Lactobacillus johnsonii N6.2 phospholipids induce immature-like dendritic cells with a migratory-regulatory-like transcriptional signature

Abstract

Shifts in the gut microbiota composition, called dysbiosis, have been directly associated with acute and chronic diseases. However, the underlying biological systems connecting gut dysbiosis to systemic inflammatory pathologies are not well understood. Phospholipids (PLs) act as precursors of both, bioactive inflammatory and resolving mediators. Their dysregulation is associated with chronic diseases including cancer. Gut microbial-derived lipids are structurally unique and capable of modulating host's immunity. Lactobacillus johnsonii N6.2 is a Gram-positive gut symbiont with probiotic characteristics. L. johnsonii N6.2 reduces the incidence of autoimmunity in animal models of Type 1 Diabetes and improves general wellness in healthy volunteers by promoting, in part, local and systemic anti-inflammatory responses. By utilizing bioassay-guided fractionation methods with bone marrow-derived dendritic cells (BMDCs), we report here that L. johnsonii N6.2 purified lipids induce a transcriptional signature that resembles that of migratory (mig) DCs. RNAseq-based analysis showed that BMDCs stimulated with L. johnsonii N6.2 total lipids upregulate maturation-mig related genes Cd86, Cd40, Ccr7, Icam1 along with immunoregulatory genes including Itgb8, Nfkbiz, Jag1, Adora2a, IL2ra, Arg1, and Cd274. Quantitative reverse transcription (qRT)-PCR analysis indicated that PLs are the bioactive lipids triggering the BMDCs response. Antibody-blocking of surface Toll-like receptor (TLR)2 resulted in boosted PL-mediated upregulation of pro-inflammatory Il6. Chemical inhibition of the IKKα kinase from the non-canonical NF-κB pathway specifically restricted upregulation of Il6 and Tnf. Phenotypically, PL-stimulated BMDCs displayed an immature like-phenotype with significantly increased surface ICAM-1. This study provides insight into the immunoregulatory capacity of Gram-positive, gut microbial-derived phospholipids on innate immune responses.

Keywords: Lactobacillus johnsonii N6.2; cellular immune response; dendritic cell; immunomodulation; innate immunity; microbial-derived lipids; phospholipid; probiotic.

Figure 1.

Global lipidomics analysis of Lactobacillus johnsonii N6.2 total lipids (TL). Total lipids from L. johnsonii N6.2 were purified and pooled from a large-scale culture and profiled by qualitative LC-MS/MS analysis. a: Percentage abundances in lipid categories. b: Percentage abundances in lipid classes. The abbreviation GCL for glycerolipids was selected to avoid confusion with GL (denotation used for the glycolipid fraction obtained after lipid fractionation of L. johnsonii N6.2 total lipids).

Figure 2.

RNAseq-based global evaluation of BMDCs responses to total lipids derived from Lactobacillus johnsonii N6.2. a: Principal component analysis of treatment groups (Lj-N6.2_TL: L. johnsonii N6.2-TL-treated group; a-GalCer: α-GalCer-treated group; and Vehicle_control). b: Heatmap of total significantly differentially expressed genes (DEG) in Lj-N6.2_TL group (p.adjust < 0.01). The data showed a specific expression signature induced by total lipids from L. johnsonii N6.2 on BMDCs. c: Gene ontology enrichment analysis of DEG in L. johnsonii N6.2 treatment with absolute values of log2FC ≥ 1. Data represent three independent assays.

Figure 3.

RNAseq-based analysis of BMDCs response to total lipids derived from L. johnsonii N6.2. Heatmap displays selected DEGs with absolute values of log2FC between 1 and 2 and greater than 2 (p.adjust < 0.01). Total lipids (TLs) from L. johnsonii N6.2 significantly upregulate several regulatory molecules involved in the regulation and suppression of immune responses. The graphic displays the results obtained from three independent assays.

Figure 4.

Gene expression of selected response-markers after fractionation of L. johnsonii N6.2 lipids into simple lipids (SL), glycolipids (GL) and phospholipids (PL). BMDCs were stimulated with total lipids (TL) at 500 ng/mL or three different fractions, at 5 µg/mL (SL, GL, and PL) for 6 h. VC: Vehicle control. The graphic displays the results obtained from three independent assays. Different letter labels denote statistically significant changes (p.value < 0.05).

Figure 5.

Relative surface expression of maturation and migration markers in BMDCs normalized to vehicle-treated cells and presented as Log2 values. BMDCs were stimulated with total lipids (TLs) at 500 ng/mL or fractions at 5 µg/mL for 6 h. SL: simple lipids, GL: glycolipids, PL: phospholipids. PLs significantly reduce surface expression of CD86 and induce a sig. increase of migration marker ICAM-1. VC: Vehicle control. The graphic displays the results obtained from three independent assays. Different letter labels denote statistically significant changes (p.value < 0.05).

Figure 6.

Antibody blocking of TLR2 on the surface of L. johnsonii N6.2 phospholipids-stimulated BMDCs. 7-day BMDCs were Ab-blocked with anti-TLR2 or isotype control Abs at 9 µg/mL for 1 h before addition of L. johnsonii N6.2 PLs at 5 µg/mL and further incubation for 6 h. Negative controls: vehicle control for PLs (Vehicle_ctrl), the Ab storage buffer (Ab_buffer) and both combined (Combined_ctrl). Ab controls: anti-TLR2_Ab and Isotype_Ctrl_Ab. Data was compared to Vehicle_Ctrl for statistical analysis. Data represent the values from three independent assays. Different letter labels denote statistically significant changes (p.value < 0.05).

Figure 7.

Blocking non-canonical NF-kB signaling in Lactobacillus johnsonii N6.2 lipid-stimulated BMDCs. 7-day BMDCs were pre incubated with the IKKα kinase inhibitor BMS-345541 at 5 µM for 1 h before addition of L. johnsonii N6.2 total lipids (TLs) or phospholipids (PLs) at 0.5 or 5 µg/mL respectively and further incubated for 6 h. Negative controls: vehicle control for L. johnsonii N6.2 lipids mixed with vehicle control for inhibitors (Vc_combined), NF- kB inhibitor alone. Data were obtained from three independent assays. Different letter labels denote statistically significant changes (p.value < 0.05).