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M2 activation state in bone marrow-derived macrophages
Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Research Article
Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages James P. Barrett 1, Aedín M. Minogue n,1, Aidan Falvey, Marina A. Lynch Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland
art ic l e i nf o
a b s t r a c t
Article history: Received 6 February 2015 Received in revised form 22 April 2015 Accepted 18 May 2015
Macrophages can be polarised to adopt the M1 or M2 phenotype and functional outcomes of activation include altered secretion of immune molecules such as insulin-like growth factor (IGF)-1 as well as upregulation of cell surface molecules specifically associated with each state. Interleukin (IL)-4 mediates its effects through two receptors, the type I and II receptors and activation of these receptors results in phosphorylation of signal transducers and activators of transcription (STAT)6. JAK3 is activated as a consequence of ligation of the type I IL-4R, which participates in Akt activation. We set out to investigate the impact of perturbation of IGF-1 tone on IL-4- and interferon (IFN)γ–induced activation, the mechanisms by which this may occur and the contribution of type I IL-4R activation to adoption of the M2 state. The data presented here indicate that IL-4-induced activation of Akt is JAK3-dependent, enhanced by release of IGF-1 and necessary for full adoption of the M2 phenotype, since blocking IGF-1 activity blunts the ability of IL-4 to induce activation of Akt and to upregulate expression of some M2-associated molecules. In addition, differential control of the expression of mannose receptor (MRC1), arginase-1 (Arg-1), chitinase-3 like 3 (Chi3l3) and found in inflammatory zone 1 (FIZZ1) was observed. The IFNγinduced decrease in IGF-1 was exacerbated by inhibition of phosphatidylinositol-3 (PI3) kinase, indicating that Akt may regulate its own activation via IGF-1. Overall, a deficit in IGF-1/Akt signalling is associated with decreased capacity to induce the M2 state and an increased responsiveness to IFNγ. & 2015 Published by Elsevier Inc.
Keywords: BMDMs; IGF-1; Akt; IL-4; IFNγ;
1. Introduction The initial discovery that macrophages adopt the so-called classical activation, M1, phenotype in response to interferon-γ (IFNγ) and the alternative activation, M2, phenotype in response to IL-4, dates back over 20 years [1] though more recent evidence suggests additional activation states also exist [2]. Emerging data also highlights the difference in functionality between M1 and M2 phenotypes [3–5]. An understanding of the phenotypes and their modulation has become increasingly important in the context of neurodegenerative disorders with the recognition that infiltration of macrophages into the brain parenchyma occurs in these diseases, and modulating these phenotypes may provide a potential Abbreviations: ANOVA, analysis of variance; Arg1, Arginase-1; BMDM, bone marrow-derived macrophages; Chi3l3, chitinase-3 like 3; EtOH, ethanol; FIZZ1, found in inflammatory zone 1; IFNγ, interferon-γ; IGF-1, insulin-like growth factor 1; IL-4, interleukin-4; MRC1, mannose receptor; NOS2, nitric oxide synthase 2; PI3K, phosphatidylinositol-3 kinase; JAK1, janus kinase 1; JAK3, janus kinase 3; STAT1, signal transducers and activators of transcription 1; STAT6, signal transducers and activators of transcription 6; TNFα, tumour necrosis factor α n Corresponding author. E-mail address: [email protected] (A.M. Minogue). 1 These authors contributed equally to the work.
strategy to limit disease progression. To maximise the potential of identifying strategies for modulation of disease progression, a comprehensive understanding of the signalling pathways that control the switch between phenotypes is imperative. The M1 and M2 phenotypes are characterised by upregulated expression of specific molecules. Increased expression of tumour necrosis factor (TNF)α and nitric oxide synthase (NOS)2 is associated with the M1 phenotype while the M2 anti-inflammatory phenotype, is characterised by upregulation of molecules such as mannose receptor (MRC1), arginase 1 (Arg1), chitinase 3-like 3 (Chi3l3) and found in inflammatory zone (FIZZ)1. IL-4 signals through the type I and type II receptors and activation of the type I receptor facilitates the phosphorylation of JAK3 while the type II receptor induces JAK1 activation [6] and results in phosphorylation of STAT6 in macrophages [6]. The presence of distinct receptor types presents a possibility by which functions of the M2 state may be distinctly regulated. It has been shown that IL-13, which engages the type II receptor and results in the same level of STAT6 phosphorylation as IL4, does not upregulate expression of Arg1, Chi3l3 and FIZZ1 to the same extent as IL-4 [7]. In addition, IL-4 stimulates activation of IRS2 and PI3K/Akt while exposure to IL-13 does not. One of the functional outcomes of macrophage activation is the secretion of immune modulatory molecules and macrophages
http://dx.doi.org/10.1016/j.yexcr.2015.05.015 0014-4827/& 2015 Published by Elsevier Inc.
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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produce IGF-1 in response to IL-4, while IFNγ downregulates IGF-1 transcription [8]. The impact of perturbation of IGF-1 tone on macrophage activation has not been investigated though IGF-1 antagonises the pro-inflammatory effects of IFNγ in microglia in vitro [9] and an inverse correlation between IGF-1 and IFNγ, associated with neuroinflammation, has been reported in the brains of aged rats [10]. IGF-1 is a potent activator of the PI3K/Akt signalling pathway and, together with its ability to inhibit the effects of IFNγ, may enhance the ability of macrophages to adopt the M2 state. We set out to investigate the role that IGF-1/Akt activity plays in modulating macrophage phenotype. The data show that, while IL-4-induced MRC1 transcription was STAT6-mediated, induction of Arg1, Chi3l3 and FIZZ1 was driven by activation of JAK3 and Akt. We show that IL-4-induced production of IGF-1, which was JAK3and PI3K/Akt-dependent, was necessary for macrophages to fully adopt the M2 phenotype while decreased IGF-1/Akt activity was associated with an exacerbated response to IFNγ stimulation, which was independent of STAT1. We propose that a disturbance in IGF-1/Akt tone may lead to a dysregulation of macrophage activation.
2. Materials and methods 2.1. Preparation and culture of BMDMs BMDMs were isolated from the marrow of the femurs and tibias of mice. The legs of the animals were sprayed with 70% EtOH and the skin and muscle tissue removed from the bones. The bones were sprayed with 70% EtOH, transferred to a sterile flow hood and cut at both ends. The marrow was flushed out into a sterile falcon tube in Dulbecco's modified Eagle's medium (DMEM; 500 ml; Invitrogen, UK) supplemented with heat-inactivated foetal bovine serum (FBS; 50 ml; 10%; Gibco, UK) and penicillin–streptomycin (5 ml; 1%; Gibco, UK). The cell suspension was triturated using a sterile Pasteur pipette, filtered through a nylon mesh filter (40 μm; BD Biosciences, US) into a sterile tube and centrifuged (400g, 5 min). The supernatant was removed and the pellet resuspended in red blood cell lysis buffer (Sigma-Aldrich, UK) for 1 min followed by the addition of DMEM to terminate the lysis. The suspension was centrifuged (400g, 5 min), the supernatant discarded, cells washed using DMEM and centrifuged once more (400g, 5 min). The pellet was resuspended in DMEM supplemented with L929 conditioned media (20%) and seeded in sterile cell culture T75 cm2 flasks. On day 2, non-adherent cells were removed from the flask and media replaced, these cells remained in culture for a further 6 days, with media being replaced on day 4. On day 6, cells were transferred to 6-well plates (0.5 106 cells per well) and remained in culture for a further 2 days. The purity of the BMDM culture was previously assessed by flow cytometric analysis. BMDMs were identified as CD11b þ CD45 þ cells, and it was found that 498% of the cells in culture were positive for both of these markers. Furthermore, 495% of these CD11b þ CD45 þ cells were also positive for CD68.
