Abstract 1263 Probiotic blend of L.plantarum and P.acidilactici (AB21) stimulates type-I interferon response in phagocytes with the participation of the IRF7 transcription factor, possibly involving both MyD88-dependent and independent mechanisms

OBJECTIVE: Gut microbiota has been shown to influence antiviral respiratory immunity via type-I interferon (IFN) responses in vivo, and some lactic acid bacteria have been shown to stimulate such responses in phagocytic cells in vitro. A probiotic formula containing Lactiplantibacillus plantarum strains KABP022, KABP023 and KABP033 plus Pediococcus acidilactici KABP021 (AB21) was recently shown to ameliorate fever and cough in a randomized, placebo-controlled trial in 300 Covid19 patients. We sought to determine if this formula induces IFN responses in phagocytic cells. METHODS: THP-1 monocytes were differentiated into macrophages with 100 ng/mL phorbol myristate acetate (PMA) in complete RPMI medium for 48h. Subsequently, macrophages were co-incubated in RPMI medium without Fetal Bovine Serum, either with the AB21 probiotic formula or the classical probiotic Lacticaseibacillus rhamnosus ATCC53103 (LrGG), at a MOI of 1:25 for 2h, then left for an additional 2h or 14h with 10 μg/mL gentamycin (to avoid bacterial over-proliferation). Dependence on MyD88 signaling was assessed by using an inhibitor (T6167923) at 10 μM (i.e. 4 times the IC50). RNA was extracted with Aurum Total RNA MiniKit, converted into cDNA with IScript Advanced cDNA Kit and type-I IFN pathway genes were analyzed by qPCR using SYBR Green SuperMix, with GAPDH as housekeeping gene. Poly I:C (TLR3 agonist) was used as positive control for the stimulation of IFN pathway gene expression. Changes in gene expression were assessed by ΔΔCt method. Significance of changes reaching >2-fold was assessed by ANOVA with Dunnet post-test on log-transformed data. RESULTS: After 4h of co-incubation, probiotics did not produce any >2-fold changes in the IFN pathway genes studied, while the positive control did. Conversely, >2-fold increases were observed after 16h of co-incubation with probiotics in most genes (high overexpression: ISG56 and IFI44L; moderate overexpression: MX1, IFNβ and IRF7), the effects being more pronounced with AB21 (p<0.05 for all five genes) than LrGG (p<0.05 for ISG56 and IFI44L only). No changes were seen on IRF3 expression under any conditions. Pharmacological inhibition of MyD88 partially modified the effects of probiotics: expression changes in ISG56 and MX1 were abrogated, IFI44L was mitigated in AB21 but boosted in LrGG, IFNβ was unchanged in LrGG and boosted in AB21, and IRF7 was unchanged in both. As expected, inhibition of MyD88 did not alter the effect of the positive control. CONCLUSIONS: The studied probiotics stimulate the expression of type-I IFN pathway genes in phagocytic cells after long (16h) but not short (4h) co-incubation, and the IRF7 transcription factor is implicated in this signaling pathway. The overexpression induced by the AB21 strain blend is more pronounced than by the LrGG strain, and possibly involves both MyD88-dependent and MyD88-independent pathways. The extent of involvement of MyD88-dependent and independent pathways may differ between probiotic strains. Of note, at the 16h timepoint, overexpression is higher in late genes involved in negative feedback (ISG56 and IFI44L), suggesting this response may be reaching a late stage. Therefore, future studies should also include intermediate timepoints and assess MyD88-independent, IRF7-dependent pathways such as MAVS or STING. This study was funded by AB-BIOTICS SA (Kaneka Corp.)