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MicroRNAs regulated by LPS target genes that contribute to the inflammatory phenotype. Here we show that Akt1, which is activated by LPS, differentially regulates miRNAs including let-7e, miR-155, miR-181c and miR125b. In silico analyses and transfection studies revealed that let-7e represses TLR4 while miR-155 represses SOCS1, two genes critical for LPS-driven TLR signalling, 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 restores 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.
Activation of macrophages by LPS initiates feedback inhibitory loops that aim in establishing tolerance to a subsequent LPS stimulus. Negative feedback signals either inhibit the expression of genes that are required for the response to LPS, such as the LPS receptor TLR4, or promote the expression of genes that inhibit the response, such as Suppressor of Cytokine Signaling 1 (SOCS1) and IL-1 Receptor-Associated Kinase-M (IRAK-M). Thus, within a few hours from the start of LPS stimulation, the expression of the LPS receptor TLR4 drops, to rise again 24 to 48 hours later (Medvedev et al., 2000; Nomura et al., 2000). Negative regulators of TLR4 signaling such as SOCS1 (Kinjyo et al., 2002; Nakagawa et al., 2002) and IRAK-M (Kobayashi et al., 2002) are upregulated within two hours from the start of the stimulation. The net effect of the downregulation of TLR4 and the induction of negative regulatory molecules is the emergence of tolerance to LPS re-stimulation. Mutations that interfere with these negative regulatory loops promote hyper-responsiveness to LPS and inhibit the development of endotoxin tolerance. One pathway that plays a critical role in feedback inhibition of LPS signals is the PI-3K/Akt pathway (Fukao and Koyasu, 2003; Guha and Mackman, 2002; Luyendyk et al., 2008; Williams et al., 2006; Zhang et al., 2007).
Akt (also known as PKB) is a family of serine/threonine protein kinases that are activated by PI-3K and play key regulatory roles in a host of cellular functions including cell survival, cell proliferation, differentiation and intermediary metabolism. Stimulation of macrophages with the TLR4 agonist LPS, activates PI-3K and its downstream targets, including Akt kinases. Thus, activation of the PI-3K/Akt pathway suppresses LPS-activated MAPK and NF-κB cascades in monocytes and dendritic cells, resulting in decreased production of TNF-α and other cytokines (Fukao et al., 2002; Guha and Mackman, 2002). Moreover, inhibition of the Akt target GSK3 in vivo results in resistance to endotoxin shock (Martin et al., 2005), suggesting that inhibition of Akt will render mice more sensitive to LPS. Consistent with these observations, Akt over-expression in lymphocytes prevents sepsis-induced apoptosis and improves survival in a cecal ligation and puncture (CLP) model of poly-microbial sepsis (Bommhardt et al., 2004). Moreover, whereas p85α deficient macrophages are hyper-responsive, PTEN deficient macrophages are hypo-responsive to signals associated with macrophage activation (Luyendyk et al., 2008).
Micro RNAs (miRNAs) play a central role in gene expression. Mechanistically, miRNAs pair with partially complementary sequences in target mRNAs and regulate their stability and/or translation. Changes in the expression of miRNAs in course of the response to extracellular signals or during differentiation or oncogenic transformation, allow miRNAs to regulate these processes. Recent studies have identified miRNAs as critical regulators of immune responses including inflammation (Baltimore et al., 2008; Chong et al., 2008; Cobb et al., 2006; Liston et al., 2008; Lu et al., 2009; Xiao and Rajewsky, 2009; Zhou et al., 2008). In addition, LPS stimulation upregulates or represses the expression of miRNAs in macrophages. Among the LPS-responsive miRNAs characterized to date, miR-155 is upregulated in response to LPS (O’Connell et al., 2007; Tili et al., 2007) promoting the expression of TNF-α and destabilization of PU.1(Vigorito et al., 2007), a key mediator of monocyte/macrophage differentiation and TLR4 transcription (Roger et al., 2005; Tsatsanis et al., 2006; Vigorito et al., 2007). MiR-155 also targets and suppresses SOCS1, a critical regulator of STAT signalling, in regulatory T-cells (Lu et al., 2009) and the inositol phosphatase SHIP1 (O’Connell et al., 2009). In contrast, miR-125b inhibits the expression of TNF-α and is down-regulated in LPS-treated macrophages (Tili et al., 2007).