supernatant concentration of IGF-1 under control conditions plateaued 18–24 h following replenishment of media. The involvement of IGF-1 in IFNγ- or IL-4-induced activation states was investigated by co-incubation of cells with either IFNγ or IL-4 in the presence or absence of a neutralising antibody to IGF-1 or the appropriate isotype control (αIGF-1; 10 mg/ml; Millipore, Ireland). Phosphorylation of Akt was assessed at 1 and 4 h for experiments involving IL-4 and IFNγ respectively and analysis of mRNA expression was carried out at 18 h in both cases. To assess the impact of JAK3 activation on IL-4-induced changes, cells were pre-treated with the JAK3 inhibitor, Janex I (50 mM; Cambridge Bioscience, UK), for 30 min followed by co-incubation with IL-4 for either 1 h for analysis of JAK3, Akt or STAT6 activation or 18 h for analysis of mRNA. For experiments involving LY290042, cells were pre-treated with LY290042 (10 μM; Millipore, Ireland) for 30 min prior to co-incubation with IL-4 or IFNγ for 24 h. 2.3. Analysis of mRNA expression by real-time PCR RNA was isolated from BMDMs using a Nucleospins RNAII kit (Macherey-Nagel GmbH, Germany) and reverse transcribed into cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems, UK) as per manufacturer's instructions. Assay ID's for the genes examined were as follows: β-actin (4352341E), TNFα (Mm00443258_m1), NOS2 (Mm0040502_m1), MRC1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm00657889_mH) and FIZZ1 (Mm00445109_m1). Real-time PCR was performed using an ABI Prism 7300 instrument (Applied Biosystems, UK) with β-actin used as the endogenous control. In all cases, relative gene expression was calculated with reference to untreated BMDMs using the ΔΔCT method with Applied Biosystems RQ software (Applied Biosystems, UK). 2.4. Analysis of proteins by western immunoblotting Western blotting was performed as previously described [11]. Cultured cells were harvested, homogenised in buffer containing Tris–HCl (0.05 M), NaCl (150 mM) and Igepal (1%), and protein (20 μg) was boiled in gel-loading buffer and separated by 7% or 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and incubated with antibodies diluted in 5% non-fat dried milk in Trisbuffered saline containing 0.05% Tween-20 (TBS–T) against the following: β-actin (1:5000; Sigma-Aldrich, UK), phospho-STAT1, STAT1, phospho-STAT6, STAT6, phospho-Akt, Akt, phospho-JAK3, JAK3 (1:1000; Cell Signalling) and Arg1 (1:1000; Pierce) for 16 h at 4 °C. Membranes were incubated with horseradish peroxidaseconjugated secondary antibodies (1:10,000 in 5% non-fat dried milk in TBS–T; Jackson ImmunoResearch, UK) and bands were visualised using WesternBright ECL Chemiluminescent substrate (Advansta, USA). Images were captured using a Fujifilm LAS-3000 (Brennan and Co, Dublin, Ireland). 2.5. Analysis of cytokine concentration by ELISA
2.2. Treatment of BMDMs In all cases, media was replenished immediately prior to initiation of experiments. Cells were incubated in the presence of IFNγ (R&D Systems, UK; 50 ng/ml) or IL-4 (R&D Systems, UK; 200 ng/ml), supernatants were collected for analysis of cytokines by ELISA and cells were harvested for analysis of markers of macrophage activation by real-time PCR and polyacrylamide gel electrophoresis followed by western immunoblotting; the concentrations of IFNγ and IL-4 were selected based on the findings of previous studies. In all cases, media was replenished immediately prior to initiation of experiments and it was noted that
Concentrations of TNFα (R&D Systems; DY410) and IGF-1 (R&D Systems; DY791) were measured in supernatant samples obtained from BMDMs by ELISA. Briefly, standards or samples (100 μl) were added to antibody-coated 96-well plates and incubated for 2 h at room temperature, plates were washed and samples were incubated in detection antibody for 2 h. Plates were washed and incubated in horseradish peroxidase-conjugated streptavidin (1:200 in PBS containing 1% Tween) for 20 min at room temperature. Substrate solution (tetramethylbenzidine; Sigma, UK) was added, incubation continued at room temperature in the dark and the reaction was stopped using H2SO4 (1 M). Absorbance
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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measurements were read at 450 nm using a microplate reader (BioTek Instruments, USA). Protein concentrations were calculated relative to the appropriate standard curve and expressed as pg/ml of supernatant. 2.6. Statistical analysis All data are expressed as mean 7standard error of the mean (SEM). Data were analysed using a Student's t-test for independent means, 1- or 2-way analysis of variance (ANOVA) where indicated followed by a Bonferroni post-hoc test. Analysis was carried out using Prism software (Graphpad, US).
3. Results We first investigated the timing of IL-4-induced changes in mRNA expression of the archetypal markers of M2 macrophages, MRC1, Arg1, Chi3l3 and FIZZ1. IL-4 increased expression of MRC1 mRNA as early as 1 h (***p o0.001; ANOVA; Fig. 1A) but expression of Arg1 mRNA was not significantly increased until 6 h following IL-4 treatment (***po 0.001; ANOVA; Fig. 1B). IL-4 significantly increased FIZZ1 and Chi3l3 mRNA expression at 24 h only (***p o0.001; ANOVA; Fig. 1C and D respectively). It is well documented that IL-4 mediates its effects through the activation of STAT6 (Gordon & Martinez, 2010). We found that pSTAT6 in BMDMs was significantly increased by IL-4 as early as 30 min (***p o0.001; ANOVA; Fig. 1E and F) and remained significantly increased for 24 h. Previous reports have demonstrated that IL-4 upregulates Akt activation and that Akt signalling may play a role in the induction of the M2 phenotype in macrophages [12]. Incubation of BMDMs with IL-4 increased pAkt at 1 h and later at 9 and 12 h (*p o0.05; ANOVA; Fig. 1G and H). The IL-4-induced increase in the expression of pAkt at 9 and 12 h correlated with enhanced release of IGF1 in comparison to unstimulated BMDMs at these times (*p o0.05; ***p o0.001; ANOVA; Fig. 2A), consistent with the known ability of IGF-1 to activate the Akt pathway. As expected, a temporal increase in IGF-1 concentration from unstimulated cells was observed due to the addition of fresh media to the cells immediately prior to the beginning of the experiment – unstimulated cells have been shown to produce high levels of IGF-1 [13]. IGF-1 concentrations, under control conditions, plateaued 18–24 h following replenishment of media (data not shown). To explore a possible role for IGF-1 in IL-4-induced Akt and M2 activation, BMDMs were stimulated with IL-4 in the presence or absence of αIGF-1 or the appropriate isotype control; the data show that αIGF-1 attenuated the IL-4-induced increase in pAkt at 18 h (*p o0.05; ANOVA; Fig. 2B). The IL-4-induced increase in MRC1 mRNA (***po 0.001; ANOVA; Fig. 2C) was enhanced in the presence of αIGF-1 ( þ p o0.05). In contrast, co-incubation of BMDMs with IL-4 and αIGF-1 attenuated the IL-4-induced increases in mRNA expression of FIZZ1 and Chi3l3 (***p o0.001, control vs IL-4; þ p o0.05; þþþ p o0.001, IL-4 vs IL-4 þ αIGF-1; ANOVA; Fig. 2D and E). While αIGF-1 had no effect on Arg1 mRNA expression at 18 h (***p o0.001, control vs IL-4; ANOVA; Fig. 2F), the IL-4-induced increase in Arg1 protein expression was attenuated in the presence of αIGF-1 (***p o0.001, control vs IL-4; þ þ po 0.01, IL-4 vs IL-4 þ αIGF-1; ANOVA; Fig. 2G and H). IL-4 mediates its effects through two receptors, the type I IL-4 receptor (composed of two subunits, IL-4Rα and IL-2Rγ) and the type II receptor (IL-4Rα and IL-13Rα1), activation of these receptors results in the phosphorylation of STAT6. JAK3 is activated as a consequence of ligation of the type I IL-4R, which has been shown to participate in the activation of Akt. To further investigate this, BMDMs were incubated in the presence of IL-4 and the JAK3 inhibitor, Janex I, for 1 h. IL-4
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increased the expression of pJAK3 and pAkt (*po0.05; ANOVA; Fig. 3A–C) and this effect was attenuated in the presence of Janex 1 ( þ po0.05; þ þ po0.01). In contrast, Janex 1 had no effect on the expression of JAK3, and Akt (Fig. 3A; 2nd, 4th and 5th panel). Similarly, inhibition of JAK3 with Janex I had no effect on the induction of pSTAT6 and this is consistent with data from the literature demonstrating that JAK3 KO had no effect on IL-13-induced STAT6 activity which is mediated by Tyk2 and JAK1 [14]. The IL-4-induced increase in release of IGF-1 (***po0.001; ANOVA; Fig. 3D) was also attenuated in the presence of Janex I ( þ þ þ po0.001). Janex 1 had no effect on the IFNγ-induced increase in pSTAT1 expression, demonstrating that it had no inhibitory effects on either JAK1 or JAK2 (data not shown). The IL-4-induced increases in expression of Arg1, FIZZ1 and Chi3l3 mRNA were similarly inhibited by Janex I (***po0.001, control vs IL-4; þþþ po0.001; IL-4 vs IL-4 þ Janex 1; ANOVA; Fig. 3F, I and J respectively). Inhibition of JAK3 also resulted in an attenuation of the IL4-induced increase in Arg1 protein expression (***po0.001, control vs IL-4; þ þ þ po0.001; IL-4 vs IL-4 þ Janex 1; ANOVA; Fig. 3G and H). However, co-incubation with Janex I enhanced the IL-4-induced increase in expression of MRC1 mRNA (***po0.001, control vs IL-4; þþþ po0.001; IL-4vs IL-4 þ Janex 1; ANOVA; Fig. 3E). Since inhibiting JAK3 activation prevented full M2 activation and this was independent of STAT6 activity, analysis was carried out to establish the effects of pAkt inhibition. As before, IL-4 increased IGF-1 release from BMDMs (***p o0.001; ANOVA; Fig. 4A), however in the presence of the PI3-kinase inhibitor, LY294002, this effect was attenuated ( þ þ þ p o0.001). Additionally, LY294002 attenuated the IL-4-induced increase of Chi3l3, FIZZ1 and Arg1 mRNA expression (***p o0.001, control vs IL-4; þ þ þ p o0.001, þ p o0.05, IL-4 vs IL-4 þ LY294002; ANOVA; Fig. 4C–E respectively), however the effect of IL-4 on MRC1 mRNA expression was enhanced by co-incubation with LY294002 (***p o0.001, control vs IL-4; þ þ þ po0.001, IL-4 vs IL-4 þ LY294002; ANOVA; Fig. 4B). Inhibition of Akt attenuated the IL-4-induced increase in Arg1 protein expression (***p o0.001, control vs IL-4; þ þ þ p o0.001, IL4 vs IL-4 þ LY294002; ANOVA; Fig. 4F) TNFα and NOS2 mRNA are archetypal markers of the M1 state and time-related analysis of the changes indicated that IFNγ significantly increased expression of TNFα mRNA at 2 h and that this increase was still evident at 9 h (***po0.001; ANOVA; Fig. 5A). IFNγ stimulation increased NOS2 mRNA expression at all times investigated, however this effect achieved statistical significance at 6 and 24 h (***po0.001; ANOVA; Fig. 1B), though not at 9 h (where the value was also not significantly different from that at 6 or 24 h). IFNγ signals through phosphorylation of STAT1 and the data presented here show that pSTAT1 expression was significantly enhanced at 30 min (***p o0.001; ANOVA; Fig. 5C and D) and remained elevated at all times examined. The regulatory role of PI3K/Akt in inflammation has been identified previously [15], in light of this, we examined the activation of this pathway in IFNγ-stimulated macrophages. We report that IFNγ led to an early increase (4 h) followed by a decrease in the expression of pAkt at 18 and 24 h (*po 0.05, **p o0.01; ANOVA; Fig. 5E). The reduction in pAkt expression was accompanied by an IFNγ-induced decrease in IGF-1 release from BMDMs (*po 0.05; ANOVA; Fig. 5F). To assess the role of IGF-1 in Akt activation and, specifically, the interplay between IGF-1 and IFNγ, we co-incubated BMDMs with IFNγ and αIGF-1 or the appropriate isotype control and show that the IFNγ-induced increase in expression of pAkt at 4 h (***p o0.001; ANOVA; Fig. 6A) was attenuated by αIGF-1 ( þ þ po 0.01; ANOVA; Fig. 6A). Additionally, the IFNγ-induced increases in expression of TNFα and NOS2 mRNA (***p o0.001; ANOVA; Fig. 6B and D) were exacerbated in the presence of αIGF-1 ( þ p o0.05; ANOVA; Fig. 6B and D). Incubation of cells in the presence of αIGF-1 enhanced the IFNγ-induced release of TNFα (***p o0.001, control vs IFNγ; þ p o0.05, IFNγ vs IFNγ þ αIGF-1;
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 1. Expression of M2 activation markers varies upon exposure to IL-4. Bone-marrow derived macrophages were incubated with IL-4 for various times. Expression of MRC1 mRNA (A) was significantly enhanced at 1 h, however Arg1 mRNA (B) was not significantly increased until 6 h, while FIZZ1 (C) and Chi3l3 (D) mRNA expression were unchanged until 24 h (***p o 0.001; ANOVA; n ¼6). Expression of phosphorylated STAT6 (F) was apparent at 30 min and still evident at 24 h (***p o0.001; **p o0.01; ANOVA; n ¼4). A representative immunoblot is shown in (E). Incubation of BMDMs with IL-4 revealed an increase in the expression of phosphorylated Akt (H) at 1 h and later at 9 and 12 h (*p o0.05; ANOVA; n¼ 4). A representative immunoblot is shown in (G). All data are expressed as mean7 SEM.
