and LPS + caeA group refers to RAW264.7 cells treated in 10 ng/mL of LPS and 20 μM of caeA for 6 h. All data of three independent trials were expressed as mean ± standard deviation

Fig. 6

Caerulomycin A inhibits STAT1 phosphorylation and thus macrophage M1 polarization. A Levels of protein phosphorylation in the JAK-STAT signaling pathway in control (LPS-treated) and caeA-treated (LPS + caeA) groups. B Detection of STAT1 phosphorylation level in control (LPS-treated) and caeA-treated groups (LPS + caeA). C Detection of STAT6 phosphorylation level in control (LPS-treated) and caeA-treated groups (LPS + caeA). LPS group refers to RAW264.7 cells treated in 10 ng/mL of LPS for 6 h, and LPS + caeA group refers to RAW264.7 cells treated in 10 ng/mL of LPS and 20 μM of caeA for 6 h. All data of three independent trials were expressed as mean ± standard deviation

Fig. 7

Mouse test further confirming that caeA inhibits macrophage M1 polarization. A Survival curve of sepsis mice after caeA treatment. B CD86 mRNA expression level in sepsis mice after caeA treatment.C Expression level of NOS2 mRNA in sepsis mice after caeA treatment. D Expression level of CD206 mRNA in sepsis mice after caeA treatment. E Expression level of ARG1 mRNA in sepsis mice after caeA treatment. F Expression level of IL-1β protein in sepsis mice after caeA treatment. G Expression level of IL-6 protein in sepsis mice after caeA treatment. H Expression level of TNF-α protein in sepsis mice after caeA treatment. I Expression level of IL-4 protein in sepsis mice after caeA treatment. J Expression level of IL-10 protein in sepsis mice after caeA treatment. Sepsis group refers to mice induced into a sepsis model by intraperitoneal injection of 10 mg/kg LPS, and Sepsis + caeA group refers to LPS induction followed by intraperitoneal injection of Caerulomycin A at 20 mg/kg. All data of three independent trials were expressed as mean ± standard deviation