Down-regulation of KLF2 in lung fibroblasts is linked with COVID-19 immunofibrosis and restored by combined inhibition of NETs, JAK-1/2 and IL-6 signaling.

Kruppel-like factor 2 (KLF2) has been linked with fibrosis and neutrophil-associated thromboinflammation; however, its role in COVID-19 remains elusive. We investigated the effect of disease microenvironment on the fibrotic potential of human lung fibroblasts (LFs) and its association with KLF2 expression. LFs stimulated with plasma from severe COVID-19 patients down-regulated KLF2 expression at mRNA/protein and functional level acquiring a pre-fibrotic phenotype, as indicated by increased CCN2/collagen levels. Pre-incubation with the COMBI-treatment-agents (DNase I and JAKs/IL-6 inhibitors baricitinib/tocilizumab) restored KLF2 levels of LFs to normal abolishing their fibrotic activity. LFs stimulated with plasma from COMBI-treated patients at day-7 expressed lower CCN2 and higher KLF2 levels, compared to plasma prior-to-treatment, an effect not observed in standard-of-care treatment. In line with this, COMBI-treated patients had better outcome than standard-of-care group. These data link fibroblast KLF2 with NETosis and JAK/IL-6 signaling, suggesting the potential of combined therapeutic strategies in immunofibrotic diseases, such as COVID-19.

Combined administration of inhaled DNase, baricitinib and tocilizumab as rescue treatment in COVID-19 patients with severe respiratory failure.

Aiming to reduce mortality in COVID-19 with severe respiratory failure we administered a combined rescue treatment (COMBI) on top of standard-of-care (SOC: dexamethasone/heparin) consisted of inhaled DNase to dissolve thrombogenic neutrophil extracellular traps, plus agents against cytokine-mediated hyperinflammation, namely anti-IL-6-receptor tocilizumab and JAK1/2 inhibitor baricitinib. Patients with PaO2/FiO2 < 100 mmHg were analysed. COMBI group (n = 22) was compared with similar groups that had received SOC alone (n = 26) or SOC plus monotherapy with either IL-1-receptor antagonist anakinra (n = 19) or tocilizumab (n = 11). COMBI was significantly associated with lower in-hospital mortality and intubation rate, shorter duration of hospitalization, and prolonged overall survival after a median follow-up of 110 days. In vitro, COVID-19 plasma induced tissue factor/thrombin pathway in primary lung fibroblasts. This effect was inhibited by the immunomodulatory agents of COMBI providing a mechanistic explanation for the clinical observations. These results support the conduct of randomized trials using combined immunomodulation in COVID-19 to target multiple interconnected pathways of immunothrombosis.