A Lacticaseibacillus rhamnosus secretome induces immunoregulatory transcriptional, functional and immunometabolic signatures in human THP-1 monocytes

Abstract

Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX₃CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.