Upregulated cytokine−mediated signalling pathway and T cell activation regulatory genes in old ECs
To clarify the possible mechanism by which ECs from young and old donors cause different effects on T cell subsets, RNA-seq analyses were performed in ECs from 3 young donors and 3 old donors that were cultured for 7 days (Fig. 4A). Among the 30,331 genes in the volcano plot (Fig. 4B), 975 genes had lower expression, and 688 genes had higher expression in the old ECs compared to the young ECs. GO term enrichment analysis indicated that genes related to neutrophil activation involved in the immune response were upregulated in old ECs (Fig. S1), suggesting that old ECs participated in more immune cell activation than young ECs. GO enrichment analysis also showed the top 10 biological processes enriched by upregulated genes in ECs from young and old donors, and the results indicated that the cytokine−mediated signalling pathway and regulation of the T cell activation process were significantly upregulated in the old group (Fig. 4C). The size of each circle indicates the ratio of differentially expressed gene counts to the total gene counts of the term. Heatmaps show that the expression of genes involved in these two biological processes, including TLR1, TLR2, costimulatory molecules such as CD86, chemokines and chemokine receptors such as C-X-C chemokine ligand 10 (CXCL10), C-C chemokine receptor (CCR) 2 and CCR5, and inflammatory cytokines such as perforin 1 (PRF1) and NOD-like receptor protein 3 (NLRP3), was upregulated in old ECs compared with young ECs (Fig. 4D).
To confirm the RNA-seq results, we further compared the relative mRNA expression of the aforementioned genes in young and old BM ECs by qRT-PCR. Table S2 shows the forward and reverse gene primers that were used for qRT-PCR. In accord with the RNA-seq results, the mRNA levels of TLR family members, including TLR2 (Fig. 4E, 1.78±0.36-fold, P=0.04) and TLR4 (Fig. 4E, 1.92±0.32-fold, P =0.01), CD80 (Fig. 4E, 1.3±0.01-fold, P =0.01), CCR5 (Fig. 4E, 3.46±1.14-fold,P =0.04), CXCL10 (Fig. 4E, 4.48±1.29-fold, P =0.02), PRF1 (Fig. 4E, 2.24±0.35-fold, P =0.004) and NLPR3 (Fig. 4E, 1.99±0.46-fold, P =0.04), were significantly higher in ECs derived from the old donors than in ECs from the young donors.