Given the importance of Akt in the negative feedback of TLR4 signaling, we examined the role of Akt1, one of the three Akt isoforms, in miRNA expression in LPS-activated macrophages. In mammals, the Akt family consists of three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, which are functionally distinct, despite their sequence similarities (Cho et al., 2001; Easton et al., 2005; Mao et al., 2007; Maroulakou et al., 2007; Yang et al., 2005). Recent studies relevant to the work presented in this paper showed that Akt1 ablation in mice, gives rise to a pro-inflammatory phenotype resulting in enhanced atherogenesis (Fernandez-Hernando et al., 2007).
In this paper, we examined the role of Akt1 in the regulation of microRNA expression in LPS-stimulated macrophages. miRNA arrays revealed, among other, that LPS-treated Akt1−/− macrophages failed to induce let-7e and expressed higher levels of miR-155. In silico analysis and transfection studies revealed that let-7e controls TLR4 and miR-155 SOCS1 and that these miRNAs are under the control of Akt1. As a result, Akt1−/− macrophages were hyper-responsive to LPS and failed to develop endotoxin tolerance in culture and in vivo. Introduction of these miRNAs in Akt1−/− macrophages restored their response to LPS, indicating that Akt1 controls macrophage responsiveness to LPS endotoxin tolerance by regulating the expression of microRNAs.
LPS-generated signals in macrophages regulate microRNAs that control the expression of molecules involved in TLR signaling (O’Connell et al., 2007; Tili et al., 2007). Since LPS signals activate Akt, we questioned whether Akt1 ablation affects the expression of microRNAs that regulate TLR signals in unstimulated and LPS-stimulated macrophages. MiRNA expression was analyzed using an array containing 365 miRNAs, as described in ‘Experimental Procedures’. Figure 1A depicts microRNAs the expression of which was affected in the presence or absence of LPS and Akt1, in thioglycollate-induced primary macrophages from Akt1+/+ and Akt1−/− mice. The first four miRNAs are the ones significantly affected. The results were confirmed by real time RT-PCR and showed that LPS promoted the expression of let-7e and miR-181c in Akt1+/+ cells only. In addition, LPS upregulated miR-155 and downregulated miR-125b, although in the absence of Akt1 LPS failed to suppress miR-125b, while induction of miR-155 was more pronounced (Figure 1B). In unstimulated Akt1−/− macrophages let-7e was expressed at higher levels. Interestingly however, the induction of let-7e was blocked in LPS-stimulated Akt1−/− macrophages (Figure 1B). Expression of let-7e and miR-155 was induced by 3 hours following LPS treatment and remained high at 24 hours following stimulation (data not shown). LPS did not induce let-7e even at 24 hours from the start of stimulation in Akt1−/− macrophages (data not shown).
MicroRNAs can be regulated at the transcriptional or post-transcriptional level. Transcriptional activation of the miRNA locus results in generation of the pri-miRNA which is subsequently processed to give rise to pre-miRNA and then to the mature miRNA. To determine if Akt1 affects let-7e, miR-155, miR-181c and miR-125b at the transcriptional or post-transcriptional level we measured expression of pri-let-7e, pri-miR-155, pri-miR-181c and pri-miR-125b in LPS-stimulated Akt1+/+ and Akt1−/− macrophages. The results showed that LPS increased the expression of pri-let-7e, pri-miR-155, and pri-miR-181c while it suppressed pri-miR-125b and ablation of Akt1 resulted in reduced pri-let-7e and pri-miR-181c, increased pri-miR-155 and abolished the suppression of pri-miR-125b (Figure 1C). We could, therefore, conclude that Akt1 regulates these miRNAs at the level of transcription.
To confirm that Akt1 controls the expression of let-7e, miR-155, miR-181c and miR-125b, primary Bone Marrow Derived Macrophages (BMDM) were infected with a retrovirus expressing myristyolated Akt1 (myrAkt1), a constitutively active mutant of Akt1, and GFP under independent promoters, to determine infection efficiency. 72 hours following infection almost all cells expressed GFP (data not shown). RNA was isolated and the expression of let-7e, miR-155, miR-181c and miR-125b was determined. Active Akt1 induced the basal expression of let-7e and mir-181c and suppressed the basal expression of miR-155 while it had no effect on the basal levels of miR-125b (Figure 1D). In LPS-stimulated cells, expression of myrAkt1 resulted in augmented let-7e and miR-181c expression, augmented suppression of miR-125b and reduced induction of miR-155 (Figure 1D), confirming that these miRNAs are under the control of Akt1. The same results were observed in the Raw264.7 macrophage cell line when infected with a myr-Akt-GFP retrovirus (data not shown) or transiently transfected with a myr-Akt1-GFP plasmid and GFP-positive cells were sorted by FACS and collected for further analysis (data not shown).
