Targeting of microRNA-142-3p in dendritic cells regulates endotoxin-induced mortality - PubMed (original) (raw)

. 2011 Jun 9;117(23):6172-83.

doi: 10.1182/blood-2010-12-325647. Epub 2011 Apr 7.

Sooryanarayana Varambally, Christopher A Maher, Qi Cao, Peter Chockley, Tomomi Toubai, Chelsea Malter, Evelyn Nieves, Isao Tawara, Yongqing Wang, Peter A Ward, Arul Chinnaiyan, Pavan Reddy

Affiliations

Targeting of microRNA-142-3p in dendritic cells regulates endotoxin-induced mortality

Yaping Sun et al. Blood. 2011.

Abstract

While miRNAs are increasingly linked to various immune responses, whether they can be targeted for regulating in vivo inflammatory processes such as endotoxin-induced Gram-negative sepsis is not known. Production of cytokines by the dendritic cells (DCs) plays a critical role in response to endotoxin, lipopolysaccharide (LPS). We profiled the miRNA and mRNA of CD11c⁺ DCs in an unbiased manner and found that at baseline, miR-142-3p was among the most highly expressed endogenous miRs while IL-6 was among the most highly expressed mRNA after LPS stimulation. Multiple computational algorithms predicted the IL-6 3' untranslated region (UTR) to be a target of miR-142-3p. Studies using luciferase reporters carrying wild-type (WT) and mutant IL-6 3'UTR confirmed IL-6 as a target for miR-142-3p. In vitro knockdown and overexpression studies demonstrated a critical and specific role for miR142-3p in regulating IL-6 production by the DCs after LPS stimulation. Importantly, treatment of only WT but not the IL-6-deficient (IL-6(⁻/⁻)) mice with locked nucleic acid (LNA)-modified phosphorothioate oligonucleotide complementary to miR 142-3p reduced endotoxin-induced mortality. These results demonstrate a critical role for miR-142-3p in regulating DC responses to LPS and provide proof of concept for targeting miRs as a novel strategy for treatment of endotoxin-induced mortality.

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Figures

Figure 1

Figure 1

Validation of DC miR profile by RT-PCR. (A) Computational prediction of miRs targeting IL-6 3′UTR by the miRanda, MicroCosm Target, and PITA Top programs The miR-142-3p and miR-223 were predicted by all 3 programs. (B) The potential miR target sequences in the 3′UTR of IL-6 mRNA that are computationally predicted in DCs are shown and the seed sequence pairings are indicated by the lines. (C) Validation of miR expression pattern at baseline and the response to LPS. DCs were treated with either LPS or diluent for 12 hours and analyzed. The levels of miRNA-142-3p, miR-223, Let-7a, miR-27a/b, miR-155, and miR-146a were analyzed by TaqMan quantitative RT-PCR. Expression of miRNAs is presented relative to snoRNA135. Data are representative of 2 to 6 separate experiments with similar results (mean ± SEM).

Figure 2

Figure 2

Endogenous miR-142-3p directly targets IL-6 3′ UTRs in DCs. (A) Schematic of the psiCHECK-2 constructs inserted with WT or truncated fragments of IL-6 3′UTR with the distinct miR target sites. (B) Differential regulatory repressions of psiCHECK-2 reporter carrying WT IL-6 3′UTR by endogenous miRNAs in DCs treated with or without LPS. Data shown are representative of results from 3 independent experiments (mean ± SEM). (C) Significantly de-repressed Luc expressions following LPS treatment. Fold changes of expression levels of WT reporters were calculated after first normalizing to empty vector control and were then compared between DCs treated with or without LPS. Data shown are representative of results from 3 separate experiments (mean ± SEM). (D) Differentially suppressed expression ratios of Luc reporters carrying WT or the various truncated IL-6 3′UTR that are mutated at various target sites for the endogenous miRs. Data are representative of 5 separate experiments (mean ± SEM). (E) Differentially regulated de-repression of Luc reporters by LPS treatment. Fold changes were calculated after normalized to empty vector first then compared between DCs treated with or without LPS. Data were obtained over 4 independent experiments (mean ± SEM). (F) Endogenous miR-142-3p predominantly targets IL-6 3′ UTR. Expression levels of Luc reporters carrying WT IL-6 3′UTR were measured after knockdown of either miR-142-3p or miR-223 and after overexpression of miR-142 in DCs treated with or without LPS. The results are from 4 separate experiments (mean ± SEM).

