The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs

Abstract

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

Figure 1. MiRNAs are differentially induced by LPS in the absence of Akt1

(A). Primary macrophages from Akt1+/+ and Akt1−/− mice were stimulated for 4 hours with LPS and differences in the miRNA profile were analyzed and validated by real time RT-PCR. (B) Comparison of let7-e, miR-155, miR-181c and miR-125b expression in stimulated and unstimulated Akt1+/+ and Akt1−/− macrophages. Results are representative of three independent experiments (±SEM); **p<0.01, ***p<0.001. (C) Akt1 regulates let-7e, miR-155, miR-181c and miR-125b at the transcriptional level. Expression of the corresponding pri-miRNAs was measured in LPS-stimulated Akt1+/+ and Akt1−/− primary BMDMs. ***p<0,001 compared to LPS-stimulated Akt1+/+ macrophages; ###p<0.001 compared to unstimulated Akt1+/+ macrophages. (D) Primary BMDMs were infected with a retrovirus expressing myrAkt1 and 72 hours later stimulated with LPS. Let-7e, miR-155 and miR-125b expression was measured 24 hours following stimulation. Results represent 2 independent experiments (±SEM). ***p<0.001 compared to mock-transfected and non-LPS-treated cells, ###p<0.001 compared to mock-transfected LPS-treated cells at the same time point. (E) miRNA targets identified by in silico analysis.

Figure 2. Akt1 regulates the expression of TLR4 and SOCS1

(A) Protein expression of TLR4 in primary thioglycollate-elicited peritoneal macrophages from Akt1−/− and Akt1+/+ mice treated with 1μg/ml LPS for 24 hours was determined by FACS analysis using a TLR4-PE conjugated Ab. Results are representative of three independent experiments. (B) Protein expression of TLR4 in primary thioglycollate-elicited peritoneal macrophages from Akt1−/− and Akt1+/+ mice measured by FACS analysis. (C) mRNA expression of TLR4 in macrophages from Akt1−/− and Akt1+/+ mice treated with LPS. (D, E) Primary thioglycollate-elicited peritoneal macrophages from Akt1−/− and Akt1+/+ mice were treated with LPS for 6, 12 or 24 hours. The LPS- induced expression levels of SOCS1 protein were measured by western blot (D) and mRNA were measured by Real-Time PCR (E). Results represent 3 independent experiments (±SEM). *p<0.05, ***p<0.001 compared to unstimulated cells of the same genotype; #p<0.05, ##p<0.01, ###p<0.001 compared to wild type LPS-treated cells at the same time point.

Figure 3. Overexpression of Akt1 affects TLR4 and SOCS1 expression

(A) BMDMs from C57BL/6 mice were infected with a retrovirus expressing myrAkt1 and GFP. The levels of TLR4 protein was measured in GFP positive, myrAkt1 overexpressing cells by FACS; control: mock transfected cells. (B, C) BMDMs overexpressing myrAkt1 were stimulated with LPS for 24 hours and TLR4 protein was measured by FACS (B) and TLR4 mRNA by real-time RT-PCR (C); control: mock transfected cells without LPS stimulation. (D, E) SOCS1 expression in myr-Akt1 transfected BMDMs in the presence or absence of LPS was measured at the protein (D) and mRNA level (E). Results represent 3 independent experiments (±SEM); *p<0.05, **p<0.01.

Figure 4. let-7e and miR-155 regulate TLR4 and SOCS1, respectively

(A) Raw264.7 macrophages were transfected with let-7e or SCR-miRNA and expression of TLR4 mRNA was measured at different time points by real-time RT-PCR. (B) Raw264.7 macrophages were transfected with as-let7e or SCR-miRNA and TLR4 mRNA was measured at different time points. (C) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′UTR of TLR4 (TLR4-UTR-luc) or with the same construct containing a mutant seed sequence at the let-7e binding site (mutTLR4-UTR-luc) in the presence or absence of let-7e or as-let-7e. (D) As-miR-155 was transfected in Raw264.7 macrophages and SOCS1 mRNA was measured by real-time RT-PCR. (E) As-miR-155 or SCR-miRNA was transefected in macrophages stimulated with LPS and SOCS1 expression was measured by real-time RT-PCR. (F) Raw264.7 macrophages were transfected with a luciferase construct containing the 3′UTR of SOCS1 (SOCS1-UTR-luc) or with the same construct containing a mutant seed sequence at the miR-155 binding site (mutSOCS1-UTR-luc) in the presence or absence of miR-155 or as-miR-155. Results represent 3 independent experiments (±SEM); **p<0.01, ***p<0.001 compared to time 0 in the mRNA quantification experiments and to SCR-miRNA-transfected cells in the luciferase experiments.