ANOVA; Fig. 6C). Furthermore, IFNγ treatment significantly decreased IGF-1 supernatant concentration (*p o0.05, control vs IFNγ; ANOVA; Fig. 6E) and this effect was exacerbated in the presence of LY294002 ( þ p o0.05, IFNγ vs IFNγ þ LY294002; 24 h; ANOVA; Fig. 6E), while co-incubation of BMDMs with IFNγ and LY294002 further increased the IFNγ-induced expression of TNFα mRNA (*p o0.05, control vs IFNγ; þ p o0.05, IFNγ vs IFNγ þ LY294002; ANOVA; Fig. 6F).
4. Discussion The significant finding of this study is that IL-4-induced activation of Akt is JAK3-dependent, is enhanced by subsequent release of IGF-1 and is necessary for full adoption of the M2 phenotype. We report that the early response of macrophages to IFNγ was to increase Akt activation and inhibition of Akt or blockade of IGF1 activity exacerbated the effects of IFNγ. This indicates that the initial IFNγ-induced phosphorylation of Akt expression may serve
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 2. M2 activation state is regulated by Akt/IGF-1 signalling. The IL-4-induced increase in the expression of pAkt at 9 and 12 h correlated with enhanced release of IGF-1 (A) at these times (*po0.05; ***po0.001; ANOVA; n¼4). Co-incubation of BMDMs with IL-4 and a neutralising antibody to IGF-1 (αIGF-1) attenuated the IL-4-induced increase in pAkt (B) at 18 h (*po0.05, control vs IL-4; ANOVA; n¼4). A representative immunoblot is also shown. The IL-4-induced increase in mRNA expression of MRC1 (C; ***po0.001; ANOVA; n¼ 4) was enhanced in the presence of αIGF-1 ( þ po0.05; ANOVA; n¼4). Co-incubation of BMDMs with IL-4 and αIGF-1 attenuated the IL-4-induced increase in mRNA expression of FIZZ1 (D) and Chi3l3 (E) (***po0.001, control vs IL-4; þ po0.05, þ þ þ po0.001, IL-4 vs IL-4 þ αIGF-1; ANOVA; n¼4). While αIGF-1 had no effect on IL-4-induced Arg1 mRNA expression (F; ***po0.001, control vs IL-4; ANOVA; n¼4), the IL-4 mediated increase in Arg1 protein expression was attenuated in the presence of αIGF-1 (G, H; ***po0.001, control vs IL-4; þ þ po0.01, IL-4 vs IL-4 þ αIGF-1; ANOVA; n¼ 4). A representative blot is shown (G). All data are expressed as mean7SEM.
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 3. Attenuation of M2 by inhibition of pJAK3 and pAkt is independent of pSTAT6. BMDMs were incubated in the presence of IL-4 and the JAK3 inhibitor, Janex I, for 1 h. Expression of phosphorylated JAK3 (A; B) and Akt (A; C) were increased by exposure to IL-4 (*po0.05, **po0.01, ANOVA; n¼3) and this effect was attenuated in the presence of Janex 1 ( þ po0.05; þ þ po0.01; ANOVA; n¼ 3). The IL-4-induced increase in expression of phosphorylated STAT6 was unaffected by Janex 1 (A; 5th panel). The presence of IL-4 or Janex 1 had no effect on expression of total JAK3 (A; 2nd panel) and Akt (A; 4th panel). Representative immunoblots are shown in (A). The IL-4-induced increase in release of IGF1 (D) was also attenuated in the presence of Janex I as were expression of Arg1 (F), Chi3l3 (I) and FIZZ1 (J) mRNA (***po0.001, control vs IL-4; þ þ po0.01, þ þ þ po0.001, IL-4 vs IL-4 þ Janex I ANOVA; n¼3). The IL-4-induced increase in Arg1 protein expression was attenuated in the presence of Janex 1 (G, H; ***po0.001, control vs IL-4; þ þ þ po0.001, IL-4 vs IL-4 þ Janex I ANOVA; n¼3), a representative blot is shown (G). However, co-incubation with Janex I enhanced the IL-4-induced increase in expression of MRC1 mRNA (E; ***po0.001, control vs Janex I, control vs IL-4; þ þ þ po0.001, IL-4 vs IL-4 þ Janex I; ANOVA; n¼ 3). All data are expressed as mean7SEM.
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Fig. 4. Inhibition of pAkt affects release of IGF-1 and M2 state in BMDMs. The IL-4-induced release of IGF-1 (A) from BMDMs was attenuated in the presence of the PI3kinase inhibitor, LY294002, as were expression of Chi3l3 (C) FIZZ1 (D) and Arg1 (E) mRNA (***p o 0.001, control vs IL-4; þ po 0.05, þ þ þ p o 0.001, IL-4 vs IL-4 þ LY294002; ANOVA; n ¼5), however the effect of IL-4 on MRC1 (B) mRNA expression was enhanced by co-incubation with LY294002 (***p o 0.001, control vs IL-4; þ þ þ p o 0.001, IL-4 vs IL-4 þ LY294002; ANOVA; n¼ 5). The IL-4-induced increase in Arg1 protein expression was attenuated in the presence of LY294002 (F; ***p o 0.001, control vs IL-4; þþþ p o 0.001, IL-4 vs IL-4 þ LY294002; ANOVA; n¼ 4). All data are expressed as mean 7 SEM.
to modulate M1 activation. Prolonged exposure to IFNγ downregulated Akt activation; this was associated with decreased IGF-1, an effect that was exacerbated by inhibition of PI3-kinase, suggesting that Akt induces its own activation via production of IGF-1. IL-4 stimulated Akt activation and this may be mediated by the IL4-induced increase IGF-1 production. We demonstrate that inhibition of PI3K/Akt activation decreased the expression of IL-4-induced genes associated with the M2 phenotype and therefore we conclude that activation of the PI3K/Akt pathway is crucial in mediating this effect of IL-4. In contrast, inhibiting the action of IGF-1 and Akt exacerbated the M1 phenotype. It is proposed that the differential effects of IL-4 and IFNγ on IGF-1 production are responsible for the observed changes in PI3K/Akt activity and that modulating PI3K/Akt signalling plays a key role in the polarisation of macrophages. Analysis of time-related IL-4-induced changes in expression of markers of M2 activation indicated that maximal upregulation of MRC1 mRNA in BMDMs occurred at 1 h, closely following the
activation of STAT6. In contrast, the induction of Arg1, Chi3l3 and FIZZ1 was much later than MRC1, and this concurs with previous findings [7]. The difference in the temporal profile of induction of M2 markers suggests differential regulation. Heller and colleagues demonstrated that IL-4 and IL-13 activated STAT6 in a similar manner but that IL-4 increased the expression of Arg1, Chi3l3 and FIZZ1 in a more robust fashion than IL-13. The effects of IL-4 and IL-13 on MRC1 mRNA expression were similar suggesting that Arg1, Chi3l3 and FIZZ1 expression may be modulated by IL-4 in a STAT6-independent manner. We found that the IL-4-induced activation of STAT6 was evident within 30 min and persisted for 24 h, confirming earlier observations [16]. Importantly, total STAT6 expression was unaffected by IL-4 treatment, indicating that the increase in activated STAT6 did not require de novo protein synthesis. It is well documented that STAT6 is necessary for the induction of the M2 phenotype, a number of studies have demonstrated that macrophages from STAT6-deficient mice do not show upregulation
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 5. Temporal response of bone-marrow derived macrophages to IFNγ. Bone-marrow derived macrophages were incubated with IFNγ for various times. Expression of TNFα mRNA (A) was significantly upregulated by 2 h and persisted until 24 h while the mRNA expression of NOS2 (B) was not significantly increased until 6 h but also persisted until 24 h (***p o 0.001; ANOVA; n ¼6). Changes in the mRNA expression of archetypal markers of the M1 activation state were preceded by enhanced expression of phosphorylated STAT1 (D) at 30 min and 1 h (***p o 0.001; ANOVA; n¼ 4). A representative immunoblot is shown (C). Exposure of BMDMs to IFNγ led to an early increase (4 h) followed by a decrease in the expression of phosphorylated Akt (E; see sample immunoblot) at 18 and 24 h (*p o 0.05, **po 0.01, control vs IL-4; ANOVA; n¼ 4). The release of IGF-1 (F) was decreased by incubation of BMDMs with IFNγ at 1 h and at all timepoints examined (*p o 0.05; ANOVA; n¼ 4, control vs IFNγ). All data are expressed as mean 7 SEM.