To identify potential targets for these microRNAs, we screened their sequences against the mouse genome database, using the microRNA target identification programs miRBase, PicTar and TargetScan v.4.0, as described in Experimental Procedures. This analysis identified TLR4 as a potential let-7e target, TNF-α as potential target of miR-125b and TNFSF11/RANKL as a potential target of miR-181c (Figure 1E). In addition to known miR155 targets, IKKε, TRAF6 and RIPK (Tili et al., 2007) this analysis identified SOCS1 as a potential miR155 target (Figure 1E). Our findings were consistent with previously published data showing that the let-7e isoform let-7i targets and promotes degradation of TLR4 mRNA in human cholangiocytes (Chen et al., 2007) and that miR-155 targets and suppresses SOCS1 in mouse regulatory T-cells (Lu et al., 2009).
TLR4 and SOCS1 are key regulators of macrophage sensitivity to LPS and their expression affects the magnitude of the response and, therefore, the levels of pro-inflammatory mediators. Since Akt1 affects let-7e and miR-155 and our predictions suggest that let-7e targets TLR4 and miR-155 targets SOCS1, we examined whether TLR4 and SOCS1 are regulated by Akt1.
We, therefore, tested whether Akt1 ablation affects TLR4 and SOCS1 expression. If the expression of TLR4 in macrophages is regulated by let-7e, one would expect that LPS stimulation would downregulate TLR4 in wild type but not in Akt1−/− macrophages. In addition, the expression of TLR4 in unstimulated Akt1−/− macrophages would be lower than in unstimulated wild type macrophages. Flow-cytometric and real-time RT-PCR analysis of unstimulated and LPS-stimulated wild type and Akt1−/− macrophages confirmed these predictions (Figure 2A, B, C), suggesting that let-7e indeed functions as a physiological inhibitor of TLR4 expression. LPS transiently enhanced TLR4 mRNA expression in Akt1−/− macrophages (Figure 2C), an effect that did not become evident at the protein level (Figure 2A).
Similarly, if the expression of SOCS1 in macrophages is regulated by miR-155, one would expect that LPS stimulation would upregulate the expression of SOCS1 more efficiently in wild type than in Akt1−/− macrophages. Western blot and real-time RT-PCR analysis revealed that LPS-treated Akt1−/− macrophages expressed lower levels of SOCS1 compared to their wild type counterparts (Figure 2D, E), suggesting that Akt1, via suppression of miR-155, augments SOCS1 expression.
To confirm that TLR4 and SOCS1 are regulated by Akt1, their expression was determined in primary BMDMs infected with a retrovirus expressing myrAkt1 and GFP, as described in Figure 1C and in the Experimental Procedures. Cells were analyzed for expression of TLR4 and SOCS1. The results indicated that myrAkt1 suppressed basal TLR4 expression (Figure 3A) and further augmented LPS-induced TLR4 suppression (Figure 3B, C). SOCS1 was induced by myrAkt1 and it further augmented LPS-induced SOCS1 protein and mRNA expression (Figure 3D, E). The same results were obtained in Raw264.7 macrophages infected with a retrovirus expressing myr-Akt1 or following transient transfection of myrAkt1 (data not shown).
Since Akt1 controls the expression of let-7e, miR-155 and their predicted targets TLR4 and SOCS1, we wanted to confirm that TLR4 and SOCS1 are indeed regulated by these miRNAs. Based on the sequence alignment of let-7e with TLR4 mRNA (Figure S1A), the miRNA programs mentioned above predicted that TLR4 is a likely target of let-7e. To validate the finding that let-7e suppresses TLR4, RAW264.7 macrophages were transfected with let-7e or with its antisense counterpart as-let7e. Control cells were transfected with scrambled miRNA (SCR-miRNA). Real time RT-PCR using total cell RNA harvested at the indicated time points revealed that whereas let-7e inhibits, as-let-7e enhances the expression of TLR4 (Figure 4A, B). Let-7e was effectively suppressed by as-let-7 within 12 hours following transfection (Figure S2). To confirm that let-7e interacted with the 3′UTR of TLR4, 383bp of the 3′UTR of TLR4 that included the let-7e binding site were cloned downstream of the luciferase gene (TLR4-UTR-luc) and co-transfected with let-7e or its antisense inhibitor as-let-7e in Raw264.7 macrophages. The results indicated that let-7e suppressed while as-let-7e enhanced luciferase expression and activity, indicating a direct interaction between let-7e and the 3′UTR of TLR4 (Figure 4C). Mutation at the seed sequence of the TLR4-3′UTR where let-7e was predicted to bind (mutTLR4-UTR-luc) abolished suppression by let-7e (Figure 4C).
