Suppressor of cytokine signaling (SOCS) - Wiley Online Library

Article

Suppressor of cytokine signaling (SOCS)1 is a key determinant of differential macrophage activation and function Claire S. Whyte,* Eileen T. Bishop,* Dominik Ru ¨ ckerl,† Silvia Gaspar-Pereira,* † Robert N. Barker,* Judith E. Allen, Andrew J. Rees,‡ and Heather M. Wilson*,1 *Division of Applied Medicine, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom; † Institute of Immunology and Infection Research, University of Edinburgh, Edinburgh, United Kingdom; and ‡Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria RECEIVED NOVEMBER 29, 2010; REVISED APRIL 27, 2011; ACCEPTED APRIL 27, 2011. DOI: 10.1189/jlb.1110644

ABSTRACT

Introduction

Macrophages become activated by their environment and develop polarized functions: classically activated (M1) macrophages eliminate pathogens but can cause tissue injury, whereas alternatively activated (M2) macrophages promote healing and repair. Mechanisms directing polarized activation, especially in vivo, are not understood completely, and here, we examined the role of SOCS proteins. M2 macrophages activated in vitro or elicited by implanting mice i.p. with the parasitic nematode Brugia malayi display a selective and IL-4dependent up-regulation of SOCS1 but not SOCS3. Using siRNA-targeted knockdown in BMDM, we reveal that the enhanced SOCS1 is crucial for IL-4-induced M2 characteristics, including a high arginase I:iNOS activity ratio, suppression of T cell proliferation, attenuated responses to IFN-␥/LPS, and curtailed SOCS3 expression. Importantly, SOCS1 was essential in sustaining the enhanced PI3K activity that drives M2 activation, defining a new regulatory mechanism by which SOCS1 controls M2 polarization. By contrast, for M1 macrophages, SOCS1 was not only an important regulator of proinflammatory mediators (IL-6, IL-12, MHC class II, NO), but critically, for M1, we show that SOCS1 also restricted IL-10 secretion and arginase I activity, which otherwise would limit the efficiency of M1 macrophage proinflammatory responses. Together, our results uncover SOCS1, not only as a feedback inhibitor of inflammation but also as a critical molecular switch that tunes key signaling pathways to effectively program different sides of the macrophage balance. J. Leukoc. Biol. 90: 845– 854; 2011.

Macrophages comprise a heterogeneous population of cells that play an important role in tissue homeostasis as well as coordinating almost all aspects of inflammation [1–3]. Infiltrating macrophages respond to microenvironmental stimuli and adopt phenotypically and functionally distinct subpopulations that can initially destroy invading pathogens and tumor cells and then, in the resolution phase of inflammation, induce tissue repair, promote healing, or in some cases, lead to fibrosis [1, 4]. Although the extent of their heterogeneity is still unclear, macrophages are generally classified into two broad activation types. Classical or M1 macrophage activation is induced by microbial agents in the presence or absence of IFN-␥ and results in proinflammatory macrophages with potent microbicidal potential. In contrast, alternative or M2 macrophage activation by IL-4 (or IL-13) gives rise to anti-inflammatory tissue-remodeling cells with antiparasitic functions. Ligation of other macrophage receptors, such as IL-10, TGF-␤, glucocorticoids, or FcgammaRs together with LPS, has defined other types of M2like, immunoregulatory macrophages [2, 5, 6]. It is not fully understood how macrophages become polarized in vivo and the degree to which they retain plasticity or remain committed to a particular activation state [7, 8]. An important feature of macrophage polarization is, however, to prevent opposing signaling pathways from being induced simultaneously. For example, exposure to IL-4 induces unresponsiveness to subsequent M1 activation stimuli, whereas M1 macrophage activation induces unresponsiveness to IL-4 [9]. Critically, the coordinated polarization of macrophages determines whether infection or injury is resolved successfully or progresses to diseases ranging from chronic inflammation to tumorigenesis, asthma, and fibrosis [3, 10]. The ability to redirect inappropriate macrophage activation, therefore, presents a novel and potentially highly effective therapeutic approach for treating macrophage-mediated diseases [6]. Elucidating the