Figure 3

Figure 3

Effect of miR-142-3p on expression of endogenous IL-6 protein and mRNA levels in DCs. (A) Knockdown of miR-142-3p de-represses IL-6 expression: IL-6 levels were measured by ELISA in the supernatants from DCs treated with or without LPS and those transfected with either miR-142-3p or miR-223 knockdown or scramble probes for 24 or 48 hours. Data shown are the pooled results from 4 separate experiments (mean ± SEM). (B) IL-6 protein levels are reduced by the overexpression of miR-142-3p: IL-6 was measured by the ELISA in the supernatants of DCs treated with LPS or diluents and ELISA in DCs transfected with either miR-142-3p knockdown or scramble probes or pre-miR-142 duplex or scramble control for 48 hours. Data shown are the pooled results from 3 separate experiments. (C) IL-6 mRNA expression is altered with either knockdown or overexpression of miR-142-3p: IL-6 mRNA levels were analyzed by quantitative RT-PCR in DCs that were transfected with either miR-142-3p knockdown or pre-miR-142 overexpression or scramble controls for 48 hours. Data are combined from 5 experiments with similar results (mean ± SEM). (D) Kinetics of IL-6 mRNA expression following miR-142-3p knockdown and LPS treatment: DCs were transfected with miR-142-3p knockdown or scramble probes for 24 hours, and then treated with LPS for the indicated time. IL-6 mRNA levels were analyzed as in panel C. Data shown are combined results from 3 similar experiments (mean ± SEM).

Figure 4

Figure 4

miR-142-3p does not affect the endogenous expression of other cytokine responses by the DCs. (A) Knockdown of either miR-142-3p and miR-223 does not affect other proinflammatory cytokines: levels of TNFα and IL-12 were analyzed by ELISA in the supernatants of DCs treated with LPS or diluents and transfected with either miR-142-3p knockdown or scramble probes or miR-223 knockdown probes for 48 hours. Data shown are combined results from 3 similar experiments (mean ± SEM). (B) The expression of cytokines TNFα, IL-12, and IL-10 are not affected by either the overexpression or knockdown of miR-142-3p: TNFα, IL-12, and IL-10 analyzed by ELISA in the supernatants of DCs treated with LPS or diluent and transfected with either miR-142-3p knockdown or scramble probes or pre-miR-142 duplex for 48 hours. Data shown are combined results from 3 similar experiments (mean ± SEM). (C) miR-142-3p has no direct impact on the expression of other immune molecules: the mRNA levels of TGFBR1, IRAK1, PCAF, and TIRAP were analyzed by quantitative RT-PCR in the DCs that were transfected with either miR-142-3p knockdown or pre-miR-142 duplex or scramble control for 48 hours. Data are combined from 3 experiments with similar results (mean ± SEM). (D) Knockdown of miR-142-3p in DCs does not affect their ability to stimulate allogeneic T cells: allogeneic (BALB/c) T cells were cultured with B6 BM DCs that were transfected with either miR-142-3p knockdown or scramble probes. T-cell proliferation was evaluated at 96 hours following pulsing with 3H for the last 18 hours. Data shown are results from 1 of 2 similar experiments (mean ± SEM).

