Long Non-Coding RNA lncTAF15:1-1 Promotes CCL5 Secretion and Cell Migration of Monocyte-Derived Dendritic Cells via PI3K/AKT/mTOR Pathway in Systemic Lupus Erythematosus

Abstract

Purpose: Systemic lupus erythematosus (SLE) is a complex autoimmune disease that seriously endangers human health. Long non-coding RNAs (lncRNAs) have been found to exhibit strong regulatory functions in cell physiology and maturation of dendritic cells (DCs). Hence, this study tried to reveal the underlying roles of one lncRNA, lncTAF15:1-1, in modulating DC functions and its involvement with CCL5 secretion in SLE pathogenesis.

Methods: The expression levels of lncTAF15:1-1 were measured using qPCR in cultivated monocyte-derived dendritic cells (moDCs). Flow cytometry, ELISA, and Transwell chamber experiments were performed to assess various biological functions of moDCs. RNA-seq analysis was conducted to investigate transcriptional alterations in cells overexpressing lncTAF15:1-1 and negative control cells. Gene Set Enrichment Analysis (GSEA) was utilized to predict potentially involved signaling pathways, which were subsequently confirmed by Western Blotting. Rescue experiments were carried out where the expression of lncTAF15:1-1 and PI3K/AKT/mTOR pathway were altered simultaneously.

Results: LncTAF15:1-1 was significantly upregulated in moDCs from SLE patients, and it exhibited a positive correlation with SLE Disease Activity Index (SLEDAI) scores. Additionally, elevated levels of CCL5 were detected in both plasma and moDC supernatants of SLE patients. Overexpression of lncTAF15:1-1 stimulated moDCs to secrete higher levels of CCL5, and it enhanced the migration ability of moDCs as well as their capacity to attract CD4+ naïve T cells. GSEA analysis of RNA profiles indicated the potential involvement of the PI3K/AKT/mTOR pathway in lncTAF15:1-1 regulation, which was further validated by Western Blotting. The rescue experiments demonstrated that the effects of lncTAF15:1-1 on multiple functions of moDCs were attenuated when the PI3K/AKT/mTOR pathway was disrupted.

Conclusion: This study elucidated the role of lncTAF15:1-1 in orchestrating DC migration and the recruitment of CD4+ T cells by enhancing CCL5 secretion through activating PI3K/AKT/mTOR pathway, which provides insights into potential molecular targets for SLE diagnosis and treatment.

Keywords: CCL5; long non-coding RNA; monocyte-derived dendritic cells; systemic lupus erythematosus.

Graphical abstract

Figure 1

LncTAF15:1–1 was highly expressed in moDCs from SLE patients and positively correlated with disease activity. (A) The expression levels of lncTAF15:1–1 in moDCs from SLE patients and healthy controls (12 vs 12) were measured by qRT-PCR. (B) Correlation of lncTAF15:1–1 expression levels with SLEDAI scores was assessed (r=0.57; p=0.03). (C) The comparison of lncTAF15:1–1 levels between SLEDAI<15 patients and SLEDAI≥15 patients was made. (D) Correlation of lncTAF15:1–1 expression levels with WBC counts was assessed (r_s=−0.54; p=0.04). Unpaired t-test (two-tailed) and Mann–Whitney test were applied. Pearson correlation (r) or Spearman correlation (r_s) was calculated to evaluate the association between two parameters. Data were shown as the mean ± SEM. *p<0.05, **p<0.01.

Figure 2

Regulation of lncTAF15:1–1 influenced CCL5 secretion in moDCs. (A and B) The expression level of lncTAF15:1–1 was artificially modulated by the transfection of adenovirus or Smart Silencer and confirmed using qRT-PCR. (C–F) Alterations of the expression of lncTAF15:1–1 resulted in corresponding changes in mRNA expressions and secretion levels of CCL5. (G and H) The secretion levels of CCL5 in the blood plasma (15 vs 15) and moDCs cell supernatant (12 vs 12) were compared between SLE patients and healthy controls. (I) The migratory ability of moDCs was assessed using Transwell chambers, and comparisons were made between adeno-V, adeno-OE, NC, and SS groups. The number of migratory moDCs was counted and normalized to the area. (J) The capacity of supernatant from moDCs to attract CD4+T cells was evaluated using Transwell assay. The migratory CD4+T cell numbers were quantified by flow cytometry. Unpaired t-test (two-tailed) or Mann–Whitney test was applied. Data were from at least three independent experiments and shown as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

Figure 3

PI3K/AKT/mTOR pathway was pivotal in the immune modulation of lncTAF15:1–1. (A and B) Volcano plot and heatmap suggested the 488 differentially expressed genes between the vector (control) and lncTAF15:1–1 overexpressed moDC groups (treat), including 140 down-regulated and 348 up-regulated genes. (C) GO function enrichment analysis indicated that these differentially expressed genes were related to different pathways. (D) GSEA plot showed that the PI3K/AKT/mTOR signaling pathway was activated in the lncTAF15:1–1 overexpressed group (FDR q-value = 0.233, nominal p-value = 0.012). (E) PI3K inhibitor LY294002 and activator 740 Y-P were used to disrupt the PI3K/AKT/mTOR pathway and the levels of AKT, p-AKT, PI3K, p-PI3K, mTOR and p-mTOR were measured and quantified by Western Blotting.

Figure 4

LncTAF15:1–1 regulated the migration of moDCs and induced CD4+ T cell recruitment through CCL5 secretion by activating the P13K/AKT/mTOR pathway. (A and B) The PI3K/AKT/mTOR pathway inhibitor LY290042 was administered in conjunction with the adenovirus, followed by the assessment of mRNA expression and secretion levels of CCL5, in comparison with the OE group. (C and D) The PI3K/AKT/mTOR pathway activator 740 Y-P was added together with the Smart Silencer and then the mRNA expression and secretion of CCL5 were measured compared with the SS group. (E) Transwell assay was performed to compare the migratory capacity of moDCs when the PI3K/AKT/mTOR signaling pathway was disrupted. The number of migratory moDCs was counted and normalized to the area. (F) The capacity of supernatant from moDCs to attract CD4+ T cells was compared when the lncTAF15:1–1 expression and the PI3K/AKT/mTOR pathway were regulated. The migratory CD4+T cell numbers were quantified by flow cytometry. (G) Intercellular sublocalization of lncTAF15:1–1 was determined by FISH. 18s and U6 were considered as the positive control of cytoplasm and nucleus. One-way ANOVA or Kruskal–Wallis test was applied. Data were from at least three independent experiments and shown as the mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.