Abbreviations: BMDM⫽bone marrow-derived macrophages, IL-4R␣–/–⫽ IL-4 receptor-alpha-deficient, M1⫽classically activated macrophages, M2⫽alternatively activated macrophages, MFI⫽median of fluorescence intensity, p-AKT⫽phosphorylated AKT, PEC⫽peritoneal exudate cells, PPD⫽purified protein derivative, RELM-␣⫽resistin-like molecule-␣, siRNA⫽short-interfering RNA, SOCS⫽suppressor of cytokine signaling The online version of this paper, found at www.jleukbio.org, includes supplemental information.

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1. Correspondence: Division of Applied Medicine, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, UK. E-mail: [email protected]

Volume 90, November 2011

Journal of Leukocyte Biology 845

molecular mechanisms that coordinate macrophage intracellular signaling and subsequent programming is thus a major goal, and here, we have examined the role of the SOCS proteins. The mammalian SOCS protein family consists of at least eight distinct members (SOCS1 to -7 and CIS (cytokine-inducible Src homology 2 protein), which regulate intracellular signaling networks. SOCS1 and -3 can be induced rapidly in macrophages [11, 12], and their expression is frequently increased in inflammatory diseases, where they have divergent and nonredundant roles [13–15]. SOCS1 inhibits macrophage responses to IFN-␥, and SOCS1-deficient mice develop symptoms of severe systemic autoimmune and inflammatory disease [11, 16 –18]. SOCS3 attenuates IL-6-induced STAT3 anti-inflammatory effects [19 –21], as well as IL-4-induced insulin receptor substrate-2/PI3K-mediated gene expression [22, 23]. SOCS1 and SOCS3 negatively regulate JAK/STAT signaling by binding to key phosphotyrosine residues in JAKs or cytokine receptor cytoplasmic domains, by inhibition of JAK tyrosine kinase activity through their kinase-inhibitory region or by targeting the signaling complex for ubiquitination and proteasomal degradation [11, 12]. They also affect MAPK and NF-␬B pathways that direct macrophage properties [23–25]. SOCS proteins, therefore, are tightly linked to macrophage polarization. Indeed, our recent work has established that the majority of macrophages infiltrating inflamed glomeruli in experimental nephritis expresses enhanced SOCS3, which is necessary for their M1-polarized phenotype [15]. Although much work has examined the effect of SOCS proteins, especially SOCS1, in regulating IFN-␥- and TLR-mediated proinflammatory responses, much less is known regarding how SOCS proteins control M2 polarization. The present study was designed to determine macrophage SOCS1 and SOCS3 expression in an M2 macrophage-activating environment in vitro and in vivo and to examine the role of SOCS1 in directing M1 and M2 polarization in vitro. We demonstrate that SOCS1, but not SOCS3, is efficiently up-regulated in M2 macrophages, and this rapid increase in SOCS1 in M2 macrophages has a critical role in sustaining specific aspects of their anti-inflammatory phenotype and function. However, for M1 macrophages, SOCS1 was shown to be an important regulator, not only of proinflammatory mediators but also of antiinflammatory effects and consequently, may act to prevent overshooting of the inflammatory response. Therefore, modulation of SOCS1 expression represents a potential strategy to control inappropriate or excessive macrophage activation in inflammatory diseases.

MATERIALS AND METHODS

Mice and infection WT BALB/c mice or IL-4R␣⫺/⫺ mice [26] were bred in-house or purchased from Harlan Laboratories (UK). IL-4R␣⫺/⫺ mice were originally a generous gift from Prof. Frank Brombacher, University of Cape Town (South Africa). All work was conducted in accordance with the UK Animals (Scientific Procedures) Act of 1986. Mice were 6 – 8 weeks old at the start of the experiment. Adult Brugia malayi parasites were removed from the peritoneal cavity of infected gerbils purchased from TRS Laboratories (Ath-

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ens, GA, USA). Mice were surgically implanted i.p. with four female and one male live adult B. malayi [26]. Six or 27 days later, mice were killed and PEC harvested by washing of the peritoneal cavity with 10 ml ice-cold RPMI. As a control, mice were sham-operated and PEC harvested as before.