of M2associated genes in response to IL-4/IL-13 stimulation [17– 19]. While it is clear that STAT6 is necessary to mediate the effects of IL-4, previous studies have demonstrated that IL-4 impacts upon other signalling pathways. For example, IL-4 activates IRS-2 [7] and consequently affects the PI3K/Akt and Ras-MAPK pathways, which activate numerous cell processes including cell proliferation [20]. Here, pAkt expression was enhanced in BMDMs after 1 h of exposure to IL-4 and was also evident at 9 and 12 h, coinciding with the IL-4-induced changes in M2 markers. Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signalling as essential for IL-4-driven murine macrophage proliferation in vivo [12]. Previous evidence has also indicated that inhibition of PI3K/Akt by wortmannin decreases the IL-4-induced expression of FIZZ1, but not Arg1, in murine macrophages [7]. IL-4 increased IGF-1 production in BMDMs, an effect that has been
reported in macrophages and microglia [13,21]. These cells also respond to IGF-1 stimulation and work from this laboratory has demonstrated that IGF-1 can attenuate the response of microglia to IFNγ stimulation [9]. Other groups have presented evidence that IGF-1 is neuroproective following ischaemia and trauma [22,23] and, as a result, IGF-1 may mediate the anti-inflammatory effects associated with IL-4. The IGF-1R is constitutively expressed in cells [24], including macrophages and microglia, while it has been demonstrated to be anti-inflammatory it has also been suggested that it may induce proliferation of macrophages [25]. These findings, coupled with those of the present study, suggest that IGF-1 produced by macrophages may act in both autocrine and paracrine manner to induce and enhance M2 activation and may lead to an increase in the number of macrophages present at the site of inflammation. While it has been previously shown that IGF-1 is a potent activator of the PI3K/Akt
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 6. Akt/IGF-1 signalling contributes to attenuation of M1 activation. Co-incubation of BMDMs with IFNγ and αIGF-1 attenuated the IFNγ-induced increase in expression of phosphorylated Akt (A; see representative immunoblot) at 4 h (***p o 0.001, control vs IFNγ; þ þ po 0.01, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼3). The IFNγ-induced increase in expression of TNFα (B) mRNA was exacerbated in the presence of αIGF-1 (***p o 0.001, control vs IFNγ; þ p o 0.05, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼4). IFNγ stimulation increased TNFα supernatant concentration (C) and this was exaggerated in the presence of αIGF-1 (***p o 0.001, control vs IFNγ; þ p o0.05, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼ 3). The effect of IFNγ on NOS2 mRNA (D) expression was enhanced in the presence of αIGF-1 (***p o 0.001, control vs IFNγ; þ p o 0.05, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼ 4). IGF-1 release was significantly decreased in the presence of IFNγ and this effect was exacerbated in the presence of LY294002 (E; *p o0.05, control vs IFNγ; þ þ p o 0.01, IFNγ vs IFNγ þ LY294002; ANOVA; n ¼4), while co-incubation of BMDMs with IFNγ and LY294002 led to an upregulation in the IFNγ-induced expression of TNFα (F) mRNA (*p o 0.05, control vs IFNγ; þ p o 0.05, IFNγ vs IFNγ þ LY294002; ANOVA; n¼ 5). All data are expressed as mean 7SEM.
pathway, the present study demonstrates its role in mediating the effect of IL-4 on pAkt expression. In the presence of αIGF-1, the IL-4induced increase in pAkt expression was significantly attenuated, suggesting that IGF-1 is necessary for the IL-4-mediated induction of pAkt expression. These findings suggest that IGF-1 expression is not
only a marker of the M2 phenotype, but induces signalling pathways that drive and maintain the M2 response. In addition to this, co-incubation of BMDMs with IL-4 and αIGF-1 attenuated the IL-4-induced FIZZ1 and Chi3l3 mRNA, identifying a role for IGF-1/Akt in the induction of the M2 phenotype. Although IL-4-induced Arg1 mRNA was
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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unaffected by αIGF-1 incubation, the effect of IL-4 on Arg1 protein expression was attenuated by αIGF-1 incubation. In contrast to the effects of αIGF-1 on the other markers of M2 activation, IL-4-induced MRC1 mRNA was significantly increased in the presence of αIGF-1. Therefore, we can conclude that IL-4-stimulated Akt activation differentially modulates expression of molecules associated with the M2 state. It has been documented that ligation of the type I, but not the type II, IL-4R can activate the PI3K/Akt pathway [20]. To establish whether the changes in pAkt occurred as a result of type 1 IL-4R activation, we set out to inhibit the activation of JAK3, a component of the type I IL-4R not utilised by type II IL-4R-associated signalling. The JAK3 inhibitor, Janex 1, attenuated the IL-4-induced increases in pAkt and IGF-1 production while STAT6 activation was unaffected. Similar to the effect of αIGF-1, inhibition of JAK3 increased IL-4-induced MRC1 mRNA, attenuated the IL-4-induced increase in Arg1, FIZZ1 and Chi3l3 mRNA and had no effect on STAT6 signalling. Together, these data indicate alternative modes of activation for specific markers of the M2 phenotype with differential responses driven through ligation of type I and type II IL-4R (Fig. 7). Interestingly, the IL-4-induced upregulation of Arg1, Chi3l3 and FIZZ1 mRNA was inhibited by LY290042, confirming earlier findings [7,12]. Therefore, PI3K/Akt appears to be crucial in the induction of at least these M2 activation markers. The effect of the SH2-containing inositol phosphatase (SHIP) on PI3K activity further highlights the role of the PI3K/Akt pathway in the induction of M2 activation; SHIP is a negative regulator of PI3K activity and Weisser and colleagues reported that macrophages from SHIPdeficient mice exhibit an exaggerated response to IL-4 treatment [26]. It should be noted that the concentration of LY290042 used in this study did not completely attenuate IL-4-induced changes in Arg1, Chi3l3 and FIZZ1 mRNA expression, indicating that while Akt activation is necessary for maximal induction of these genes, IL-4 can induce their expression through other pathways. In complete contrast to the effect of LY290042 on IL-4-induced changes in Arg1, Chi3l3 and FIZZ1 mRNA, MRC1 mRNA was enhanced by LY290042 mimicking the effect of both α-IGF-1 and Janex 1; this suggests that the IL-4-induced increase in MRC1 mRNA expression is not dependent on PI3K/Akt activation [7]. On the basis of our
findings and work by others, we conclude that activation of STAT6 is primarily involved in modulation of the expression of MRC1. One objective of this study was to investigate the differential effects of IL-4 and IFNγ on the PI3K/Akt pathway. The effect of IFNγ on Akt activation is complex and time-dependent, with no evidence of an immediate effect but an increase at 4 h. We propose that the early activation of Akt reflects a negative feedback control on IFNγ-induced changes. Consistent with this, we demonstrate that αIGF-1 decreased pAkt expression and enhanced the response to IFNγ, while the effect of IFNγ on TNFα and NOS2 mRNA expression was exacerbated in the presence of α-IGF. The mechanism by which IFNγ induced an increase in pAkt expression at 4 h may be IGF-1-independent since IFNγ induced a decrease in IGF-1 production at this time. However, blocking the interaction of IGF-1 with IGF-1R (even at this lower IGF-1 concentration) decreased tonic pAkt expression and may thus have contributed to the IFNγinduced increases in TNFα and NOS2. Data from numerous studies have indicated that activation of the PI3K/Akt pathway promotes anti-inflammatory effects and attenuates TLR-induced cytokine production [15,27]. Similarly, decreased signalling through this pathway has been associated with an increased response to inflammatory stimuli; specifically Luyendyk and colleagues demonstrated that macrophages, in which PI3K signalling was decreased, exhibit an enhanced response to LPS stimulation [15]; in contrast, macrophages cultured from PTEN / mice exhibit enhanced Akt activation and this was associated with a decrease in responsiveness to LPS stimulation. Like PTEN, SHIP also inhibits the activity of PI3K, and LPS-induced cytokine release is also impaired in macrophages prepared from SHIP-deficient mice [26]. Thus, the PI3K/Akt pathway plays a part in restricting inflammatory events, and evidence from experiments in vivo have demonstrated that inhibition of PI3K increased inflammation in mouse models of endotoxemia and sepsis [28,29]. We conclude that a deficit in IGF-1/Akt signalling is associated with a decreased capacity to induce the M2 state and an increase in responsiveness to IFNγ. This is interesting in the context of our earlier findings that hippocampal expression of IGF-1, as well as activation of Akt, is decreased in the brain with age and that these changes are associated with increased IFNγ concentration [10].
Fig. 7. Schematic. IL-4 ligates the type II IL-4R to recruit and activate Tyk2 and JAK1 leading to the subsequent activation of STAT6 and upregulation of M2 markers. IL-4 interacts with the type I IL-4R to induce recruitment and activation of JAK1 and JAK3, leading to activation of PI3K/Akt signalling and upregulation of IGF-1 expression. IGF-1 induces further activation of Akt and contributes to upregulated expression of M2 markers of activation.
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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These age-related changes probably provide an explanation for the increased propensity of microglia to adopt an M1 phenotype, and their limited ability to adopt the M2 phenotype, in aged animals [30].
Author contribution JPB, AMM and ML designed the work. JPB, AMM and AF prepared the tissue for analysis; the analysis and preparation of figures was carried out by JPB and AMM. JPB and AMM prepared the initial draft of the manuscript and ML critically assessed the content and approved the final manuscript. ML supervised the work.
Conflict of interest disclosure The authors declare no conflict of interest.
Acknowledgements This work was funded by Science Foundation Ireland (07/IN.1/ B949). JPB was the recipient of a Trinity College Postgraduate student award.