Sequence alignment of miR-155 with SOCS1 mRNA predicted that SOCS1 is a likely target of miR-155 (Supplemental Figure S1B). Earlier studies indicated that miR-155 and SOCS1 interacted in regulatory T-cells and in 293T cells (Lu et al., 2009). To confirm that miR-155 also regulates SOCS1 in macrophages, RAW264.7 macrophages were transfected with as-miR-155 or SCR-miRNA. Real time RT-PCR using total cell RNA harvested at the indicated time points, revealed that as-miR-155 enhanced the expression of SOCS1 both in naïve (Figure 4D) and LPS-activated (Figure 4E) RAW264.7 cells. MiR-155 was effectively suppressed by as-miR-155 within 24 hours following transfection (Figure S3). To confirm that miR-155 interacted with the 3′UTR of SOCS1, a 397 bp fragment of the SOCS1 3′UTR that contained the miR-155 binding site was cloned downstream the luciferase gene (SOCS1-UTR-luc) and co-transfected with miR-155 or as-miR-155 in Raw264.7 macrophages. The results indicated that miR-155 suppressed while as-miR-155 enhanced luciferase expression and activity while mutation of the seed sequence for miR-155 at the 3′UTR of SOCS1 (mutSOCS1-UTR-luc) abolished the suppression (Figure 4F).
To determine whether differential expression of the microRNAs let-7e and miR-155 affects the magnitude of macrophage response to LPS, Raw264.7 macrophages were transfected with an as-let-7e, let-7e, miR-155 as-miR-155 or with combinations of these, prior to LPS stimulation and the pro-inflammatory mediators TNF-α, IL-6, IL-17, IP10, MCP1, MIP1a, and PGE2 were measured and compared to SCR-miRNA-transfected cells. Efficacy of let-7e and miR-155 suppression by as-let-7e and as-miR-155 was evaluated by real-time RT-PCR (data not shown). The results showed that introduction of as-let-7e augmented while let-7e suppressed the response to LPS. In contrast, introduction of as-miR-155 suppressed while miR-155 augmented the LPS response. Combination of as-let-7e and miR-155 exhibited the highest increase in pro-inflammatory mediators while combination of let-7e and as-miR155 significantly reduced all pro-inflammatory mediators (Figure 5A–G).
The preceding data suggest that differential expression of let-7e, miR-155 and their target genes TLR4 and SOCS1, may contribute to enhanced induction of pro-inflammatory cytokines by LPS in Akt1−/− macrophages. Indeed, stimulation of Akt1+/+ and Akt1−/− thioglycollate-elicited peritoneal macrophages with LPS for 24 hours resulted in increased expression of pro-inflammatory mediators. Thus, ablation of Akt1 resulted in increased secretion of the pro-inflammatory cytokines TNFα, IL-6 and IL-17 (Figure 5H, I, J), the pro-inflammatory chemokines IP10, MIP1a and MCP1 (Figure 5K, L, M) as well as increased production of PGE2 and NO (Figure 5N, O). Similar results were obtained using BMDMs from Akt1+/+ and Akt1−/− mice (data not shown), confirming that homologous deletion of Akt1 confers increased sensitivity to LPS.
Since Akt1 regulated let-7e and miR-155, and LPS-activated Akt1−/− macrophages expressed increased levels of TLR4 and reduced levels of SOCS1, we wanted to confirm that the Akt1 phenotype was mediated by these miRNAs. To this end, we transfected let-7e and as-miR155 or SCR-miRNA in primary BMDMs from Akt1−/− mice and expression of TLR4 and SOCS1 was determined upon LPS stimulation. The results showed that transfection of let-7e in Akt1−/− macrophages restored their ability to suppress TLR4 expression in response to LPS (Figure 6A, B). Similarly, transfection of as-miR-155 in Akt1−/− macrophages resulted in comparable levels of SOCS1 to those observed in Akt1+/+ mice (Figure 6C, D).
