Ariadne Androulidaki

The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.

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.

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2−/− mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1−/− mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2−/− macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2−/− macrophages, and its target C/EBPβ appear to play a key role in this process. C/EBPβ, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2

Macrophages undergo apoptosis as a mechanism of regulating their activation and the inflammatory reaction. Macrophages express the Corticotropin‐Releasing Factor Receptor‐2 (CRFR2) the endogenous agonists of which, the urocortins, are also present at the site of inflammation. We have found that urocortins induced macrophage apoptosis in a dose‐ and time‐dependent manner via CRFR2. In contrast to lipopolysaccharide (LPS)‐induced apoptosis, the pro‐apoptosis pathway activated by urocortins involved the pro‐apoptotic Bax and Bad proteins and not nitric oxide, JNK and p38MAPK characteristic of LPS. In conclusion, our data suggest that endogenous CRFR2 ligands exert an anti‐inflammatory effect via induction of macrophage apoptosis.

Corticotropin releasing factor receptor 1 (CRF1) and CRF2 agonists exert an anti-inflammatory effect during the early phase of inflammation suppressing LPS-induced TNF-α release from macrophages via induction of COX-2 and PGE2†

Corticotropin‐releasing factor (CRF), the principal regulator of the hypothalamus‐pituitary‐adrenal (HPA) axis, also modulates the inflammatory response directly, via its effect on mast cells and macrophages. On macrophages, it augments production of lipopolysaccharide (LPS)‐induced pro‐inflammatory cytokines. CRF and its related peptides may also act as anti‐inflammatory agents. Aim of the present work was to examine the role of macrophages on the anti‐inflammatory effects of CRF‐peptides and the mechanism involved. Thus, we examined if CRF receptor 1 (CRF1) and CRF2 agonists exert any anti‐inflammatory effect on primary mouse macrophages. We have found that: (a) CRF, Urocortin (UCN)1 and UCN2 transiently suppressed the release of Tumor Necrosis Factor‐alpha (TNF‐α) in LPS‐activated macrophages, an effect peaking at 4 h. This effect did not involve changes on TNF‐α transcription. (b) CRF peptide‐induced suppression of TNF‐α release depended on induction of COX‐2 and PGE2 synthesis. (c) Use of specific CRF1 and CRF2 antagonists suggested that this effect involved both CRF receptor types. (d) The effect of CRF‐peptides on COX‐2 was mediated via PI3K and p38MAPK. (e) Longer exposure of macrophages to CRF‐peptides resulted in induction of TNF‐α production via enhancement of its transcription. In conclusion, this is the first report suggesting that CRF1 and CRF2 agonists exert a biphasic effect on macrophages. During the early stages of the inflammatory response, they suppress TNF‐α release via induction of COX‐2/PGE2 while later on they induce TNF‐α transcription. Hence, the reported anti‐inflammatory effect of CRF‐peptides appears to involve macrophages and is confined at the early stage of inflammation. J. Cell. Physiol. 210: 774–783, 2007.

Corticotropin-Releasing Factor and the Urocortins Induce the Expression of TLR4 in Macrophages via Activation of the Transcription Factors PU.1 and AP-1

Corticotropin-releasing factor (CRF) augments LPS-induced proinflammatory cytokine production from macrophages. The aim of the present study was to determine the mechanism by which CRF and its related peptides urocortins (UCN) 1 and 2 affect LPS-induced cytokine production. We examined their role on TLR4 expression, the signal-transducing receptor of LPS. For this purpose, the murine macrophage cell line RAW 264.7 and primary murine peritoneal macrophages were used. Exposure of peritoneal macrophages and RAW 264.7 cells to CRF, UCN1, or UCN2 up-regulated TLR4 mRNA and protein levels. To study whether that effect occurred at the transcriptional level, RAW 264.7 cells were transfected with a construct containing the proximal region of the TLR4 promoter linked to the luciferase gene. CRF peptides induced activation of the TLR4 promoter, an effect abolished upon mutation of a proximal PU.1-binding consensus or upon mutation of an AP-1-binding element. Indeed, all three peptides promoted PU.1 binding to the proximal PU.1 site and increased DNA-binding activity to the AP-1 site. The effects of CRF peptides were inhibited by the CRF2 antagonist anti-sauvagine-30, but not by the CRF1 antagonist antalarmin, suggesting that CRF peptides mediated the up-regulation of TLR4 via the CRF2 receptor. Finally, CRF peptides blocked the inhibitory effect of LPS on TLR4 expression. In conclusion, our data suggest that CRF peptides play an important role on macrophage function. They augment the effect of LPS by inducing Tlr4 gene expression, through CRF2, via activation of the transcription factors PU.1 and AP-1.

Adiponectin Promotes Endotoxin Tolerance in Macrophages by Inducing IRAK-M Expression

High levels of plasma adiponectin are associated with low levels of inflammatory markers and cardioprotection. The mechanism via which adiponectin exerts its anti-inflammatory effect is yet unknown. In the present study, we demonstrate that globular adiponectin (gAd) induces the expression of the inactive isoform of IL-1R-associated kinases (IRAK), IRAK-M. Homologous deletion of IRAK-M in IRAK-M−/− mice abolished the tolerogenic properties of gAd because pretreatment of IRAK-M−/− macrophages with gAd did not suppress LPS-induced proinflammatory cytokine production. GAd activated the MAPKs MEK1/2 and ERK1/2 in macrophages via their upstream regulator Tpl2. Activation of ERK1/2 via Tpl2 appeared necessary for the induction of IRAK-M because gAd did not induce IRAK-M in Tpl2−/− macrophages or in macrophages pretreated with the MEK1/2 inhibitor UO126. In addition, activation of PI3K and Akt1 also appeared necessary for the induction of IRAK-M by gAd, because treatment of Akt1−/− macrophages or pretreatment of macrophages with the PI3K inhibitor wortmannin abolished gAd-induced IRAK-M expression. Analysis of IRAK-M expression in human peripheral blood cells confirmed that serum adiponectin was negatively associated with IRAK-M and responsiveness to LPS. In conclusion, our data demonstrate that IRAK-M is a major mediator of gAd-induced endotoxin tolerance in primary macrophages, expression of which depends on the activation of Tpl2/ERK and PI3K/Akt1 signaling pathways.