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Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Research Article
Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages James P. Barrett 1, Aedín M. Minogue n,1, Aidan Falvey, Marina A. Lynch Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland
art ic l e i nf o
a b s t r a c t
Article history: Received 6 February 2015 Received in revised form 22 April 2015 Accepted 18 May 2015
Macrophages can be polarised to adopt the M1 or M2 phenotype and functional outcomes of activation include altered secretion of immune molecules such as insulin-like growth factor (IGF)-1 as well as upregulation of cell surface molecules specifically associated with each state. Interleukin (IL)-4 mediates its effects through two receptors, the type I and II receptors and activation of these receptors results in phosphorylation of signal transducers and activators of transcription (STAT)6. JAK3 is activated as a consequence of ligation of the type I IL-4R, which participates in Akt activation. We set out to investigate the impact of perturbation of IGF-1 tone on IL-4- and interferon (IFN)γ–induced activation, the mechanisms by which this may occur and the contribution of type I IL-4R activation to adoption of the M2 state. The data presented here indicate that IL-4-induced activation of Akt is JAK3-dependent, enhanced by release of IGF-1 and necessary for full adoption of the M2 phenotype, since blocking IGF-1 activity blunts the ability of IL-4 to induce activation of Akt and to upregulate expression of some M2-associated molecules. In addition, differential control of the expression of mannose receptor (MRC1), arginase-1 (Arg-1), chitinase-3 like 3 (Chi3l3) and found in inflammatory zone 1 (FIZZ1) was observed. The IFNγinduced decrease in IGF-1 was exacerbated by inhibition of phosphatidylinositol-3 (PI3) kinase, indicating that Akt may regulate its own activation via IGF-1. Overall, a deficit in IGF-1/Akt signalling is associated with decreased capacity to induce the M2 state and an increased responsiveness to IFNγ. & 2015 Published by Elsevier Inc.
Keywords: BMDMs; IGF-1; Akt; IL-4; IFNγ;
1. Introduction The initial discovery that macrophages adopt the so-called classical activation, M1, phenotype in response to interferon-γ (IFNγ) and the alternative activation, M2, phenotype in response to IL-4, dates back over 20 years [1] though more recent evidence suggests additional activation states also exist [2]. Emerging data also highlights the difference in functionality between M1 and M2 phenotypes [3–5]. An understanding of the phenotypes and their modulation has become increasingly important in the context of neurodegenerative disorders with the recognition that infiltration of macrophages into the brain parenchyma occurs in these diseases, and modulating these phenotypes may provide a potential Abbreviations: ANOVA, analysis of variance; Arg1, Arginase-1; BMDM, bone marrow-derived macrophages; Chi3l3, chitinase-3 like 3; EtOH, ethanol; FIZZ1, found in inflammatory zone 1; IFNγ, interferon-γ; IGF-1, insulin-like growth factor 1; IL-4, interleukin-4; MRC1, mannose receptor; NOS2, nitric oxide synthase 2; PI3K, phosphatidylinositol-3 kinase; JAK1, janus kinase 1; JAK3, janus kinase 3; STAT1, signal transducers and activators of transcription 1; STAT6, signal transducers and activators of transcription 6; TNFα, tumour necrosis factor α n Corresponding author. E-mail address: [email protected] (A.M. Minogue). 1 These authors contributed equally to the work.
strategy to limit disease progression. To maximise the potential of identifying strategies for modulation of disease progression, a comprehensive understanding of the signalling pathways that control the switch between phenotypes is imperative. The M1 and M2 phenotypes are characterised by upregulated expression of specific molecules. Increased expression of tumour necrosis factor (TNF)α and nitric oxide synthase (NOS)2 is associated with the M1 phenotype while the M2 anti-inflammatory phenotype, is characterised by upregulation of molecules such as mannose receptor (MRC1), arginase 1 (Arg1), chitinase 3-like 3 (Chi3l3) and found in inflammatory zone (FIZZ)1. IL-4 signals through the type I and type II receptors and activation of the type I receptor facilitates the phosphorylation of JAK3 while the type II receptor induces JAK1 activation [6] and results in phosphorylation of STAT6 in macrophages [6]. The presence of distinct receptor types presents a possibility by which functions of the M2 state may be distinctly regulated. It has been shown that IL-13, which engages the type II receptor and results in the same level of STAT6 phosphorylation as IL4, does not upregulate expression of Arg1, Chi3l3 and FIZZ1 to the same extent as IL-4 [7]. In addition, IL-4 stimulates activation of IRS2 and PI3K/Akt while exposure to IL-13 does not. One of the functional outcomes of macrophage activation is the secretion of immune modulatory molecules and macrophages
http://dx.doi.org/10.1016/j.yexcr.2015.05.015 0014-4827/& 2015 Published by Elsevier Inc.
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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produce IGF-1 in response to IL-4, while IFNγ downregulates IGF-1 transcription [8]. The impact of perturbation of IGF-1 tone on macrophage activation has not been investigated though IGF-1 antagonises the pro-inflammatory effects of IFNγ in microglia in vitro [9] and an inverse correlation between IGF-1 and IFNγ, associated with neuroinflammation, has been reported in the brains of aged rats [10]. IGF-1 is a potent activator of the PI3K/Akt signalling pathway and, together with its ability to inhibit the effects of IFNγ, may enhance the ability of macrophages to adopt the M2 state. We set out to investigate the role that IGF-1/Akt activity plays in modulating macrophage phenotype. The data show that, while IL-4-induced MRC1 transcription was STAT6-mediated, induction of Arg1, Chi3l3 and FIZZ1 was driven by activation of JAK3 and Akt. We show that IL-4-induced production of IGF-1, which was JAK3and PI3K/Akt-dependent, was necessary for macrophages to fully adopt the M2 phenotype while decreased IGF-1/Akt activity was associated with an exacerbated response to IFNγ stimulation, which was independent of STAT1. We propose that a disturbance in IGF-1/Akt tone may lead to a dysregulation of macrophage activation.
2. Materials and methods 2.1. Preparation and culture of BMDMs BMDMs were isolated from the marrow of the femurs and tibias of mice. The legs of the animals were sprayed with 70% EtOH and the skin and muscle tissue removed from the bones. The bones were sprayed with 70% EtOH, transferred to a sterile flow hood and cut at both ends. The marrow was flushed out into a sterile falcon tube in Dulbecco's modified Eagle's medium (DMEM; 500 ml; Invitrogen, UK) supplemented with heat-inactivated foetal bovine serum (FBS; 50 ml; 10%; Gibco, UK) and penicillin–streptomycin (5 ml; 1%; Gibco, UK). The cell suspension was triturated using a sterile Pasteur pipette, filtered through a nylon mesh filter (40 μm; BD Biosciences, US) into a sterile tube and centrifuged (400g, 5 min). The supernatant was removed and the pellet resuspended in red blood cell lysis buffer (Sigma-Aldrich, UK) for 1 min followed by the addition of DMEM to terminate the lysis. The suspension was centrifuged (400g, 5 min), the supernatant discarded, cells washed using DMEM and centrifuged once more (400g, 5 min). The pellet was resuspended in DMEM supplemented with L929 conditioned media (20%) and seeded in sterile cell culture T75 cm2 flasks. On day 2, non-adherent cells were removed from the flask and media replaced, these cells remained in culture for a further 6 days, with media being replaced on day 4. On day 6, cells were transferred to 6-well plates (0.5 106 cells per well) and remained in culture for a further 2 days. The purity of the BMDM culture was previously assessed by flow cytometric analysis. BMDMs were identified as CD11b þ CD45 þ cells, and it was found that 498% of the cells in culture were positive for both of these markers. Furthermore, 495% of these CD11b þ CD45 þ cells were also positive for CD68.
supernatant concentration of IGF-1 under control conditions plateaued 18–24 h following replenishment of media. The involvement of IGF-1 in IFNγ- or IL-4-induced activation states was investigated by co-incubation of cells with either IFNγ or IL-4 in the presence or absence of a neutralising antibody to IGF-1 or the appropriate isotype control (αIGF-1; 10 mg/ml; Millipore, Ireland). Phosphorylation of Akt was assessed at 1 and 4 h for experiments involving IL-4 and IFNγ respectively and analysis of mRNA expression was carried out at 18 h in both cases. To assess the impact of JAK3 activation on IL-4-induced changes, cells were pre-treated with the JAK3 inhibitor, Janex I (50 mM; Cambridge Bioscience, UK), for 30 min followed by co-incubation with IL-4 for either 1 h for analysis of JAK3, Akt or STAT6 activation or 18 h for analysis of mRNA. For experiments involving LY290042, cells were pre-treated with LY290042 (10 μM; Millipore, Ireland) for 30 min prior to co-incubation with IL-4 or IFNγ for 24 h. 2.3. Analysis of mRNA expression by real-time PCR RNA was isolated from BMDMs using a Nucleospins RNAII kit (Macherey-Nagel GmbH, Germany) and reverse transcribed into cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems, UK) as per manufacturer's instructions. Assay ID's for the genes examined were as follows: β-actin (4352341E), TNFα (Mm00443258_m1), NOS2 (Mm0040502_m1), MRC1 (Mm00485148_m1), Arg1 (Mm00475988_m1), Chi3l3 (Mm00657889_mH) and FIZZ1 (Mm00445109_m1). Real-time PCR was performed using an ABI Prism 7300 instrument (Applied Biosystems, UK) with β-actin used as the endogenous control. In all cases, relative gene expression was calculated with reference to untreated BMDMs using the ΔΔCT method with Applied Biosystems RQ software (Applied Biosystems, UK). 2.4. Analysis of proteins by western immunoblotting Western blotting was performed as previously described [11]. Cultured cells were harvested, homogenised in buffer containing Tris–HCl (0.05 M), NaCl (150 mM) and Igepal (1%), and protein (20 μg) was boiled in gel-loading buffer and separated by 7% or 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and incubated with antibodies diluted in 5% non-fat dried milk in Trisbuffered saline containing 0.05% Tween-20 (TBS–T) against the following: β-actin (1:5000; Sigma-Aldrich, UK), phospho-STAT1, STAT1, phospho-STAT6, STAT6, phospho-Akt, Akt, phospho-JAK3, JAK3 (1:1000; Cell Signalling) and Arg1 (1:1000; Pierce) for 16 h at 4 °C. Membranes were incubated with horseradish peroxidaseconjugated secondary antibodies (1:10,000 in 5% non-fat dried milk in TBS–T; Jackson ImmunoResearch, UK) and bands were visualised using WesternBright ECL Chemiluminescent substrate (Advansta, USA). Images were captured using a Fujifilm LAS-3000 (Brennan and Co, Dublin, Ireland). 2.5. Analysis of cytokine concentration by ELISA
2.2. Treatment of BMDMs In all cases, media was replenished immediately prior to initiation of experiments. Cells were incubated in the presence of IFNγ (R&D Systems, UK; 50 ng/ml) or IL-4 (R&D Systems, UK; 200 ng/ml), supernatants were collected for analysis of cytokines by ELISA and cells were harvested for analysis of markers of macrophage activation by real-time PCR and polyacrylamide gel electrophoresis followed by western immunoblotting; the concentrations of IFNγ and IL-4 were selected based on the findings of previous studies. In all cases, media was replenished immediately prior to initiation of experiments and it was noted that
Concentrations of TNFα (R&D Systems; DY410) and IGF-1 (R&D Systems; DY791) were measured in supernatant samples obtained from BMDMs by ELISA. Briefly, standards or samples (100 μl) were added to antibody-coated 96-well plates and incubated for 2 h at room temperature, plates were washed and samples were incubated in detection antibody for 2 h. Plates were washed and incubated in horseradish peroxidase-conjugated streptavidin (1:200 in PBS containing 1% Tween) for 20 min at room temperature. Substrate solution (tetramethylbenzidine; Sigma, UK) was added, incubation continued at room temperature in the dark and the reaction was stopped using H2SO4 (1 M). Absorbance
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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measurements were read at 450 nm using a microplate reader (BioTek Instruments, USA). Protein concentrations were calculated relative to the appropriate standard curve and expressed as pg/ml of supernatant. 2.6. Statistical analysis All data are expressed as mean 7standard error of the mean (SEM). Data were analysed using a Student's t-test for independent means, 1- or 2-way analysis of variance (ANOVA) where indicated followed by a Bonferroni post-hoc test. Analysis was carried out using Prism software (Graphpad, US).
