Probiotic peptidoglycan skeleton enhances vaccine efficacy against MRSA by inducing trained immunity via the TLR2/JAK-STAT3 pathway

Abstract

Trained immunity refers to the ability of trained innate immune cells to generate an immune memory that produces rapid, broad-spectrum, and long-lasting protection against heterologous stimuli. Based on the rapid and broad-spectrum protection that the peptidoglycan backbone from lactic acid bacteria, bacterium-like particles (BLPs), offers, we hypothesized that BLPs enhance protection through trained immunity. Here, we found that combining BLP with a vaccine significantly improves protective efficacy against methicillin-resistant Staphylococcus aureus (MRSA) infection, accompanied by changes in trained immunity markers. We demonstrate that BLP-induced trained immunity macrophages exhibit increased cytokine secretion and phagocytic activity in vitro. In an in vivo model, BLP confers protection against S. aureus 26003 even without specific antigens. In an ex vivo model, BLP induces increased markers of trained immunity. Transcriptome analysis suggests that BLP may induce trained immunity by activating the IL-6-JAK-STAT3 pathway through TLR2 receptor activation, thereby modulating macrophage metabolic reprogramming and function. In summary, our study establishes that BLP induction of trained immunity, along with regulated metabolic reprogramming and macrophage function, may contribute to enhancing vaccine efficacy. Our findings elucidate a novel mechanism for BLP-mediated immune enhancement, critical for the application of BLP as a vaccine vector to construct a vaccine that combines specific immune response with innate immune response.

Keywords: MRSA; bacterium-like particles; innate immunity; peptidoglycan backbone; trained immunity.

Figure 1

Bacterium-like particles (BLP) combined with the multi-epitope subunit vaccine-SAMEA provide better protection for mice. (A) Experimental design of BLP combined with two-dose subunit vaccine immunization against MRSA. (B) Specific IgG antibody levels in serum on days 28, 35, and 42 after immunization. (C) IgG1 antibody levels, IgG2a antibody levels, and the ratio of IgG2a/IgG in serum on days 28, 35, and 42. (D) The survival of mice after MRSA challenge. (E) The body weight change in mice after MRSA challenge. (F) The bacterial load of MRSA in the kidney on day 63. (G) Histological HE image of mice in the kidney on day 63. Scale bars, 200 and 40 µm. (H) The mRNA relative expression levels of IL-6, IL-1β, TNF-α, IL-10, IL-12, IFN-β, TGF-β, CCL2, CCL7, TLR2, TLR4, HDAC7, mTORC2, and HIF1-α on day 63 in kidney was measured by RT-qPCR. (I) The levels of glucose (GLU) and lactic acid (LA) in serum on day 63 (****/#### p<0.0001, *** p<0.001, ** p<0.01, * p<0.05). NS indicates that there is no significant difference between the two groups.. The results of qPCR were expressed using the 2^−ΔΔCT value.

Figure 2

BLP combined with one dose of SAMEA also provides better protection for mice against MRSA infection. (A) Experimental design of BLP combined with one-dose subunit vaccine immunization against MRSA. (B) Specific IgG antibody levels in serum on days 7, 14, and 21. (C) IgG1 and IgG2a antibody levels and the ratio of IgG2a/IgG1 in serum on days 7, 14, and 21 after immunization. (D) The survival of mice after MRSA challenge. (E) The body weight change in mice after MRSA challenge. (F) The bacterial load of MRSA in the kidney on day 42. (G) Relative expression levels of cytokines and trained immunity-related indices in the mouse kidney on day 42 of IL-6, IL-1β, TNF-α, IL-10, IL-12, IFN-β, TGF-β, CCL2, CCL7, TLR2, TLR4, HDAC7, mTORC2, and HIF1-α were measured by RT-qPCR. (H) The levels of GLU and LA in serum on day 42 (**** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05). NS indicates that there is no significant difference between the two groups. The results of qPCR were expressed using the 2^−ΔΔCT value.

