Harnessing the microbiome to regulate systemic innate immunity I
Mechanisms through which the microbiome communicates with the systemic immune system remain unclear. We have identified a family of microbiome Bacteroidota-derived lipopeptides – the serine-glycine (S/G) lipids, and specifically L654 – that are TLR2 ligands, access the systemic circulation, and potentially link the microbiome and systemic innate immunity. We have previously postulated that L654 and the S/G lipids regulate systemic innate immunity by entering the systemic circulation and mediating weak TLR2 interactions that maintain “normal” levels of innate immune signaling feedback inhibitors. In proof-of-concept studies, we reported that increasing systemic L654 levels in mice by administering exogenous L654 intravenously significantly diminishes systemic innate immune responses and attenuates murine autoimmunity. In the present study, our goal was to confirm the role of the microbiome in mediating this mode of immunoregulation by decreasing the microbiome-based production of S/G lipids. We now report that decreasing microbiome Bacteroidota in mice using a specific oral antibiotic/rest protocol reduces fecal and plasma S/G lipids levels and significantly enhances systemic innate immune responses. Replenishing systemic levels of S/G lipids after antibiotic treatment through exogenous administration of L654 returns innate immune responses to normal levels, confirming the regulatory role of S/G lipids in this mode of microbiome regulation. Finally, RNAseq analysis of splenic monocytes derived from antibiotic-treated and control mice prior to ex vivo stimulation demonstrates that the antibiotic/rest protocol and the concomitant decrease in microbiome S/G lipids and Bacteroidota has significant downregulatory effects on normal homeostatic pro-inflammatory pathways. These effects are also associated with downregulation of specific proinflammatory pathway inhibitors, which may suggest a potential mechanism underlying the enhancement in ex vivo innate immune stimulated responses of these monocytes. Overall, our results suggest that S/G lipids are microbiome-derived bacterial factors capable of regulating systemic innate immunity and as such are manipulatable targets with therapeutic potential for enhancing or decreasing innate immunity in the context of infectious, malignant, and autoimmune diseases. Overall design: We performed RNA-Seq analysis on splenic monocytes from vancomycin or water gavaged mice to determine gene expression dynamics driving enhanced pro-inflammatory response following vancomycin treatment. Splenic monocytes were isolated via EasySep Mouse Monocyte Isolation Kit (Stemcell) from 3 biological replicates per treatment. RNA-Seq libraries were generated using Illumina TruSeq Stranded mRNA library prep kit and sequenced on Illumina NovaSeq S4 v1.5 for 200 cycles at 10M total pair end reads of length 100 base-pairs. Reads in Fastq files were aligned using HISAT2 v2.2.1 on the GRCm38 index, with SAMtools v1.9 for BAM conversion. A matrix of fragments per kilobase of transcript per million fragments mapped (FPKM) was calculated using Stringtie v2.1.3 with Ballgown on the GRCm38 index. FPKM data was converted to gene count data matrix using tximport v1.26.1 R package.