Suppression of LPS-induced Interferon-gamma and nitric oxide in splenic lymphocytes by select estrogen-regulated microRNAs: a novel mechanism of immune modulation

Abstract

MicroRNAs (miRNAs), recently identified noncoding small RNAs, are emerging as key regulators in homeostasis of the immune system. Therefore, aberrant expression of miRNAs may be linked to immune dysfunction, such as in chronic inflammation and autoimmunity. In this study, we investigated the potential role of miRNAs in estrogen-mediated regulation of innate immune responses, as indicated by up-regulation of lipopolysaccharide (LPS)-induced interferon-gamma (IFNgamma), inducible nitric oxide synthase (iNOS), and nitric oxide in splenic lymphocytes from estrogen-treated mice. We found that miR-146a, a negative regulator of Toll-like receptor (TLR) signaling, was decreased in freshly isolated splenic lymphocytes from estrogen-treated mice compared with placebo controls. Increasing the activity of miR-146a significantly inhibited LPS-induced IFNgamma and iNOS expression in mouse splenic lymphocytes. Further, miRNA microarray and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that estrogen selectively up-regulates/down-regulates the expression of miRNAs in mouse splenic lymphocytes. miR-223, which is markedly enhanced by estrogen, regulates LPS-induced IFNgamma, but not iNOS or nitric oxide in splenic lymphocytes. Inhibition of miR-223 activity decreased LPS-induced IFNgamma in splenic lymphocytes from estrogen-treated mice. Our data are the first to demonstrate the selective regulation of miRNA expression in immune cells by estrogen and are indicative of an important role of miRNAs in estrogen-mediated immune regulation.

Figure 1

LPS-induced IFNγ in mouse splenic lymphocytes is promoted by estrogen at the posttranscriptional level. (A) ELISA analysis of IFNγ levels in supernatants from 5 × 10⁶ freshly isolated (t0) splenocytes and those stimulated with LPS (500 ng/mL, the same concentration was used in all the experiments in this study) for 6 hours. The graphs show the means plus or minus SEM (n = 4 each). (B) Total RNA was prepared from freshly isolated splenocytes and from those stimulated with LPS for 6 hours. IFNγ mRNA expression levels were determined by real-time RT-PCR. The graphs show the relative mRNA expression levels with the means plus or minus SEM (n ≥ 3). *P < .05.

Figure 2

LPS-induced IFNγ in mouse splenic lymphocytes is regulated by the estrogen-regulated miRNA, miR-146a. (A) Total RNA was isolated from freshly isolated splenic lymphocytes using mirVana miRNA isolation kits. Relative expression levels of miR-146a and miR-146b between splenic lymphocytes from placebo- and estrogen-treated mice were analyzed using the Taqman miRNA assay system. The graph shows the means plus or minus SEM (n ≥ 6 each). (B-D) Freshly isolated splenic lymphocytes (1.5 × 10⁷) from placebo- and estrogen-treated mice were transfected with either a negative mimic (control) or miR-146a mimic. Twenty-four hours after transfection, cells were left unstimulated (B) or stimulated with LPS for 24 hours (C,D), and then the supernatants and cell pellets were collected for analysis. (B) Western blot analysis of the expression of IRAK-1 in unstimulated cells at 48 hours after transfection. (C) The level of IFNγ in supernatants from LPS-stimulated miR-146a mimic transfected cells is shown as the percentage expression of negative control mimic transfected cells. Graph shows mean plus or minus SEM (n ≥ 6 each). (D) Western blot analysis of the expression of IFNγ in cell extracts from negative mimic and miR-146a mimic transfected cells stimulated with LPS. Representative Western blot images are shown from at least 3 independent experiments. Densitometry analysis of the IFNγ signal in blot images was performed using Kodak molecular imaging software (version 4.5) and normalized to the loading control β-actin. The graph shows relative density with means plus or minus SEM (n = 3 each). *P < .05; ***P < .001.

