miR-146a is critical for endotoxin-induced tolerance: IMPLICATION IN INNATE IMMUNITY

Abstract

The human toll-like receptor 4 (TLR4) pathway is activated in response to lipopolysaccharide (LPS), and subsequent signal transductions lead to the production of cytokines such as tumor necrosis factor-alpha (TNF-alpha) by innate immune cells. Defects in innate immune response may contribute to the overproduction of TNF-alpha leading to systemic inflammation and diseases. Thus, the innate immune response needs to be tightly regulated by elaborate mechanisms to control its onset and termination. LPS tolerance is a state of hyporesponsiveness to subsequent LPS challenge and is achieved by monocytic cells after prolonged exposure to LPS. In this report, kinetics of endotoxin-responsive microRNAs expression analysis revealed a unique pattern of gradual increase for miR-146a starting 4 h after LPS stimulation in THP-1 cells and continued up to 35-fold over 24 h. Conversely, TNF-alpha increased up to 4 h and then decreased gradually implicating a negative correlation with miR-146a progression. The characteristic up-regulation of miR-146a toward subsequent LPS challenge in THP-1 cells was studied. Strikingly, microRNA expression analysis during the tolerized state of THP-1 cells showed only miR-146a overexpression suggesting its important role in LPS tolerance. In addition, LPS tolerance was dependent on a LPS-priming dose and associated miR-146a up-regulation. LPS-tolerized cells were observed to regain responsiveness in TNF-alpha production 22 h after LPS removal correlating with a decrease in miR-146a level. Transfection of miR-146a into THP-1 cells mimicked LPS priming, whereas transfection of miR-146a inhibitor largely abolished LPS tolerance. Thus our studies demonstrated that miR-146a is critical for the in vitro monocytic cell-based endotoxin tolerance.

FIGURE 1.

Dose- and time-dependent expression of TNF-α and adaptors TRAF6 and IRAK1 mRNA preceded that of miR-146a in LPS-stimulated monocytic THP-1 cells. THP-1 cells were treated with 0, 10, 100, or 1000 ng/ml LPS and incubated for 2, 4, 8, 12, or 24 h. Culture supernatants were collected at the indicated time points from 2 to 24 h for TNF-α protein analysis using ELISA (A). Total RNA were purified from the respective cell pellets and analyzed by qRT-PCR for the expression of TNF-α mRNA (B), miR-146a (C), miR-155 (D), miR-132 (E), miR-16 (F), IRAK-1 mRNA (G), and TRAF6 mRNA (H). mRNA and miRNA expression were normalized with control 18 S RNA and RNU44, respectively. All results are expressed as mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01 compared with untreated cells.

FIGURE 2.

High level of miR-146a may account for LPS tolerance in THP-1 cell model. THP-1 cells primed with 10 ng/ml LPS continuously for 18 h (tolerized, ■) and untreated controls incubated for the same time period (untolerized, □) were washed twice with PBS and challenged with 0, 10, 100, or 1000 ng/ml LPS for 5 h. Culture supernatants and total RNA were analyzed as described under “Experimental Procedures.” A–H, all results are expressed as mean ± S.D. from three independent experiments. *, p < 0.05; **, p < 0.01 compared with untolerized THP-1 cells. Lysates of the tolerized and untolerized cells 2 h after LPS challenge were analyzed for IRAK-1, TRAF6, and tubulin expression by Western blot (I).

FIGURE 3.

Higher priming dose of LPS induced higher miR-146a levels and more efficient suppression of TNF-α production in subsequent LPS challenge. THP-1 cells were primed with 0, 10, 100, or 1000 ng/ml LPS continuously for 18 h, washed twice with PBS, and challenged with 1000 ng/ml LPS. Supernatants and cell pellets were collected 5 h later as described under “Experimental Procedures” for TNF-α protein determined by ELISA (A), miR-146a expression analysis in total RNA (B). Data points and error bars represent mean ± S.D. of three independent experiments. *, p < 0.01 compared with untreated cells. Western blot analysis for IRAK-1, TRAF6, and tubulin in the cell lysates collected 2 h after LPS challenge (C).

FIGURE 4.

Reduction in miR-146a expression in LPS tolerized THP-1 cells inversely correlated with TNF-α production. THP-1 cells were cultured with (tolerized) or without (untolerized) 10 ng/ml LPS continuously for 18 h. Cells were then washed twice with PBS and cultured in complete growth medium for an additional 0, 12, or 22 h (LPS withdrawal). At each time point, 8 × 10⁵ cells were challenged with 1000 ng/ml LPS for 5 h prior to analysis of TNF-α protein production in culture supernatant by ELISA (A) and qRT-PCR analysis of total RNA for miR-146a expression (B). Values are expressed as mean ± S.D. from three independent experiments. *, p < 0.01 compared with LPS untreated cells.

FIGURE 5.

Transfected miR-146a alone could mimic LPS priming in LPS tolerance assay. THP-1 cells transfected with 40 nm miR-146a mimic followed by incubation for 24 h, mock transfected cells, and 10 ng/ml LPS primed cells (tolerance positive control) were washed with complete growth medium and challenged with 1000 ng/ml LPS for 3 h. A, RNA isolates were prepared from cell pellets of mock and transfected cells followed by qRT-PCR for miR-146a expression normalized with RNU44. B, ELISA analysis of TNF-α protein in supernatants tolerance-positive control, mock transfected, and miR-146a mimic transfected cells. C and D, multiplex analysis of IL-1β and IL-6 proteins in supernatants of tolerance-positive control, mock transfected, and miR-146a mimic-transfected cells. E, the same RNA samples were also analyzed by qRT-PCR for mRNA expression of miR-146a targets TRAF6, IRAK-1, and an unrelated gene lamin A/C (LMNA, negative control). Data are representative of three independent experiments and expressed as mean ± S.D. *, p < 0.01 compared with mock transfected cell.

FIGURE 6.

Blockade of miRNA-146a expression abrogated LPS-induced tolerance in THP-1 cells. THP-1 cells transfected with 40 nm miR-146a inhibitor for 24 h, along with mock transfected, were washed with complete growth medium and primed with 10 ng/ml LPS for 16 h. Cells were then washed with complete growth medium and challenged with 1000 ng/ml LPS for 3 h. Additional controls included were cells transfected with miR-146a inhibitor alone and cells treated with 1000 ng/ml LPS alone for 3 h (LPS alone, TNF-α-positive control). A, total RNA from cell pellets of mock and miR-146a inhibitor-transfected cells, primed with 10 ng/ml LPS for 16 h, were analyzed by qRT-PCR for miR-146a expression. B, TNF-α protein in supernatants of TNF-α (+) control, miR-146a inhibitor alone (without LPS), mock transfected, and miR-146a inhibitor-transfected, were measured by ELISA. C, total RNA were analyzed for TRAF6, IRAK-1, and an unrelated gene lamin A/C (LMNA) expression in both mock and miR-146a inhibitor-transfected cells by qRT-PCR. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.01 compared with mock transfected cells.

FIGURE 7.

A model of the role of miR-146a in LPS-TLR4-mediated signal transduction in tolerized (A) versus untolerized (B) cells. See text under “Discussion.” MD2, myeloid differentiation protein-2.