Figure 5. let-7e and miR-155 and Akt1 control macrophage sensitivity to LPS

Raw264.7 cells were transfected with let-7e, mir-155, as-let-7e, as-miR-155 or combinations of these. TNF-α (A), IL-6 (B), IL-17 (C), IP-10 (D), MCP1 (E), MIP1a (F) and PGE2 (G) were measured at 12 and 24 hours following LPS stimulation. Results represent 3 independent experiments; (±SEM) *p<0,05, **p<0,01 and ***p<0,001 compared to control-transfected cells stimulated with LPS for the same time period. Thioglycollate elicited macrophages from Akt1−/− and Akt1+/+ mice were stimulated with LPS for 24 hours; TNF-α (H), IL-6 (I), IL-17 (J), IP-10 (K), MCP1 (L), MIP1a (M), PGE2 (N) and NO (O) were measured in the culture supernatants. Results represent 5 independent experiments; (±SEM) **p<0.01; ***p<0.001 compared to Akt1+/+ cells.

Figure 6

Altered LPS-response of Akt1−/− macrophages depends on let-7e and miR-155. Akt1−/− macrophages were transfected with let-7e, as-miR-155 or combination of the two and stimulated with LPS. Expression of TLR4 mRNA (A) and protein (B) and SOCS1 mRNA (C) and protein (D) was measured. (±SEM) **p<0.01; ***p<0.001 compared to SCR-miRNA-transfected Akt1−/− cells at the same time point; ###p<0.001 compared to Akt1+/+ macrophages at time 0. (E, F) Production of TNF-α (E) and IL-6 (F) was measured by ELISA in the culture supernatant. Results are representative of three independent experiments; (±SEM) **p<0.01; ***p<0.001 compared to SCR-miRNA-transfected Akt1−/− cells at the same time point.

Figure 7. Akt1 controls the development of endotoxin tolerance

(A, B) BMDMs from Akt1−/− mice were transfected with SCR-miRNA or with let-7e and as-miR-155, stimulated with LPS for 6 hours and then re-stimulated with LPS. TNF-α (A) and IL-6 (B) was measured in the culture supernatant. Results are representative of three independent experiments; (±SEM) **p<0,01, ***p<0,001 compared to Akt1−/− macrophages transfected with let-7e and as-miR-155, and to Akt1+/+ macrophages. (C) Akt1−/− and Akt1+/+ mice were treated once with 1.5 mg/25gr LPS and survival was monitored. TNF-α (D) and IL-6 (E) were measured in the serum 2 and 4 hours following LPS administration, respectively. Results represent measurements of 6 mice per group. (F) Akt1−/− and Akt1+/+ mice were treated with 500μg/25gr for 24h followed by a second LPS stimulation of 1.5mg/25gr. Survival of animals was monitored. Serum TNF-α (G) and IL-6 (H) was measured 1 and 4 hours following LPS re-stimulation, respectively. Results represent measurements from 5 mice per group; *p<0.05, **p<0.01. (I) F4/80⁺ macrophages were isolated from the spleen of LPS-injected (500μg/25g) Akt1+/+ and Akt1−/− mice 6 hours following injection and let-7e, miR-155, pri-let-7e and pri-miR-155 were measured. (J) TLR4 and SOCS1 mRNA expression was measured in the same samples. (K) TLR4 expression was measured by FACS analysis in F4/80⁺ cells and SOCS1 was measured by Western blot in spleens from the same animals. Results represent the average of three independent experiments.