Previous reports have shown that Gal-3BP induces IL-6 expression and secretion in multiple different cell types12,13. To evaluate whether induction of IL-6 also occurs in cells permissive to SARS-CoV-2 infection, human colon adenocarcinoma Caco-2 cells were cultured with different concentrations of D2 and the levels of IL-6 in the cellular supernatant was collected for IL-6 quantification. Treatment of the cells with D2 (5, 10 or 20 µg/mL) dose-dependently increased the level of IL-6 at 24 hours (p = 0.0435 and 48 hours (p = 0.0020) compared to untreated cells (Fig. 1A). The stimulatory effect of D2 was also observed when IL-6 expression was analysed by real-time PCR (p = 0.0254; Fig. 1B).
IL-6 expression induced by Gal-3BP requires β-galactoside-mediated interaction between Gal-3BP and Gal-317. Then, we analyzed IL-6 secretion in the presence of lactose (Galβ1-4glucose) in Caco-2 cells and two additional SARS-CoV-2 susceptible cell lines derived from human lung adenocarcinoma, A549 and HCC827. As expected, treatment of the cells with 20 µg/ml of D2 for 48 hours elicited a significant increase of IL-6 in the supernatant of all three cell lines compared to untreated controls (Fig. 1C). On the contrary, treatment with D2 in the presence of lactose (25 mM) leads to a significant decrease of IL-6 secretion compared to cells treated with D2 alone (p = 0.0222 for Caco-2 cells; p = 0.0260 for A549 cells; p = 0.0008 for HCC827 cells; Fig. 1C). The extent of inhibitory effect of lactose was proportional to the levels of Gal-3 expressed by the cells, being greater in HCC827 than A549 and Caco-2 cells (Fig. 1D),
We later verified whether the ability of D2 to stimulate IL-6 secretion could be blocked by an anti-Gal-3BP monoclonal antibody (1959). To this end, Caco-2, HCC827 and A549 cells were pre-treated with 40 µg/ml of 1959 for 20 min before adding 10 µg/ml of D2 and continuing incubation for 48 hours. As shown in Fig. 1D, the augmentation of IL-6 secretion upon D2 stimulation was significantly reduced by treatment with 1959 (p = 0.0222 for Caco-2 cells; p = 0.0026 for A549 cells; p = 0.0488 for HCC827 cells; Fig. 1E).
Several studies revealed increased levels of Gal-3BP in the plasma of patients with COVID-1918–20. Consistently, these studies showed that Gal-3BP was significantly overexpressed in all symptomatic COVID-19 cohorts, with highest levels associated with severe disease. Thus, this increased production of Gal-3BP may induce a higher activation of its endogenous ligand Gal-3, promoting the release of inflammatory cytokines, which may contribute or even exacerbate pathologic proinflammatory signalling cascades in the lung and other organs21. Indeed, Gal-3 was recently recognized as a potential prognostic biomarker of severe COVID-19 and its inhibition has been suggested as a potential therapeutic approach to attenuate the hyperinflammation observed in these patients22,23.
In conclusion, we showed that D2, a highly glycosylated, lectin binding fragment of Gal-3BP stimulated secretion of IL-6 in SARS-CoV-2 susceptible epithelial cell lines in a Gal-3 dependent manner. Further, we showed that the stimulatory effect could be partially reverted by the anti-Gal-3BP monoclonal antibody 1959.
Overall, these findings show that targeting Gal-3BP-Gal-3 interaction with a specific antibody may represent a potential approach to reduce massive inflammation and organ damage in severe COVID-19 patients.