To confirm that the LPS-hyper-responsiveness observed in Akt1−/− macrophages was due to altered expression of let-7e and miR-155, Akt1−/− BMDMs were simultaneously transfected with let-7e and as-miR-155 or SCR-miRNA and stimulated with LPS for 12 or 24 hours. TNF-α and IL-6 were measured in the culture supernatants and showed that transfection of let-7e and as-miR-155 restored the response of Akt1−/− macrophages to LPS (Figure 6E, F), suggesting that Akt1 controls the responsiveness of macrophages to LPS via let-7e and miR-155. As-miR-155 effectively suppressed miR-155 in BMDMs (data not shown).
Given the increased response of Akt1−/− macrophages to LPS, reduced induction of SOCS1 and absence of TLR4 suppression, hallmark events for the development of endotoxin tolerance, we questioned whether ablation of Akt1 interferes with the development of endotoxin tolerance. When macrophages are exposed to an initial LPS stimulus they initiate tolerance mechanisms and upon re-stimulation they fail to effectively produce pro-inflammatory cytokines. BMDMs were, therefore, tolerized with an initial LPS treatment for 6 hours, culture media were changed and cells were exposed to a second LPS stimulus for different time periods. The results showed that while Akt1+/+ macrophages became tolerant to subsequent LPS stimulation and produced low amounts of pro-inflammatory cytokines, Akt1−/− macrophages did not and continued to produce pro-inflammatory cytokines in response to the second LPS stimulus (Figure 7A, B). To evaluate the contribution of let-7e and miR-155 in the abrogation of LPS-tolerance observed in Akt1−/− macrophages, Akt1−/− BMDMs were transfected with let-7e and as-miR-155 or SCR-miRNA, tolerized with a first LPS stimulus and then re-stimulated with LPS for different time periods. Pro-inflammatory cytokines were measured in culture supernatants and showed that introduction of let-7e and suppression of miR-155 in Akt1−/− macrophages restored tolerance to LPS (Figure 7A, B).
LPS-induced cytokine production in culture is the result of continuous stimulation since LPS remains active throughout the stimulation period, thus the differences in cytokine production observed in LPS-treated Akt1−/− macrophages in culture are, at least in part due to the defects in negative regulatory mechanisms. In vivo, LPS is rapidly neutralized by serum lipoproteins (Flegel et al., 1993; Freudenberg et al., 1980), allowing us to address the role of Akt1 at the early stages of macrophage activation. Intraperitoneal inoculation of a cohort of 12 wild type and 9 Akt1−/− mice with 1.5 mg LPS per 25g of body weight, being the minimal lethal dose as determined by titration experiments (see Experimental Procedures), revealed no significant differences in LPS sensitivity between the two genotypes (Figure 7C). Measuring TNF-α and IL-6 in the serum revealed no statistically significant differences between the two groups (Figure 7D, E). This result suggested that Akt1 may be redundant during the initial phase of macrophage activation but is required at later stages for the induction of miRNAs and development of endotoxin tolerance. Earlier studies have shown that overexpression of Akt1 suppresses ERK1/2 and NFkB activation in LPS-stimulated macrophages. We, thus, treated Akt1−/− and Akt1+/+ macrophages with LPS and measured ERK1/2 phosphorylation and IkBα degradation. LPS-activated Akt1−/− macrophages exhibited increased ERK1/2 activation (Figure S4) but no change in IkBα degradation (Figure S5), suggesting that Akt1 plays an inhibitory role in early TLR4 signals but its ablation is not sufficient to suppress pro-inflammatory cytokine production at the early stages of endotoxemia.
To determine if Akt1 is essential for the development of endotoxin tolerance in mice, another cohort of 9 wild type and 11 Akt1−/− mice was inoculated with a sublethal dose of LPS (500 μg/25g body weight) IP and 24 hours later, with a lethal dose of LPS (1.5mg/25g body weight), also IP. The results showed a significant delay in endotoxin-induced mortality in wild type, but not in Akt1−/− mice (Figure 7F) accompanied by increased TNF-α and IL-6 in the serum of Akt1−/− mice (Figure 7G, H), suggesting that the ablation of Akt1 inhibits the induction of endotoxin tolerance in vivo.