3. Results We first investigated the timing of IL-4-induced changes in mRNA expression of the archetypal markers of M2 macrophages, MRC1, Arg1, Chi3l3 and FIZZ1. IL-4 increased expression of MRC1 mRNA as early as 1 h (***p o0.001; ANOVA; Fig. 1A) but expression of Arg1 mRNA was not significantly increased until 6 h following IL-4 treatment (***po 0.001; ANOVA; Fig. 1B). IL-4 significantly increased FIZZ1 and Chi3l3 mRNA expression at 24 h only (***p o0.001; ANOVA; Fig. 1C and D respectively). It is well documented that IL-4 mediates its effects through the activation of STAT6 (Gordon & Martinez, 2010). We found that pSTAT6 in BMDMs was significantly increased by IL-4 as early as 30 min (***p o0.001; ANOVA; Fig. 1E and F) and remained significantly increased for 24 h. Previous reports have demonstrated that IL-4 upregulates Akt activation and that Akt signalling may play a role in the induction of the M2 phenotype in macrophages [12]. Incubation of BMDMs with IL-4 increased pAkt at 1 h and later at 9 and 12 h (*p o0.05; ANOVA; Fig. 1G and H). The IL-4-induced increase in the expression of pAkt at 9 and 12 h correlated with enhanced release of IGF1 in comparison to unstimulated BMDMs at these times (*p o0.05; ***p o0.001; ANOVA; Fig. 2A), consistent with the known ability of IGF-1 to activate the Akt pathway. As expected, a temporal increase in IGF-1 concentration from unstimulated cells was observed due to the addition of fresh media to the cells immediately prior to the beginning of the experiment – unstimulated cells have been shown to produce high levels of IGF-1 [13]. IGF-1 concentrations, under control conditions, plateaued 18–24 h following replenishment of media (data not shown). To explore a possible role for IGF-1 in IL-4-induced Akt and M2 activation, BMDMs were stimulated with IL-4 in the presence or absence of αIGF-1 or the appropriate isotype control; the data show that αIGF-1 attenuated the IL-4-induced increase in pAkt at 18 h (*p o0.05; ANOVA; Fig. 2B). The IL-4-induced increase in MRC1 mRNA (***po 0.001; ANOVA; Fig. 2C) was enhanced in the presence of αIGF-1 ( þ p o0.05). In contrast, co-incubation of BMDMs with IL-4 and αIGF-1 attenuated the IL-4-induced increases in mRNA expression of FIZZ1 and Chi3l3 (***p o0.001, control vs IL-4; þ p o0.05; þþþ p o0.001, IL-4 vs IL-4 þ αIGF-1; ANOVA; Fig. 2D and E). While αIGF-1 had no effect on Arg1 mRNA expression at 18 h (***p o0.001, control vs IL-4; ANOVA; Fig. 2F), the IL-4-induced increase in Arg1 protein expression was attenuated in the presence of αIGF-1 (***p o0.001, control vs IL-4; þ þ po 0.01, IL-4 vs IL-4 þ αIGF-1; ANOVA; Fig. 2G and H). IL-4 mediates its effects through two receptors, the type I IL-4 receptor (composed of two subunits, IL-4Rα and IL-2Rγ) and the type II receptor (IL-4Rα and IL-13Rα1), activation of these receptors results in the phosphorylation of STAT6. JAK3 is activated as a consequence of ligation of the type I IL-4R, which has been shown to participate in the activation of Akt. To further investigate this, BMDMs were incubated in the presence of IL-4 and the JAK3 inhibitor, Janex I, for 1 h. IL-4
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increased the expression of pJAK3 and pAkt (*po0.05; ANOVA; Fig. 3A–C) and this effect was attenuated in the presence of Janex 1 ( þ po0.05; þ þ po0.01). In contrast, Janex 1 had no effect on the expression of JAK3, and Akt (Fig. 3A; 2nd, 4th and 5th panel). Similarly, inhibition of JAK3 with Janex I had no effect on the induction of pSTAT6 and this is consistent with data from the literature demonstrating that JAK3 KO had no effect on IL-13-induced STAT6 activity which is mediated by Tyk2 and JAK1 [14]. The IL-4-induced increase in release of IGF-1 (***po0.001; ANOVA; Fig. 3D) was also attenuated in the presence of Janex I ( þ þ þ po0.001). Janex 1 had no effect on the IFNγ-induced increase in pSTAT1 expression, demonstrating that it had no inhibitory effects on either JAK1 or JAK2 (data not shown). The IL-4-induced increases in expression of Arg1, FIZZ1 and Chi3l3 mRNA were similarly inhibited by Janex I (***po0.001, control vs IL-4; þþþ po0.001; IL-4 vs IL-4 þ Janex 1; ANOVA; Fig. 3F, I and J respectively). Inhibition of JAK3 also resulted in an attenuation of the IL4-induced increase in Arg1 protein expression (***po0.001, control vs IL-4; þ þ þ po0.001; IL-4 vs IL-4 þ Janex 1; ANOVA; Fig. 3G and H). However, co-incubation with Janex I enhanced the IL-4-induced increase in expression of MRC1 mRNA (***po0.001, control vs IL-4; þþþ po0.001; IL-4vs IL-4 þ Janex 1; ANOVA; Fig. 3E). Since inhibiting JAK3 activation prevented full M2 activation and this was independent of STAT6 activity, analysis was carried out to establish the effects of pAkt inhibition. As before, IL-4 increased IGF-1 release from BMDMs (***p o0.001; ANOVA; Fig. 4A), however in the presence of the PI3-kinase inhibitor, LY294002, this effect was attenuated ( þ þ þ p o0.001). Additionally, LY294002 attenuated the IL-4-induced increase of Chi3l3, FIZZ1 and Arg1 mRNA expression (***p o0.001, control vs IL-4; þ þ þ p o0.001, þ p o0.05, IL-4 vs IL-4 þ LY294002; ANOVA; Fig. 4C–E respectively), however the effect of IL-4 on MRC1 mRNA expression was enhanced by co-incubation with LY294002 (***p o0.001, control vs IL-4; þ þ þ po0.001, IL-4 vs IL-4 þ LY294002; ANOVA; Fig. 4B). Inhibition of Akt attenuated the IL-4-induced increase in Arg1 protein expression (***p o0.001, control vs IL-4; þ þ þ p o0.001, IL4 vs IL-4 þ LY294002; ANOVA; Fig. 4F) TNFα and NOS2 mRNA are archetypal markers of the M1 state and time-related analysis of the changes indicated that IFNγ significantly increased expression of TNFα mRNA at 2 h and that this increase was still evident at 9 h (***po0.001; ANOVA; Fig. 5A). IFNγ stimulation increased NOS2 mRNA expression at all times investigated, however this effect achieved statistical significance at 6 and 24 h (***po0.001; ANOVA; Fig. 1B), though not at 9 h (where the value was also not significantly different from that at 6 or 24 h). IFNγ signals through phosphorylation of STAT1 and the data presented here show that pSTAT1 expression was significantly enhanced at 30 min (***p o0.001; ANOVA; Fig. 5C and D) and remained elevated at all times examined. The regulatory role of PI3K/Akt in inflammation has been identified previously [15], in light of this, we examined the activation of this pathway in IFNγ-stimulated macrophages. We report that IFNγ led to an early increase (4 h) followed by a decrease in the expression of pAkt at 18 and 24 h (*po 0.05, **p o0.01; ANOVA; Fig. 5E). The reduction in pAkt expression was accompanied by an IFNγ-induced decrease in IGF-1 release from BMDMs (*po 0.05; ANOVA; Fig. 5F). To assess the role of IGF-1 in Akt activation and, specifically, the interplay between IGF-1 and IFNγ, we co-incubated BMDMs with IFNγ and αIGF-1 or the appropriate isotype control and show that the IFNγ-induced increase in expression of pAkt at 4 h (***p o0.001; ANOVA; Fig. 6A) was attenuated by αIGF-1 ( þ þ po 0.01; ANOVA; Fig. 6A). Additionally, the IFNγ-induced increases in expression of TNFα and NOS2 mRNA (***p o0.001; ANOVA; Fig. 6B and D) were exacerbated in the presence of αIGF-1 ( þ p o0.05; ANOVA; Fig. 6B and D). Incubation of cells in the presence of αIGF-1 enhanced the IFNγ-induced release of TNFα (***p o0.001, control vs IFNγ; þ p o0.05, IFNγ vs IFNγ þ αIGF-1;
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 1. Expression of M2 activation markers varies upon exposure to IL-4. Bone-marrow derived macrophages were incubated with IL-4 for various times. Expression of MRC1 mRNA (A) was significantly enhanced at 1 h, however Arg1 mRNA (B) was not significantly increased until 6 h, while FIZZ1 (C) and Chi3l3 (D) mRNA expression were unchanged until 24 h (***p o 0.001; ANOVA; n ¼6). Expression of phosphorylated STAT6 (F) was apparent at 30 min and still evident at 24 h (***p o0.001; **p o0.01; ANOVA; n ¼4). A representative immunoblot is shown in (E). Incubation of BMDMs with IL-4 revealed an increase in the expression of phosphorylated Akt (H) at 1 h and later at 9 and 12 h (*p o0.05; ANOVA; n¼ 4). A representative immunoblot is shown in (G). All data are expressed as mean7 SEM.
