IL-23 receptor regulation by Let-7f in human CD4+ memory T cells

Abstract

CD4(+) memory T cells include the Th17 cell population, which has been shown to be implicated in autoimmune and inflammatory diseases. These memory T cells express higher IL-23R and produce more IL-17 compared with their naive counterparts. However, the molecular mechanisms that regulate IL-23R expression in human T cells are not completely understood. MicroRNAs play important roles in a wide range of biological events through posttranscriptional suppression of target mRNAs. In this article, we provide evidence that a specific microRNA, Let-7f, inhibits IL-23R expression in human CD4(+) memory T cells. Endogenous expression of Let-7f in memory T cells is significantly lower when compared with naive T cells, and Let-7f blocks IL-23R expression through its complementary target sequence within 3' untranslated region of target gene. Furthermore, exogenous transfection of a Let-7f mimic into memory T cells results in downregulation of IL-23R and its downstream cytokine, IL-17. Our findings reveal a novel mechanism in regulating the IL-23/IL-23R pathway and subsequent downstream IL-17 production, which may provide novel therapeutics for human inflammatory and autoimmune diseases.

FIGURE 1

Human memory T cells produce significantly more IL-17. Isolated naive and memory CD4⁺ T cells were cultured with anti-CD3, anti-CD2, and CD28 beads for 3 d and then culture for 5 d. RNA and culture supernatant were harvested. A, IL-17 secretion in cell culture supernatant was measured by ELISA. n = 16. *p < 0.05. B, IL-17 mRNA expression were assessed by qRT-PCR between naive and memory T cells. n = 16. *p < 0.05. C, Intracellular cy-tokine staining and flow cytometry were used to measure IL-17–producing cells after PMA and ionomycin stimulation for 5 h (representative of two experiments). D and E, IL-1β, IL-23, or both were added into the memory or naive T cell culture system, and IL-17 production was detected by ELISA. n = 7. *p < 0.05.

FIGURE 2

miRNA Let-7f, miR-17, and miR-936 inversely correlated with IL-23R expression. A, IL-23R mRNA expression in naive and memory T cells were assessed by qRT-PCR. n = 16. *p < 0.05. B–E, miRNAs with putative binding sites within 3′UTR of IL-23R gene were quantified in naive and memory T cells by qRT-PCR. n = 16. *p < 0.05.

FIGURE 3

IL-23R and putative associated miRNA expression in human cell lines. A, IL-23R mRNA expression was assessed in human myeloid and lymphoid cell lines by qRT-PCR. B, miRNA expression was assessed in human and lymphoid cell lines by qRT-PCR.

FIGURE 4

Exogenous transfection of Let-7f mimic inhibits IL-23R expression in human K562 cells. A, Let-7f expression in cell lines was measured by qRT-PCR. B, Let-7e expression in cell lines was measured by qRT-PCR. C, IL-23R mRNA level was qualified in K562 cells after Let-7e, Let-7f, and miR-17 mimics (25 and 100 nM) transfection by qRT-PCR. **p < 0.01. D, MIP-2α mRNA level was measured in K562 cells after miRNA mimic transfection by qRT-PCR. *p < 0.05.

FIGURE 5

IL-23R miRNA binding site mutations’ effect on reporter gene expression. A, The IL-23R 3′UTR contains complementarities to miRNA Let-7f, Let-7e, and miR-17. Using in silico computational target prediction analysis, we identified that Let-7f, Let-7e, and miR-17 have five- to seven-nucleotide seed regions complementary to the 3′UTR of IL-23R mRNA. B, Luciferase reporter activity in the pMIR-IL-23R 37prime;UTR reporter constructs and associated miRNA binding site mutants. Data shown are relative to the wild type pMIR-IL-23R 3′UTR report construct. n = 9. *p < 0.05.

FIGURE 6

Let-7f inhibits IL-23R and IL-17 expression in human primary memory T cells. A, Memory T cells were transfected with control, Let-7f, Let-7e, and miR-17 mimics for 24 h. RNA was extracted and mRNA level of genes was detected by qRT-PCR. IL-23R mRNA expression was significantly reduced in memory T cells transfected with Let-7f mimic. **p < 0.01. Control mimic, Let-7e, and miR-17 had no effect. B, Memory T cells were transfected with control and Let-7f mimics for 24 h, and protein extracts were isolated. Western blot analysis demonstrated reduced IL-23R protein expression in Let-7f mimic transfected cells (representative of two individual donors). C, IL-17 mRNA expression was also significantly decreased in memory T cells after Let-7f mimic transfection. **p < 0.01. D, Decreased IL-17–producing cells were detected by intracellular staining after Let-7f mimic transfection. E, TNF-α mRNA expression was not inhibited by the Let-7f mimic and other mimics.

FIGURE 7

Let-7f, but not Let-7e, is decreased in IL-1β–stimulated human primary memory T cells. Primary memory T cells were treated in the presence or absence of IL-1β for 48 h, RNA extracted, and miRNA expression assessed by qRT-PCR. Let-7f was significantly decreased in IL-1β–treated memory T cells, whereas Let-7e was unchanged. *p < 0.05. n = 10.