To address the involvement of let-7e and miR-155 in endotoxin tolerance in vivo, we treated Akt1+/+ and Akt1−/− mice with a tolerogenic sublethal dose of LPS. The expression of let-7e, miR-155 and their pri-miRNA precursors was measured in macrophages, which were isolated by cell sorting 6 hours later from the spleens of these animals. The results showed that let-7e was induced in Akt1+/+, but not in Akt1−/− macrophages and that miR-155 was induced in Akt1+/+ and super-induced in Akt1−/− macrophages (Figure 7I). Expression of TLR4 was measured in macrophages from the same mice at both the mRNA and protein levels. The results showed that TLR4 was downregulated in Akt1+/+, but not in Akt1−/− macrophages (Figure 7J, K). The expression of SOCS1 at the mRNA and protein levels was measured in macrophages and spleen lysates respectively. The results showed that SOCS1 was induced at higher levels in Akt1+/+ than in Akt1−/− macrophages (Figures 7J and 7K). These results confirmed that let7e, miR-155 and their targets are differentially regulated in vivo, in Akt1+/+ and Akt1−/− mice inoculated with a sublethal, tolerogenic dose of LPS.
To confirm that let7e and miR-155 were responsible for Akt1-dependent endotoxin tolerance in vivo, we first depleted C57BL/6 mice of macrophages, using liposome-encapsulated clodronate. The macrophage-depleted mice were reconstituted with Akt1+/+ or Akt1−/− bone marrow derived macrophages (BMDMs), transfected with let-7e and as-miR-155 or SCR-miRNA. To monitor the reconstitution of macrophage-depleted animals, donor BMDMs were transfected with FAM-labeled SCR-RNA, which rendered them fluorescent (see Supplemental figure S6). Experimental mice were inoculated with a sublethal dose of LPS and 24 hours later with the LD50 dose, as determined by titration experiments (see Supplemental Experimental Procedures). The results showed that mice reconstituted with Akt1−/− macrophages, or Akt1−/− macrophages transfected with SCR-miRNA were not tolerant to the second dose of LPS. However, mice reconstituted with BMDMs transfected with let-7e and as-miR-155 became tolerant, as indicated by their resistance to LPS determined by survival and cytokine production (Figure S7). We, therefore, conclude that Akt1 contributes to endotoxin tolerance in vivo by regulating the expression of let-7e and miR-155.
LPS stimulation of macrophages results in differential expression of miRNAs that contribute to the inflammatory response. Evidence presented in this paper confirmed these observations and identified miRNAs that lie under the control of Akt1. Moreover, we provide evidence associating miRNA expression with the regulation of molecules controlling macrophage sensitivity and tolerance to LPS.
The present study identified let-7e, miR-125b and miR-181c to be positively and miR-155 to be negatively regulated by Akt1 in LPS-activated macrophages. Earlier studies had shown that let-7i, an isoform of let-7e, targets TLR4 in human cholangiocytes (Chen et al., 2007), miR-155 targets TNF-α, PU.1, SHIP1 and SOCS1 (Lu et al., 2009; Tili et al., 2007; Vigorito et al., 2007; O’Connell et al., 2009) and that miR-125b targets TNF-α (Tili et al., 2007). Our silico analysis showed that let-7e targets TLR4, miR-155 targets SOCS1 and that miR-181c targets TNFSF11/RANKL. MiR-181c is suppressed in leukemia cells (Yu et al., 2006) and in response to TPA in HL-60 cells (Chen et al., 2008). The present work showed that miR-181c is induced by LPS, possibly contributing to LPS-induced RANKL suppression (Maruyama et al., 2006). Akt1 deletion resulted in inhibition of miR-181c induction, while RANKL was further inhibited in the absence of Akt1 (data not shown) suggesting that Akt1 controls RANKL through a more complex mechanism. LPS also suppressed miR-125b in an Akt1-dependent manner, while expression of the miR-125b target TNFα was augmented in Akt1−/− macrophages. It is likely that the higher levels of miR-125b in Akt1−/− macrophages cannot effectively suppress TNF-α and other targets of the same miRNA may positively contribute in LPS signaling. Our studies focused on let-7e and miR-155 since they control genes that regulate macrophage responsiveness to LPS. Indeed, Akt1 ablation resulted in hyper-responsiveness to LPS.
Previous studies have shown that PI-3K and its downstream target Akt are activated by TLR4 signals and that they may function in a feedback loop that inhibits the response to both the primary and secondary exposure to TLR4 activators (Fukao and Koyasu, 2003). These inhibitory effects may be mediated by PI3K targets other than Akt, or by Akt. We, therefore, examined the phenotypic consequences of a single Akt isoform ablation. Akt1 is only partly redundant during the early stages of macrophage activation since its deletion does not affect primary endotoxemia and NFkB activation but it augments ERK1/2 activation during the initial response to LPS, while it appears to be essential at later stages for the development of endotoxin tolerance. As a result ablation of Akt1, similar to the inhibition of PI3K, gives rise to a pro-inflammatory phenotype. This suggests that the pro-inflammatory phenotype of PI3K inhibition is, at least partly, due to Akt1 because despite the fact that Akt2 and Akt3 are also regulated by PI3K, they are functionally inert with regard to negative feedback signals in macrophages. All Akt isoforms are expressed in macrophages with Akt1 and Akt2 being expressed at higher levels than Akt3 (Shiratsuchi and Basson, 2007), but hyper-responsiveness to LPS appeared to be a specific attribute of Akt1−/− macrophages since the ablation of Akt2 was associated with hypo-responsiveness to the same stimulus both in culture and in vivo (Androulidaki et al, unpublished results).