ANOVA; Fig. 6C). Furthermore, IFNγ treatment significantly decreased IGF-1 supernatant concentration (*p o0.05, control vs IFNγ; ANOVA; Fig. 6E) and this effect was exacerbated in the presence of LY294002 ( þ p o0.05, IFNγ vs IFNγ þ LY294002; 24 h; ANOVA; Fig. 6E), while co-incubation of BMDMs with IFNγ and LY294002 further increased the IFNγ-induced expression of TNFα mRNA (*p o0.05, control vs IFNγ; þ p o0.05, IFNγ vs IFNγ þ LY294002; ANOVA; Fig. 6F).
4. Discussion The significant finding of this study is that IL-4-induced activation of Akt is JAK3-dependent, is enhanced by subsequent release of IGF-1 and is necessary for full adoption of the M2 phenotype. We report that the early response of macrophages to IFNγ was to increase Akt activation and inhibition of Akt or blockade of IGF1 activity exacerbated the effects of IFNγ. This indicates that the initial IFNγ-induced phosphorylation of Akt expression may serve
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 2. M2 activation state is regulated by Akt/IGF-1 signalling. The IL-4-induced increase in the expression of pAkt at 9 and 12 h correlated with enhanced release of IGF-1 (A) at these times (*po0.05; ***po0.001; ANOVA; n¼4). Co-incubation of BMDMs with IL-4 and a neutralising antibody to IGF-1 (αIGF-1) attenuated the IL-4-induced increase in pAkt (B) at 18 h (*po0.05, control vs IL-4; ANOVA; n¼4). A representative immunoblot is also shown. The IL-4-induced increase in mRNA expression of MRC1 (C; ***po0.001; ANOVA; n¼ 4) was enhanced in the presence of αIGF-1 ( þ po0.05; ANOVA; n¼4). Co-incubation of BMDMs with IL-4 and αIGF-1 attenuated the IL-4-induced increase in mRNA expression of FIZZ1 (D) and Chi3l3 (E) (***po0.001, control vs IL-4; þ po0.05, þ þ þ po0.001, IL-4 vs IL-4 þ αIGF-1; ANOVA; n¼4). While αIGF-1 had no effect on IL-4-induced Arg1 mRNA expression (F; ***po0.001, control vs IL-4; ANOVA; n¼4), the IL-4 mediated increase in Arg1 protein expression was attenuated in the presence of αIGF-1 (G, H; ***po0.001, control vs IL-4; þ þ po0.01, IL-4 vs IL-4 þ αIGF-1; ANOVA; n¼ 4). A representative blot is shown (G). All data are expressed as mean7SEM.
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 3. Attenuation of M2 by inhibition of pJAK3 and pAkt is independent of pSTAT6. BMDMs were incubated in the presence of IL-4 and the JAK3 inhibitor, Janex I, for 1 h. Expression of phosphorylated JAK3 (A; B) and Akt (A; C) were increased by exposure to IL-4 (*po0.05, **po0.01, ANOVA; n¼3) and this effect was attenuated in the presence of Janex 1 ( þ po0.05; þ þ po0.01; ANOVA; n¼ 3). The IL-4-induced increase in expression of phosphorylated STAT6 was unaffected by Janex 1 (A; 5th panel). The presence of IL-4 or Janex 1 had no effect on expression of total JAK3 (A; 2nd panel) and Akt (A; 4th panel). Representative immunoblots are shown in (A). The IL-4-induced increase in release of IGF1 (D) was also attenuated in the presence of Janex I as were expression of Arg1 (F), Chi3l3 (I) and FIZZ1 (J) mRNA (***po0.001, control vs IL-4; þ þ po0.01, þ þ þ po0.001, IL-4 vs IL-4 þ Janex I ANOVA; n¼3). The IL-4-induced increase in Arg1 protein expression was attenuated in the presence of Janex 1 (G, H; ***po0.001, control vs IL-4; þ þ þ po0.001, IL-4 vs IL-4 þ Janex I ANOVA; n¼3), a representative blot is shown (G). However, co-incubation with Janex I enhanced the IL-4-induced increase in expression of MRC1 mRNA (E; ***po0.001, control vs Janex I, control vs IL-4; þ þ þ po0.001, IL-4 vs IL-4 þ Janex I; ANOVA; n¼ 3). All data are expressed as mean7SEM.
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Fig. 4. Inhibition of pAkt affects release of IGF-1 and M2 state in BMDMs. The IL-4-induced release of IGF-1 (A) from BMDMs was attenuated in the presence of the PI3kinase inhibitor, LY294002, as were expression of Chi3l3 (C) FIZZ1 (D) and Arg1 (E) mRNA (***p o 0.001, control vs IL-4; þ po 0.05, þ þ þ p o 0.001, IL-4 vs IL-4 þ LY294002; ANOVA; n ¼5), however the effect of IL-4 on MRC1 (B) mRNA expression was enhanced by co-incubation with LY294002 (***p o 0.001, control vs IL-4; þ þ þ p o 0.001, IL-4 vs IL-4 þ LY294002; ANOVA; n¼ 5). The IL-4-induced increase in Arg1 protein expression was attenuated in the presence of LY294002 (F; ***p o 0.001, control vs IL-4; þþþ p o 0.001, IL-4 vs IL-4 þ LY294002; ANOVA; n¼ 4). All data are expressed as mean 7 SEM.
to modulate M1 activation. Prolonged exposure to IFNγ downregulated Akt activation; this was associated with decreased IGF-1, an effect that was exacerbated by inhibition of PI3-kinase, suggesting that Akt induces its own activation via production of IGF-1. IL-4 stimulated Akt activation and this may be mediated by the IL4-induced increase IGF-1 production. We demonstrate that inhibition of PI3K/Akt activation decreased the expression of IL-4-induced genes associated with the M2 phenotype and therefore we conclude that activation of the PI3K/Akt pathway is crucial in mediating this effect of IL-4. In contrast, inhibiting the action of IGF-1 and Akt exacerbated the M1 phenotype. It is proposed that the differential effects of IL-4 and IFNγ on IGF-1 production are responsible for the observed changes in PI3K/Akt activity and that modulating PI3K/Akt signalling plays a key role in the polarisation of macrophages. Analysis of time-related IL-4-induced changes in expression of markers of M2 activation indicated that maximal upregulation of MRC1 mRNA in BMDMs occurred at 1 h, closely following the
activation of STAT6. In contrast, the induction of Arg1, Chi3l3 and FIZZ1 was much later than MRC1, and this concurs with previous findings [7]. The difference in the temporal profile of induction of M2 markers suggests differential regulation. Heller and colleagues demonstrated that IL-4 and IL-13 activated STAT6 in a similar manner but that IL-4 increased the expression of Arg1, Chi3l3 and FIZZ1 in a more robust fashion than IL-13. The effects of IL-4 and IL-13 on MRC1 mRNA expression were similar suggesting that Arg1, Chi3l3 and FIZZ1 expression may be modulated by IL-4 in a STAT6-independent manner. We found that the IL-4-induced activation of STAT6 was evident within 30 min and persisted for 24 h, confirming earlier observations [16]. Importantly, total STAT6 expression was unaffected by IL-4 treatment, indicating that the increase in activated STAT6 did not require de novo protein synthesis. It is well documented that STAT6 is necessary for the induction of the M2 phenotype, a number of studies have demonstrated that macrophages from STAT6-deficient mice do not show upregulation
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 5. Temporal response of bone-marrow derived macrophages to IFNγ. Bone-marrow derived macrophages were incubated with IFNγ for various times. Expression of TNFα mRNA (A) was significantly upregulated by 2 h and persisted until 24 h while the mRNA expression of NOS2 (B) was not significantly increased until 6 h but also persisted until 24 h (***p o 0.001; ANOVA; n ¼6). Changes in the mRNA expression of archetypal markers of the M1 activation state were preceded by enhanced expression of phosphorylated STAT1 (D) at 30 min and 1 h (***p o 0.001; ANOVA; n¼ 4). A representative immunoblot is shown (C). Exposure of BMDMs to IFNγ led to an early increase (4 h) followed by a decrease in the expression of phosphorylated Akt (E; see sample immunoblot) at 18 and 24 h (*p o 0.05, **po 0.01, control vs IL-4; ANOVA; n¼ 4). The release of IGF-1 (F) was decreased by incubation of BMDMs with IFNγ at 1 h and at all timepoints examined (*p o 0.05; ANOVA; n¼ 4, control vs IFNγ). All data are expressed as mean 7 SEM.
