The JAK1/2 Inhibitor Baricitinib Mitigates the Spike-Induced Inflammatory Response of Immune and Endothelial Cells In Vitro

Abstract

The purpose of this study was to examine the effect of the JAK-STAT inhibitor baricitinib on the inflammatory response of human monocyte-derived macrophages (MDM) and endothelial cells upon exposure to the spike S1 protein from SARS-CoV-2. The effect of the drug has been evaluated on the release of cytokines and chemokines from spike-treated MDM, as well as on the activation of endothelial cells (HUVECs) after exposure to conditioned medium collected from spike-activated MDM. Results obtained indicate that, in MDM, baricitinib prevents the S1-dependent phosphorylation of STAT1 and STAT3, along with the induction of IP-10- and MCP-1 secretion; the release of IL-6 and TNFα is also reduced, while all other mediators tested (IL-1β, IL-8, RANTES, MIP-1α and MIP-1β) are not modified. Baricitinib is, instead, poorly effective on endothelial activation when HUVECs are exposed to supernatants from S1-activated macrophages; the induction of VCAM-1, indeed, is not affected by the drug, while that of ICAM-1 is only poorly inhibited. The drug, however, also exerts protective effects on the endothelium by limiting the expression of pro-inflammatory mediators, specifically IL-6, RANTES and IP-10. No effect of baricitinib has been observed on IL-8 synthesis and, consistently, on neutrophils chemiotaxis. Our in vitro findings reveal that the efficacy of baricitinib is limited, with effects mainly focused on the inhibition of the IL-6-mediated inflammatory loop.

IRF1 Mediates Growth Arrest and the Induction of a Secretory Phenotype in Alveolar Epithelial Cells in Response to Inflammatory Cytokines IFNγ/TNFα

“…Since this transcription factor is a downstream target gene of STAT1 in the JAK/STAT signaling pathway, the molecular mechanisms underneath cytomix-dependent cell growth arrest were investigated by employing the JAK1/2 inhibitor baricitinib ( Figure 5 ). To this concern, we have also recently demonstrated that the exposure of A549 cells to conditioned medium from spike S1-activated human macrophages triggers IRF1 expression through the JAK1/2–STATs axis [ 33 , 34 ]. Results shown in Panel A demonstrated that the exposure to IFNγ + TNFα caused the phosphorylation of STAT1 in both WT and IRF1 KO cells, an effect completely prevented, as expected, by baricitinib.…”

“…In a previous study we demonstrated that the exposure of A549 cells to conditioned medium (CM) obtained from SARS-CoV-2-activated human macrophages caused proliferative arrest [ 33 ]; by analyzing the cytokine content of this conditioned medium, we concluded that the IFNγ–IRF1 axis likely plays a central role in the induction of this cytostatic effect [ 34 ]. Based on these findings, the present study aimed to deepen this issue by assessing the contribution of single and combined inflammatory cytokines IFNγ, TNFα, IL-1β and IL-6 on growth arrest and on the secretory phenotype of A549 alveolar epithelial cells; in addition, we clarified the role of IRF1 by taking advantage of A549 cells knockout for this transcription factor.…”

Growth Arrest of Alveolar Cells in Response to Cytokines from Spike S1-Activated Macrophages: Role of IFN-γ

“…Recently, we have shown that the exposure of alveolar A549 cells to conditioned medium (CM) from spike S1-activated macrophages causes the activation of the epithelial cells; the reason is the massive presence of cytokines released from treated macrophages that, in turn, provokes a further release of inflammatory mediators from epithelial cells [ 18 , 24 ]. Given the relevance of the epithelial alveolar damage in COVID-19 disease, we examine here whether this treatment causes, in addition, signs of cell injury.…”

“…The JAK/STAT pathway is currently recognized as a signalling mechanism central to the response and secretion of cytokines and chemokines in COVID-19 [ 24 , 35 , 36 ]; thus, it is now generally accepted that targeting JAKs represents a valid therapeutic strategy for the treatment of the disease [ 37 ]. Among the JAK-STAT inhibitors, baricitinib proved effective in preventing the progression to a severe form of COVID-19 with reduced hospitalization and mortality [ 38 , 39 , 40 , 41 , 42 ].…”

“…Among the JAK-STAT inhibitors, baricitinib proved effective in preventing the progression to a severe form of COVID-19 with reduced hospitalization and mortality [ 38 , 39 , 40 , 41 , 42 ]. We have recently described that baricitinib in vitro limits the secretion of different cytokines and chemokines by macrophages and endothelial cells [ 24 ]; in particular, the release of IFN-γ-Induced Protein 10 (IP10), which depends upon the activity of IRF-1 [ 43 , 44 ], was completely abolished. Here we demonstrate that baricitinib is able to decrease the synthesis of IRF-1 in A549 cells and to eliminate almost completely the expression of its downstream targets TRIM22, UbD and PSMB8.…”