The unique role of Akt1 is also supported by earlier studies which had shown that Akt1−/−/APOE−/− mice produce high levels of pro-inflammatory mediators which promote the development of atherosclerosis (Fernandez-Hernando et al., 2007). However, this study suggested that the pro-atherogenic phenotype promoted by Akt1 ablation, primarily depends on endothelial cell dysfunction and production of pro-inflammatory mediators. Here we show that macrophages are also affected by Akt1 ablation and contribute to a pro-inflammatory phenotype. Moreover, we demonstrate that miRNAs mediate the augmented response to TLR4 signals and genetic manipulation of these miRNAs restores the Akt1−/− pro-inflammatory phenotype. Whether the pro-inflammatory phenotype in endothelial cells depends on the same miRNAs remains to be determined.
In conclusion, our data suggest that systemic inhibition of Akt1, drugs for which are currently under development, may contribute to a pro-inflammatory phenotype via differential regulation of miRNAs. Understanding the mechanism through which PI3K and Akt1 modulates the inflammatory response will allow us to selectively target molecules controlling PI3-K/Akt1 signals to positively or negatively regulate the inflammatory response. Given the importance of Akt and miRNAs in cancer and other human diseases these findings may have significant translational implications.
C57BL/6 mice were purchased from the Hellenic Pasteur Institute (Athens, Greece). Akt1−/− and Akt1+/+ mice (Mao et al., 2007; Maroulakou et al., 2007) were housed at the University of Crete School of Medicine, Greece. All procedures described below were approved by the Animal Care Committee of the University of Crete School of Medicine, Heraklion, Crete, Greece and from the Veterinary Department of the Heraklion Prefecture, Heraklion, Crete, Greece, and by the DLAM at TUFTS-Medical Center, Boston, USA.
The murine macrophage cell line RAW264.7 and primary murine peritoneal macrophages were cultured as previously described (Arranz et al., 2008). E. coli-derived LPS (1μg/ml) (O111:B4; catalogue no. L2630; Sigma-Aldrich) was used as described at the results section.
Total cellular RNA was isolated using TRIzol Reagent (Invitrogen). cDNA was prepared by reverse transcription (Thermoscript RT; Invitrogen) and amplified by PCR using the following primers: β-actin, 5′-TCAGAAGAACTCCTATGTGG-3′/5′-TCTCTTTGATGTCACGCA CG-3′; SOCS1, 5′-TCTATTGGGGACCCCTGAG-3′/5′-GAAGCCATCTTCACGCTGAG-3′. Detection of TLR4 mRNA was performed as previously described (Medvedev et al., 2000; Nomura et al., 2000; Tsatsanis et al., 2006). Amplification was performed as in an ABI PRISM 7000 Real-Time PCR engine. The amplification efficiency of the TLR4 and SOCS1 was the same as the one of β-actin as indicated by the standard curves of amplification, allowing us to use the formula: fold difference = 2−(ΔCtA −ΔCtB), where Ct is the cycle threshold. Reactions were performed in triplicate to allow statistical evaluation.
Cells were washed twice with PBS containing 1% BSA. Non specific binding was blocked using Fc-block (BD Biosciences). Washed cells were resuspended in a 1:200 dilution of PE-conjugated anti-mouse TLR4 antibody (clone MTS510; e-Bioscience) or the anti-mouse pan-macrophage marker F4/80 (Clone BM8- e-Bioscience). Stained cells were washed twice with PBS containing 1% BSA and they were analyzed by flow cytometry (FACSCalibur, BD Biosciences and CyAn, DAKO). Sorting of F4/80 stained macrophages was performed in a MoFlo cell sorter (DAKO).