of M2associated genes in response to IL-4/IL-13 stimulation [17– 19]. While it is clear that STAT6 is necessary to mediate the effects of IL-4, previous studies have demonstrated that IL-4 impacts upon other signalling pathways. For example, IL-4 activates IRS-2 [7] and consequently affects the PI3K/Akt and Ras-MAPK pathways, which activate numerous cell processes including cell proliferation [20]. Here, pAkt expression was enhanced in BMDMs after 1 h of exposure to IL-4 and was also evident at 9 and 12 h, coinciding with the IL-4-induced changes in M2 markers. Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signalling as essential for IL-4-driven murine macrophage proliferation in vivo [12]. Previous evidence has also indicated that inhibition of PI3K/Akt by wortmannin decreases the IL-4-induced expression of FIZZ1, but not Arg1, in murine macrophages [7]. IL-4 increased IGF-1 production in BMDMs, an effect that has been
reported in macrophages and microglia [13,21]. These cells also respond to IGF-1 stimulation and work from this laboratory has demonstrated that IGF-1 can attenuate the response of microglia to IFNγ stimulation [9]. Other groups have presented evidence that IGF-1 is neuroproective following ischaemia and trauma [22,23] and, as a result, IGF-1 may mediate the anti-inflammatory effects associated with IL-4. The IGF-1R is constitutively expressed in cells [24], including macrophages and microglia, while it has been demonstrated to be anti-inflammatory it has also been suggested that it may induce proliferation of macrophages [25]. These findings, coupled with those of the present study, suggest that IGF-1 produced by macrophages may act in both autocrine and paracrine manner to induce and enhance M2 activation and may lead to an increase in the number of macrophages present at the site of inflammation. While it has been previously shown that IGF-1 is a potent activator of the PI3K/Akt
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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Fig. 6. Akt/IGF-1 signalling contributes to attenuation of M1 activation. Co-incubation of BMDMs with IFNγ and αIGF-1 attenuated the IFNγ-induced increase in expression of phosphorylated Akt (A; see representative immunoblot) at 4 h (***p o 0.001, control vs IFNγ; þ þ po 0.01, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼3). The IFNγ-induced increase in expression of TNFα (B) mRNA was exacerbated in the presence of αIGF-1 (***p o 0.001, control vs IFNγ; þ p o 0.05, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼4). IFNγ stimulation increased TNFα supernatant concentration (C) and this was exaggerated in the presence of αIGF-1 (***p o 0.001, control vs IFNγ; þ p o0.05, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼ 3). The effect of IFNγ on NOS2 mRNA (D) expression was enhanced in the presence of αIGF-1 (***p o 0.001, control vs IFNγ; þ p o 0.05, IFNγ vs IFNγ þ αIGF-1; ANOVA; n¼ 4). IGF-1 release was significantly decreased in the presence of IFNγ and this effect was exacerbated in the presence of LY294002 (E; *p o0.05, control vs IFNγ; þ þ p o 0.01, IFNγ vs IFNγ þ LY294002; ANOVA; n ¼4), while co-incubation of BMDMs with IFNγ and LY294002 led to an upregulation in the IFNγ-induced expression of TNFα (F) mRNA (*p o 0.05, control vs IFNγ; þ p o 0.05, IFNγ vs IFNγ þ LY294002; ANOVA; n¼ 5). All data are expressed as mean 7SEM.
pathway, the present study demonstrates its role in mediating the effect of IL-4 on pAkt expression. In the presence of αIGF-1, the IL-4induced increase in pAkt expression was significantly attenuated, suggesting that IGF-1 is necessary for the IL-4-mediated induction of pAkt expression. These findings suggest that IGF-1 expression is not
only a marker of the M2 phenotype, but induces signalling pathways that drive and maintain the M2 response. In addition to this, co-incubation of BMDMs with IL-4 and αIGF-1 attenuated the IL-4-induced FIZZ1 and Chi3l3 mRNA, identifying a role for IGF-1/Akt in the induction of the M2 phenotype. Although IL-4-induced Arg1 mRNA was
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
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unaffected by αIGF-1 incubation, the effect of IL-4 on Arg1 protein expression was attenuated by αIGF-1 incubation. In contrast to the effects of αIGF-1 on the other markers of M2 activation, IL-4-induced MRC1 mRNA was significantly increased in the presence of αIGF-1. Therefore, we can conclude that IL-4-stimulated Akt activation differentially modulates expression of molecules associated with the M2 state. It has been documented that ligation of the type I, but not the type II, IL-4R can activate the PI3K/Akt pathway [20]. To establish whether the changes in pAkt occurred as a result of type 1 IL-4R activation, we set out to inhibit the activation of JAK3, a component of the type I IL-4R not utilised by type II IL-4R-associated signalling. The JAK3 inhibitor, Janex 1, attenuated the IL-4-induced increases in pAkt and IGF-1 production while STAT6 activation was unaffected. Similar to the effect of αIGF-1, inhibition of JAK3 increased IL-4-induced MRC1 mRNA, attenuated the IL-4-induced increase in Arg1, FIZZ1 and Chi3l3 mRNA and had no effect on STAT6 signalling. Together, these data indicate alternative modes of activation for specific markers of the M2 phenotype with differential responses driven through ligation of type I and type II IL-4R (Fig. 7). Interestingly, the IL-4-induced upregulation of Arg1, Chi3l3 and FIZZ1 mRNA was inhibited by LY290042, confirming earlier findings [7,12]. Therefore, PI3K/Akt appears to be crucial in the induction of at least these M2 activation markers. The effect of the SH2-containing inositol phosphatase (SHIP) on PI3K activity further highlights the role of the PI3K/Akt pathway in the induction of M2 activation; SHIP is a negative regulator of PI3K activity and Weisser and colleagues reported that macrophages from SHIPdeficient mice exhibit an exaggerated response to IL-4 treatment [26]. It should be noted that the concentration of LY290042 used in this study did not completely attenuate IL-4-induced changes in Arg1, Chi3l3 and FIZZ1 mRNA expression, indicating that while Akt activation is necessary for maximal induction of these genes, IL-4 can induce their expression through other pathways. In complete contrast to the effect of LY290042 on IL-4-induced changes in Arg1, Chi3l3 and FIZZ1 mRNA, MRC1 mRNA was enhanced by LY290042 mimicking the effect of both α-IGF-1 and Janex 1; this suggests that the IL-4-induced increase in MRC1 mRNA expression is not dependent on PI3K/Akt activation [7]. On the basis of our
findings and work by others, we conclude that activation of STAT6 is primarily involved in modulation of the expression of MRC1. One objective of this study was to investigate the differential effects of IL-4 and IFNγ on the PI3K/Akt pathway. The effect of IFNγ on Akt activation is complex and time-dependent, with no evidence of an immediate effect but an increase at 4 h. We propose that the early activation of Akt reflects a negative feedback control on IFNγ-induced changes. Consistent with this, we demonstrate that αIGF-1 decreased pAkt expression and enhanced the response to IFNγ, while the effect of IFNγ on TNFα and NOS2 mRNA expression was exacerbated in the presence of α-IGF. The mechanism by which IFNγ induced an increase in pAkt expression at 4 h may be IGF-1-independent since IFNγ induced a decrease in IGF-1 production at this time. However, blocking the interaction of IGF-1 with IGF-1R (even at this lower IGF-1 concentration) decreased tonic pAkt expression and may thus have contributed to the IFNγinduced increases in TNFα and NOS2. Data from numerous studies have indicated that activation of the PI3K/Akt pathway promotes anti-inflammatory effects and attenuates TLR-induced cytokine production [15,27]. Similarly, decreased signalling through this pathway has been associated with an increased response to inflammatory stimuli; specifically Luyendyk and colleagues demonstrated that macrophages, in which PI3K signalling was decreased, exhibit an enhanced response to LPS stimulation [15]; in contrast, macrophages cultured from PTEN / mice exhibit enhanced Akt activation and this was associated with a decrease in responsiveness to LPS stimulation. Like PTEN, SHIP also inhibits the activity of PI3K, and LPS-induced cytokine release is also impaired in macrophages prepared from SHIP-deficient mice [26]. Thus, the PI3K/Akt pathway plays a part in restricting inflammatory events, and evidence from experiments in vivo have demonstrated that inhibition of PI3K increased inflammation in mouse models of endotoxemia and sepsis [28,29]. We conclude that a deficit in IGF-1/Akt signalling is associated with a decreased capacity to induce the M2 state and an increase in responsiveness to IFNγ. This is interesting in the context of our earlier findings that hippocampal expression of IGF-1, as well as activation of Akt, is decreased in the brain with age and that these changes are associated with increased IFNγ concentration [10].
Fig. 7. Schematic. IL-4 ligates the type II IL-4R to recruit and activate Tyk2 and JAK1 leading to the subsequent activation of STAT6 and upregulation of M2 markers. IL-4 interacts with the type I IL-4R to induce recruitment and activation of JAK1 and JAK3, leading to activation of PI3K/Akt signalling and upregulation of IGF-1 expression. IGF-1 induces further activation of Akt and contributes to upregulated expression of M2 markers of activation.
Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i
J.P. Barrett et al. / Experimental Cell Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
These age-related changes probably provide an explanation for the increased propensity of microglia to adopt an M1 phenotype, and their limited ability to adopt the M2 phenotype, in aged animals [30].
Author contribution JPB, AMM and ML designed the work. JPB, AMM and AF prepared the tissue for analysis; the analysis and preparation of figures was carried out by JPB and AMM. JPB and AMM prepared the initial draft of the manuscript and ML critically assessed the content and approved the final manuscript. ML supervised the work.
Conflict of interest disclosure The authors declare no conflict of interest.
Acknowledgements This work was funded by Science Foundation Ireland (07/IN.1/ B949). JPB was the recipient of a Trinity College Postgraduate student award.
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Please cite this article as: J.P. Barrett, et al., Involvement of IGF-1 and Akt in M1/M2 activation state in bone marrow-derived macrophages, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.05.015i