Figure 3

BLP induced trained immunity in Raw264.7 cells. (A) Experimental design of the trained immunity model in vitro. (B) The mRNA relative express levels of IL-6, IL-1β, and TNF-α on D1, D7, and D8 (n = 5). (C) Heatmap of the mRNA relative expression levels of IL-10, IL-12, IFN-β, TGF-β, TLR2, CCL2, and CCL7 on D8 (n = 5). (D) The mRNA relative expression levels of HDAC7, mTORC2, and HIF1-α on D8 (n = 5). The levels of (E) GLU and LA; (G) ROS; (H) NO in the cell supernatant on D8 (n = 3). (F) Raw264.7 cells on D7 were obtained and rested overnight before being co-cultured with E. coli pET28a-GFP/Rosetta for 1 h. A fluorescence microscope determines phagocytosis of Raw264.7 cells from each group. Scale bars, 100 µm. (I). Inhibition experimental design of the trained immunity model in vitro. (J) After the action of an inhibitor, the mRNA relative expression levels of IL-6, IL-1β, and TNF-α on D8 were measured by RT-qPCR (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). The results of qPCR were expressed using the 2^−ΔΔCT value.

Figure 4

Trained immunity induced by BLP rapidly protected mice infected with S. aureus. (A) Experimental design of trained immunity model in vivo. (B) The survival of mice after S. aureus challenge. (C) The body weight change in mice after S. aureus challenge. (D) The injury score of mice after S. aureus challenge. (E) The bacterial load of S. aureus in the spleen, kidney, and liver on D12. (F) Histological HE image of the mouse spleen on D12. Scale bars, 100 µm. (G) Histological HE image of the mouse kidney on D12. Scale bars, 100 µm. (H) Heatmap of the mRNA relative expression levels of IL-10, IL-12, IFN-β, TGF-β, TLR2, CCL2, and CCL7 on D12 in the spleen and kidney (n = 5). (I) The mRNA relative expression level of HDAC7, mTORC2, and HIF1-α on D12 in the spleen and kidney was measured by RT-qPCR. (J) The levels of GLU and LA in serum on D12 (****/#### p<0.0001, *** p<0.001, ** p<0.01, */# p<0.05). NS indicates that there is no significant difference between the two groups.. The results of qPCR were expressed using the 2^−ΔΔCT value.

Figure 5

(A) Schematic showing trained immunity in the stimulated with LPS in mouse peritoneal macrophages. The mRNA relative expression levels of (B) IL-6, IL-1β, and TNF-α; (C) HDAC7, mTORC2, and HIF1-α in 1, 6, 12, and 24 h were measured by RT-qPCR (n = 5). The levels of (D) GLU and LA in the cell supernatant at 1, 6, 12, and 24 h (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). The results of qPCR were expressed using the 2^−ΔΔCT value.

Figure 6

Mouse peritoneal macrophages stimulated with LPS for 6 h were analyzed for RNA-seq. (A) Heatmap plot of differentially expressed genes between the Control group and the BLP group. (B) Volcano plot of differentially expressed genes between the Control group and the BLP group. Differential expression of epigenetically related genes is labeled. (C) Heatmap of related upregulated genes of proinflammatory cytokines, chemokines, PRRs, and metabolism. (D) Immunity and phagocytosis-related GO function analysis histogram of differentially expressed genes. Heatmap of related upregulated genes of (E) phagocytosis and (F) antigen presentation-related genes. (G) Metabolism-related pathway KEGG enrichment. (H) Comparative transcription factor enrichment analysis of differentially expressed genes using the HOMER package (* indicates transcription factors involved in the epigenetic reprogramming, # indicates transcription factors involved in the innate immune response). (I) Gene set enrichment analysis (GSEA) using the Molecular Signatures Database (MSigDB) Hallmark gene set collection. Differentially expressed gene sets in BLP-treated monocytes versus controls.