Peripheral Factors in the Metabolic Syndrome The Pivotal Role of Adiponectin

Abstract:  Several recently published reports, including ours, suggest that adiponectin is a strong proinflammatory agent. Indeed, exposure of human placenta and adipose tissue to adiponectin induces the production of interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor α (TNF‐α), and prostaglandin E2 (PGE2). We have previously shown that adiponectin is a powerful inducer of proinflammatory cytokines production by macrophages. The reported anti‐inflammatory effect of adiponectin may be due to the induction of macrophage tolerance to further adiponectin exposure or to other proinflammatory stimuli including the Toll‐like receptor (TLR) 3 ligand polyI:C and the TLR4 ligand lipopolysaccharide (LPS). We now present additional data supporting the hypothesis that adiponectin is a strong proinflammatory adipokine. More specifically, we demonstrate that adiponectin induces IL‐1β and IL‐8 from THP‐1 macrophage cell line. The effect of adiponectin is not restricted to differentiated THP‐1 macrophages but it is evident at lower levels in undifferentiated THP‐1 monocytes promoting TNF‐α, IL‐6, and IL‐8 production. Thus, its high levels in the circulation of lean subjects render their macrophages resistant to several proinflammatory stimuli including its own thus acting in effect as an anti‐inflammatory agent. Lowering of its high levels, as a consequence of increased body mass index (BMI), renders macrophages sensitive to any proinflammatory insult.

Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells

The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2−/− genetic background. Surprisingly, the TCR2Ctg/tg/Tpl2−/− mice developed T cell lymphomas with a latency of 4–6 months. The tumor cells were consistently TCR2C+CD8+CD4−, suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8+ ISP cells was not expanded in the thymus of TCR2Ctg/tg/Tpl2−/− mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2−/− mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

Vasoactive intestinal peptide suppresses toll-like receptor 4 expression in macrophages via Akt1 reducing their responsiveness to lipopolysaccharide

Toll-like receptor 4 (TLR4) recognizes and initiates signals from Gram-negative bacterial lipopolysaccharide (LPS) triggering the inflammatory response. Expression levels of TLR4 on macrophages partly regulate the magnitude of the response to LPS. Vasoactive Intestinal Peptide (VIP) is known to block inflammatory responses by inhibiting pro-inflammatory cytokine production from activated macrophages. In the present report we demonstrate that VIP directly suppressed TLR4 expression on naïve primary mouse macrophages utilizing signalling cascades that control TLR4 transcription. VIP-induced suppression of TLR4 occurred at the transcriptional level by decreasing PU.1 DNA binding. Mutation of the proximal PU.1 but not the AP-1-binding site on the TLR4 promoter abrogated VIP-induced suppression of TLR4 transcription. Moreover, inhibition of PI3K by wortmannin or homologous deletion of the Akt1 isoform, a pathway known to act as a negative regulator of macrophage activation, alleviated the suppressive action of VIP on TLR4 expression. To evaluate the biological significance of VIP effect on TLR4 expression, Raw264.7 macrophages were pre-treated with VIP for 24h and then exposed to LPS. Pre-treatment with VIP rendered macrophages hypo-responsive to LPS resulting in reduced pro-inflammatory cytokine production. Moreover, in vivo administration of VIP in C57BL/6 mice resulted in lower IL-6 production upon treatment with LPS. Overall, the results indicate that VIP promotes endotoxin tolerance by downregulating TLR4 expression via Akt1.

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness

IntroductionCancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells.MethodsIn the present study we report our findings regarding the impact of CRF on breast cancer cell motility and invasiveness. For this purpose we used the MCF7 breast cancer cell line and evaluated the effect of CRF on motility and invasiveness using the wound-healing and boyden-chamber assays. In addition, we measured the effect of CRF on molecules that mediate motility by western blot, immunofluorescence, ELISA and RT-PCR.ResultsOur findings show that: 1. CRF transiently inhibited the apoptosis of MCF7 cells. 2. CRF enhanced MCF7 cell motility in a wound healing assay and their invasiveness through extracellular matrix. 3. CRF increased actin polymerization, phosphorylation of Focal Adhesion Kinase (FAK), providing a potential mechanism for the observed induction of MCF7 motility. 4. CRF induced the expression of Cox-1 but not Cox-2 in MCF7 cells as well as the production of prostaglandins, factors known to promote invasiveness and metastasis.ConclusionOverall, our data suggest that CRF stimulates cell motility and invasiveness of MCF7 cells most probably via induction of FAK phosphorylation and actin filament reorganization and production of prostaglandins via Cox1. Based on these findings we postulate that the stress neuropeptide CRF present in the vicinity of tumors (either produced locally by the tumor cells themselves or by nearby normal cells or secreted from the innervations of surrounding tissues) may play an important role on breast tumor growth and metastatic capacity, providing a potential link between stress and tumor progression.