Figure 5

Figure 5

miR-142-3p regulates IL-6 expression in vivo. To analyze the effect of miR-142-3p, B6 mice were injected with either high-affinity LNA-anti–miR-142-3p with phosphorothioate modifications or scrambled control at various doses. The splenocytes were then harvested and used for the analyses of the expression of miR-142-3p as described in “Methods.” (A) Dose-dependent in vivo knockdown of miR-142-3p by anti–miR-142-3p in splenocytes measured by TaqMan quantitative RT-PCR. Data shown are from 1 of 2 experiments with 3-4 mice/group (mean ± SEM). (B) anti–miR-142-3p specifically knocked down miR-142-3p but did not affect off-target miRs. Expression levels of miR-142-3p and other miRs such as miR-142-5p or miR-155 were measured by TaqMan quantitative RT-PCR. Data shown are from 1 of 2 experiments with 3 mice/group (mean ± SEM). (C) In vivo knockdown of miR-142-3p by anti–miR-142-3p de-repressed LPS-induced IL-6 expression at both mRNA and protein levels: IL-6 mRNA levels in splenocytes were determined by quantitative RT-PCR and the protein levels was measured in the sera by ELISA. Data shown are from 1 of 2 experiments with 4-5 mice/group. (D) In vivo knockdown of miR-142-3p by anti–miR-142-3p does not affect LPS-induced TNFα expression at both mRNA and protein levels: TNFα mRNA levels in splenocytes were determined by quantitative RT-PCR and the protein levels was measured in the sera by ELISA of IL-6−/− and WT B6 mice following administration of LPS. Data shown are from 2 experiments with 6 mice/group. (E) Silencing of mir-142-3p mitigated endotoxin-induced mortality. Treatment of mice with LNA-modified oligonucleotide complementary to miR-142-3p (anti–miR-142-3p) followed by LPS injection (■) significantly reduced mortality compared with animals that were treated with scrambled anti-miR and LPS (●). P = .029. Data shown are combined from 2 different experiments with similar results. (F) Silencing of mir-142-3p does not alter endotoxin-induced mortality in IL-6−/− mice. Treatment of IL-6−/− mice with LNA-modified oligonucleotide complementary to miR-142-3p followed by LPS injection (dotted line, n = 6) did not affect the rate and overall mortality compared with IL-6−/− animals that were treated with scrambled anti-miR and LPS (solid line, n = 6). P = NS.

Figure 6

Figure 6

Human and murine hematopoietic cells express miR-142. (A) Endogenous expression of miR-142 in CD11c DC subsets: basal expression levels of miRNA-142-3p and 142-5p was analyzed by in purified FACS sorted CD11c+/CD8+ DC and CD11c+/CD8− DC subsets with quantitative RT-PCR. Data shown are from 1 of 3 similar experiments (mean ± SEM). (B) miR-142s are specific for hematopoietic cells in mice. miR-142 expression was analyzed in the brain, whole splenocytes, DCs harvested from spleen, and the T and B lymphocytes harvested from the spleen or lymphoid nodes of naive mice. Data were obtained from 1 of 2 experiments with n = 3/group. (C) The miR-142-3p target sequence in the 3′UTR of IL-6 mRNA is conserved across several mammalian species. Seed sequence pairing is indicated by lines. (D) miR-142-3p and miR-142-5p are highly expressed in human hematopoietic cells: DCs and T lymphocytes were harvested from the PBMCs that were freshly isolated from normal healthy human volunteers (n = 3). The human cell lines—such as the endothelial cells, fibroblasts, and some cancers cells such as MCF7 and PC3—were used for controls as the nonhematopoietic cells. miR-142-3p and miR-142-5p expression was analyzed by quantitative RT-PCR. Results shown are from 1 of 2 experiments. (E) Control miR expression analyses in human cells: The above cells were also concomitantly analyzed for expression of miR-744, 668, and 720 as additional controls. miR-744 was extensively expressed in hematopoietic, nonhematopoietic, and cancer cells. Both miR-668 and miR-720 were expressed in nonhematopoietic and cancer cells but not in hematopoietic cells.

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