To titrate the dose of LPS, mice received i.p injections of 1, 1.5 or 2 mg of LPS per 25g of body weight. LPS was dissolved prior to injection in phosphate-buffered saline at a concentration of 10 mg/ml. Injected animals were monitored for a 24 hour period and they were sacrificed when moribund. The results showed that the minimal lethal dose of LPS was 1.5 mg (data not shown), which was used in the subsequent experiments. Cytokine concentration in serum was determined by enzyme-linked immunoabsorbent assay (ELISA) at the indicated time points. ELISA assays measuring cytokine concentration in serum samples and in culture supernatants were carried out using ELISA kits (R&D Systems) or a bead-based ELISA assay (Lincoplex-Millipore).
The expression profile of 365 microRNAs was addressed using TaqMan microRNA microarrays as previously described (Thum et al., 2007). The results were validated with the mirVana qRT–PCR miRNA Detection Kit and qRT–PCR Primer Sets, according to the manufacturer’s instructions (Ambion). Expression of RNU48 was used as an internal control. Expression of pri-miRNAs was determined using the following primers: for pri-let-7e: forward: 5′-TTCTGGTCTCCCATCAATCC-3′ and reverse 5′-CAGAGAAGACACCCCAGCTC-3′; for pri-miR-155: forward 5′ ACCCTGCTGGATGAACGTAG-3′ and reverse 5′-CATGTGGGCTTGAAGTTGAG-3′; for pri-miR-181c: forward: 5′-ACCCTGGAGGGTTAGGTGTT-3′ and reverse: 5′-CAGACTTCATTCCCGCTCAT-3′; for pri-miR-125b: forward: 5′-AGCAAGGGTTTCACCATGAC-3′ and reverse: 5′-CACACCCAAAGCGATGTTTA-3′; 18S was used as internal control. Potential miRNA targets were identified using the miRBase (http://microrna.sanger.ac.uk/), PicTar (http://pictar.bio.nyu.edu/) and TargetScan version 4.0 (http://www.targetscan.org/index.html) search engines. To optimize the accuracy of prediction, a given target should be predicted by a minimum of 2 out of 3 programs and the targeted sequence should be conserved among species.
Detection of SOCS1 protein was performed by western blot analysis as previously described (Dumitru et al., 2000). SOCS1 was detected using a polyclonal anti-SOCS1 antibody (ZYMED-Invitrogen) and protein loading was determined using a monoclonal anti-tubulin antibody (SIGMA). Proteins were visualized using the ECL Western blotting kit (ECL Amersham Biosciences) and blots were exposed to Kodak X-Omat AR films.
BMDMs were differentiated as previously described (Dumitru et al., 2000). Following differentiation cells were seeded in 6-well plates and infected with a retrovirus expressing myristyolated Akt1 and GFP under the control of two distinct CMV promoters. 72 hours later almost all cells (>90%) expressed Akt1 and GFP as determined by fluorescence microscopy and FACS analysis. Raw264.7 cells were infected with the myr-Akt1-GFP retrovirus using the same procedure. In parallel experiments a plasmid expressing Akt1 and GFP under CMV promoters was transfected in Raw264.7 cells using lipofectamine 2000 (Invitrogen) and GFP expressing cells were sorted and analyzed (Dako cytomation sorter).
For miRNA tranfection studies, Raw264.7 cells were seeded in 6-well plates and they were transfected with 50nM of miRNAs let-7e and miR-155, or with antisense miRNAs as-let-7e and as-miR-155 (Ambion) or with a Scramble miRNA (SCR-miRNA, Ambion Cat#AM17110), using the transfection agent siPORT NeoFX. RNA was extracted at different time points (0, 6, 12, 24 and 48 hours) after microRNA transfection and Real-time PCR analysis was performed as described above.
The 3′UTR of murine TLR4 and murine SOCS1 was cloned in the pMIR-REPORT-luciferase vector (Ambion) as described in Supplemental Experimental Procedures. Constructs were transfected in Raw264.7 and luciferase activity was measured using the Luciferase Reporter Assay System (Promega). Each sample was assayed in triplicate.
All values are expressed as the mean±SEM of data obtained from at least three experiments. Comparison between groups was made using the Student’s t-test and ANOVA test. Log Rank analysis was used for survival experiments. P< 0.05 was the significance level.
This work was partly supported from the Association for International Cancer Research (AICR07-0072) and UICC ICRETT (ICR-08-054) with Federal funds from the NCI, NIH under Contract #2-CO-41101 to CT, from the Hellenic Secretariat for Research and Technology (ΠENEΔ 03EΔ372) to ANM and from the National Institutes of Health (R01-CA057436) to PNT.
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