The fungal-derived compound AM3 modulates pro-inflammatory cytokine production and skews the differentiation of human monocytes

Abstract

The proper functioning of the immune system depends on an appropriate balance between pro-inflammation and anti-inflammation. When the balance is disrupted and the system is excessively biased towards inflammation, immune responses cannot return within the normal range, which favors the onset of diseases of autoimmune or inflammatory nature. In this scenario, it is fundamental to find new compounds that can help restore this balance and contribute to the normal functioning of the immune system in humans. Here, we show the properties of a fungal compound with a strong safety profile in humans, AM3, as an immunomodulatory molecule to decrease excessive cytokine production in human cells. Our results present that AM3 treatment of human peripheral blood mononuclear cells and monocytes decreased their pro-inflammatory cytokine secretion following the challenge with bacterial lipopolysaccharide. Additionally, AM3 skewed the differentiation profile of human monocytes to macrophages towards a non-inflammatory phenotype without inducing tolerance, meaning these cells kept their capacity to respond to different stimuli. These effects were similar in young and elderly individuals. Thus, the fungal compound, AM3 may help reduce excessive immune activation in inflammatory conditions and keep the immune responses within a normal homeostatic range, regardless of the age of the individual.

Keywords: AM3; cytokines; disease; homeostasis; immune response; inflammation; macrophages; monocytes.

Figure 1

AM3 treatment limits LPS-driven cytokine response in human PBMCs (A) Experimental design: adherent PBMCs were stimulated with primary stimuli and supernatants were collected for cytokine detection at 24h, then cells were washed with control medium to remove any residue of the primary stimulation before secondary stimuli were added to the cultures following which the supernatants collected after an additional 24h (48h in total). (B) Cytokine production from PBMCs at 24 hours and at 48 hours after stimulation with medium control (RPMI), AM3 and LPS, eventually combined as by (A) in a number of healthy donors (n=18). Friedman test with Dunn’s multiple comparison was applied to compare experimental groups, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Bars without pattern: cytokine levels measured 24 hours after first stimulation, Patterned bars: cytokine levels measured 48 hours after first stimulation. Values for the groups RPMI (1st) + AM3 (2nd), RPMI (1st) + LPS (2nd), AM3 (1st) + RPMI (2nd), and LPS (1st) + RPMI (2nd) are presented in Figure S2B .

Figure 2

AM3 is similarly effective in cells from both young and elderly individuals. Cytokine production measured in supernatants from PBMCs after 24 hours and after 48 hours following primary stimulation (RPMI, AM3 and LPS) and combination treatments, in cultures derived from young (n=9, pink data points) and elderly (n=9, purple data points) healthy donors. Mann-Whitney test used to compare elderly and young individuals. *p<0.05, **p<0.01. The gray line depicts the median whereas colored lines represent the borders of interquartile range.

Figure 3

AM3 treatment of human monocytes skews the differentiation process towards an anti-inflammatory phenotype (A) Microscope images of cells at day 0 and day 6 of the differentiation process, magnification: 20X, scale bar: 100 µM. The areas highlighted in a black rectangle show an area of the same image with a larger magnification to better appreciate the cell morphology. (B) Flow cytometry analysis of macrophage markers at day 6 after exposure to AM3, M-CSF or GM-CSF. (C) Comparisons of MFIs by marker (CD68, CD80, CD86, and CD163) are depicted on the charts. n=9, Friedman test with Dunn’s multiple comparisons. *p<0.05, **p<0.01, ***p<0.001. For (B) and (C) all cells were gated as CD45+ CD206+ live cells.

Figure 4

AM3 treatment does not induce innate immune memory in human monocyte. (A) Experimental design: First 48 hours were conducted as depicted in Figure 1A . Afterwards, adherent cells were left for rest in the base medium supplemented with 10% Human pooled serum for 5 days followed by LPS restimulation. After 24 hours of LPS stimulaion, the supernatants were collected for cytokine measurement. (B) Microscope images at day 6 after the first stimulation, magnification:10x, scale bar: 200 µM. The areas highlighted with a black rectangle show an area of the same image with a larger magnification to better appreciate the cell morphology. (C) Cytokine production after LPS restimulation at day 7 in the innate immune memory model, n=8, Wilcoxon matched-paired test comparing each condition to RPMI. (D) Cytokine production after LPS restimulation at day 7 in the tolerance model. n=18, Friedman test with Dunn’s multiple comparison. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.