Supplementary Material for: Interactions of probiotics with the immune cells of patients with COVID-19 pneumonia

Introduction: In severe COVID-19, excessive cytokine release may be driven by SARS-CoV-2. We investigated the modulatory effect of probiotics which may have direct interaction with the immune gut cells. Methods: Fifty-five patients with confirmed COVID-19 infection were classified by the presence of acute respiratory distress syndrome (ARDS) or not. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with lipopolysaccharide (LPS), a preparation of four probiotics (LactoLevure® containing Saccharomyces boulardii, Bifidobacterium lactis BB-12, Lactobacillus acidophilus LA-5 and L.plantarum) and/or recombinant human interferon gamma (rhIFNγ) and Tocilizumab. Cytokine concentrations were measured in cell supernatants. Gene expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) was performed by quantitative real-time polymerase chain reaction (RT-PCR). Results were associated with the level of viremia. Results: Probiotics decreased tumor necrosis factor alpha (TNFα) production by the PBMCs of both ARDS and non-ARDS patients. LPS-stimulated the production of interleukin (IL)-1β, IL-6 in non-ARDS patients. IL-6 production was maintained in the presence of probiotics. rhIFNγ enhanced LPS-stimulated cytokine production by PBMCs; this was not the case when PBMCs were stimulated by probiotics. Probiotics upregulated TLR2 and LPS downregulated TLR4 in the PBMCs of patients with ARDS. PBMCs from patients with viremia had more cytokine production by probiotic stimulation. Conclusion: Probiotics interact with the immune system of COVID-19 patients by modulating the production of TNFα, IL-1β and IL-6 in an IFNγ-independent mechanism.