Figure 3

LPS-induced iNOS and nitric oxide in mouse splenic lymphocytes is enhanced by estrogen and regulated by miR-146a. An aliquot of 2.5 × 10⁶ splenic lymphocytes (5 × 10⁶/mL) from placebo- and estrogen-treated mice (n = 4 each) were stimulated with LPS or left unstimulated for 24 hours (medium only; A,B). Supernatants and cell pellets were collected for further analysis. (A) The production of nitric oxide in culture supernatants was determined with Griess assays. The graph shows means plus or minus SEM (n = 4 each). (B) Western blot analysis of the expression of iNOS in whole cell extracts. (C) The expression of iNOS mRNA in freshly isolated and 6 hours of LPS stimulated spenic lymphocytes was analyzed as indicated for Figure 1B. The graphs show the relative mRNA expression level with the means plus or minus SEM (n ≥ 3 each). (D) Freshly isolated splenic lymphocytes were transfected and stimulated with LPS as described for Figure 2B-D. The level of nitric oxide in supernatants from LPS-stimulated transfected cells was determined with Greiss assays. The graph shows means plus or minus SEM (n ≥ 6 each). (E) Western blot analysis of the expression of iNOS in cell extracts from negative mimic and miR-146a mimic transfected cells stimulated with LPS. Representative Western blot images are shown from at least 3 independent experiments. Densitometry analysis of iNOS signal detected by Western blotting was performed using Kodak molecular imaging software (version 4.5) and normalized to the loading control β-actin. The graph shows relative density with means plus or minus SEM (n = 4). *P < .05; **P < .01.

Figure 4

Microarray data analysis. (A) The heat map was generated using hierarchical cluster analysis to show distinct miRNA expression patterns in splenic lymphocytes between placebo- and estrogen-treated mice. The intensity values were Log2 transformed, centered by the mean of individual genes across all 4 samples, and then subjected to cluster analysis for generating the heat map. The color bar was extracted to show the color contrast level of the heat map. Red and green indicate high expression level and low expression level, respectively. (B) miRNAs that demonstrated statistically different expression levels between freshly isolated splenic lymphocytes from placebo- and estrogen-treated mice (P < .05) are listed. Fold-changes in miRNA expression were calculated as the ratio of mean intensity values between estrogen- and placebo-treated mice.

Figure 5

Real-time RT-PCR analysis of miRNA expression in splenic lymphocytes. The expression level of selected estrogen-regulated miRNA in freshly isolated splenic lymphocytes between placebo- and estrogen-treated mice were further quantified using Taqman miRNA assays. (A,B) The graphs show miRNA that were confirmed to be significantly up-regulated or down-regulated by estrogen treatment, respectively. Means plus or minus SEM (n ≥ 4 each) are shown in the graphs. *P < .05; **P < .01; and ***P < .001.

Figure 6

miR-223, but not miR-451 and miR-486, regulate LPS-induced IFNγ in splenic lymphocytes from estrogen-treated mice. Freshly isolated splenic lymphocytes (1.5 × 10⁷) from estrogen-treated mice were transfected with either a negative inhibitor (control), miR-223, miR-451, or miR-486 inhibitors. Twenty-four hours after transfection, cells were stimulated with LPS for 24 hours, and the supernatants and cell pellets were collected for analysis. (A,B) The level of IFNγ and nitric oxide in culture supernatants of LPS-stimulated cells were determined by ELISAs (A) and Griess assays (B), respectively. The level of IFNγ and nitric oxide in supernatants from specific inhibitor transfected cells were presented as the percentage level of negative control inhibitor transfected cells. The graphs show the means plus or minus SEM (n = 5 each). (C) Western blot analysis of the expression of IFNγ protein in miRNA inhibitor transfected cells. Representative Western images are shown from at least 3 independent experiments. Densitometry analysis of IFNγ signal was performed as described for Figure 2. The graph shows relative density with means plus or minus SEM (n = 4 each). *P < .05.