Molecular Mechanism of the Anti-Inflammatory Action of Heparin

Abstract

Our objective is to reveal the molecular mechanism of the anti-inflammatory action of low-molecular-weight heparin (LMWH) based on its influence on the activity of two key cytokines, IFNγ and IL-6. The mechanism of heparin binding to IFNγ and IL-6 and the resulting inhibition of their activity were studied by means of extensive molecular-dynamics simulations. The effect of LMWH on IFNγ signalling inside stimulated WISH cells was investigated by measuring its antiproliferative activity and the translocation of phosphorylated STAT1 in the nucleus. We found that LMWH binds with high affinity to IFNγ and is able to fully inhibit the interaction with its cellular receptor. It also influences the biological activity of IL-6 by binding to either IL-6 or IL-6/IL-6Rα, thus preventing the formation of the IL-6/IL-6Rα/gp130 signalling complex. These findings shed light on the molecular mechanism of the anti-inflammatory action of LMWH and underpin its ability to influence favourably conditions characterised by overexpression of these two cytokines. Such conditions are not only associated with autoimmune diseases, but also with inflammatory processes, in particular with COVID-19. Our results put forward heparin as a promising means for the prevention and suppression of severe CRS and encourage further investigations on its applicability as an anti-inflammatory agent.

Keywords: COVID-19; IFNγ; IL-6; cytokine storm; inflammation; low-molecular-weight heparin (LMWH); molecular dynamics; molecular modelling; signalling pathway.

Figure 1

Initial (A) and final (B–D) configurations of the three IFNγ–LMWH hexasaccharide binding simulations. The protein is shown in gray ribbon, and the hexasaccharide chains are coloured as follows: dp6_1—green, dp6_2 –orange, dp6_3—blue, and dp6_4—red.

Figure 2

Pair contacts between IFNγ and the hexasaccharides. Number of contacts (averaged over the three independent simulations) as a function of the simulation time between any pair of atoms of IFNγ and any of the four hexasaccharides within 0.6 nm, with the standard deviation band (in grey) and number-of-contact frequencies.

Figure 3

Contact map of the IFNγ/hexasaccharide complex. Contacts within 0.6 nm formed between each of the four hexasaccharides and the two monomers of IFNγ. Contact occupancy within the last 250 ns of the three simulations (upper, middle, and lower panels, resp.) ranges from 0 (cyan) to 1 (purple).

Figure 4

Inhibitory effect of LMWH on the production of IDO induced by 100 IU/mL recombinant IFNγ. After subtraction of the blank value, the absorbance obtained from cells treated with a mixture of IFNγ and LMWH is related to that obtained from cells treated with IFNγ only, taken as 100%. The bars represent the statistical errors.

Figure 5

Translocation of the phosphorylated STAT1 into the cell nucleus. Translocation of the phosphorylated STAT1 into the cell nucleus. (A) WISH cells were treated with LMWH or IFNγ only, or with IFNγ after pretreatment with LMWH at the indicated concentrations, and fixed and stained with an antibody against pSTAT1 (Tyr701). The nuclei were counterstained with DAPI. Representative images are shown; scale bar, 10 µm; (B) Distribution of nuclear intensity of pSTAT1 (Tyr701) of individual nuclei from (A). Data are from 3 independent experiments (n > 200 per condition per experiment); NS—non-significant (two-tailed unpaired Student’s t-test).

Figure 6

The IL-6/LMWH complex. (a) IL-6 in yellow, heparin in red; (b) IL-6 molecule presented with its SAS (hydrophobic regions in green, positively charged regions in blue, and negatively charged regions in red), heparin coloured by atom type.

Figure 7

Average SASA values for the most affected through LMWH binding residues (SASA change exceeds the standard deviation). IL6 (top panel); IL-6/IL-6Rα complex (bottom panel).

Figure 8

The IL-6/IL-6Rα/LMWH/2Mg²⁺ complex. (a) IL-6 in yellow, heparin in red, IL-6Rα in green; Mg²⁺ ions presented as orange spheres; (b) The same complex, with gp130 receptor (in blue) superimposed.