Bifidobacterium animalis subsp. lactis inl1 cell-free supernatant attenuates the inflammatory response of lps-stimulated macrophages

Abstract

The inflammatory response protects the body against pathogens;however its persistence can lead to inflammatory diseases. Thiswork aimed to explore the effect of the cell-free supernatant (CFS)of the human milk-derived strain Bifidobacterium animalis subsp. lactis INL1 (B. lactis INL1) (transfer agreement UNS-UNL NoREC-1092496-2) in an in vitro model of inflammation, specificallylipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Toevaluate the mitochondrial integrity of these macrophages, theywere pretreated with CFS for 3 hours and then exposed to LPSfor 24 hours. By JC-1 assay we observed that LPS maintains mitochondrial health but it increases due CFS pretreatment followedLPS treatment (p˂0,05), suggesting that CFS from B. lactis INL1 hasbeneficial properties for cell physiology. Next, an in silico analysiswas performed to identify the signaling pathways associated withthe documented effect of the B. lactis bacteria on macrophages. Using Cytoscape software, the gene interaction network was obtained.JUN, which encodes the c-jun transcription factor involved in the inflammatory response, was found to be the core gene of the networkby betweenness and closeness. Functional enrichment showed thatthe genes obtained are associated with the inflammatory response,the LPS response, and inflammatory bowel diseases. In addition,we analyzed gene expression data from RAW264.7 macrophagemicroarrays exposed or not to LPS (GSE21548-GEO). Differentially expressed genes (Log2FC≥1 or ≤-1) showed that the JUN genewas overexpressed in LPS-exposed macrophages (Log2FC=1,435;padj˂0,05). Based on these data, we study c-jun protein levels status by Western blot in our experimental model. The expression ofc-jun increases by LPS action but this effect is attenuated by CFSpretreatment of B. lactis INL1 in these cells (p˂0.05), suggesting thatc-jun pathway could be a potential mediator of the effects of B. lactisINL1 in the inflammatory context.