Cells and reagents Harvested PEC were washed and adjusted to 1 ⫻ 106 cells/ml in complete medium and cytocentrifuge preparations made using a Shandon Cytospin (Shandon, PA, USA). Cytocentrifuge preparations of PEC were air-dried and fixed for 5 min in 70% methanol prior to immunofluorescent staining. Macrophages accounted for at least 70% of cells and were easily identified by microscopy. For PCR analyses, isolated PEC were washed in PBS containing 1% FCS and 0.1% sodium azide and stained with F4/80 and Siglec F antibodies, after which, macrophages were purified from total PEC by cell sorting based on positivity for F4/80 and negativity for Siglec F using a BD FACSAria cell sorting system (BD Immunocytometry Systems, Oxford, UK). Rat BMDM were isolated by flushing BM from the femur and tibia, as described previously [27]. Cells were suspended in culture medium comprising DMEM, supplemented with 10% heat-inactivated FCS, 200 U/ml penicillin, and 200 ␮g/ml streptomycin with the addition of 20% L929 conditioned medium produced using a standard protocol [28]; this yields a population of ⬎95% macrophages. BMDM were stimulated with rat cytokines, as indicated in the text: IL-4, 20 ng/ml (PeproTech, London, UK); and IFN-␥, 2.5 ng/ml, and LPS, 100 ng/ml (Sigma-Aldrich, London, UK).

Immunofluorescent microscopy For SOCS1 and SOCS3 detection, cytospin preparations of PEC were rehydrated and permeabilized in 0.5% Triton X-100/PBS, blocked with 1% glycine/PBS, then incubated with goat anti-rat SOCS1 (Abcam, Cambridge, UK, 1:200) or rabbit anti-rat SOCS3 (Abcam; 1:200), and detected with Alexa fluor 488-conjugated chicken anti-goat and Alexa fluor 594-conjugated donkey anti-rabbit secondary antibody, respectively. The nonspecific background was determined by use of an isotype control antibody and the secondary antibody alone. Images were captured under fluorescence using an Olympus IX70 inverted microscope, ColorView 12 digital camera, and Soft Imaging Systems (SIS GmbH, Germany).

RNA extraction and RT-PCR For mouse peritoneal cells, RNA was recovered by resuspension in TRIzol reagent (Invitrogen, Paisley, UK). Total RNA was extracted, according to the manufacturer’s instructions. Following DNase I treatment (Ambion, Huntingdon, UK), 1 ␮g RNA was used for the synthesis of cDNA using Moloney murine leukemia virus RT (Stratagene, Cambridge, UK). Relative quantification of the genes of interest was measured by real-time PCR, using a Roche LightCycler 480. Five serial, 1/4 dilutions of a positive control sample of cDNA were used as a standard curve in each reaction, and the expression levels were estimated from the curve. Real-time PCR of the housekeeping gene GAPDH was used to normalize expression. Primer sequences and PCR conditions were as published previously [29, 30]. For BMDM, RNA was extracted as above, followed, in some cases, by RNA clean-up, using an RNeasy mini kit (Qiagen, Crawley, UK), according to the manufacturer’s instructions. RNA was reverse-transcribed, using the first-strand cDNA synthesis kit and oligo (dT) primer, as recommended by the manufacturer (Invitrogen); rat primers were as described previously [15]. PCR was performed semiquantitatively and PCR products separated on 1% (w/v) agarose gel and visualized by staining with ethidium bromide. In this case, the level of mRNA expression in each sample was determined by densitometric image analysis and standardized against the GAPDH measurement.

Transfection of siRNA Three predesigned SOCS1 siRNA duplexes (ID: 196356, 286837, 47137) and a nontargeting, siRNA sequence-negative control, containing at least four mismatches to any mouse, human, or rat gene, were purchased from

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Whyte et al. SOCS1 directs macrophage activation and function Ambion. The sequence giving maximum knockdown and minimal off-target effects (286837) was used in all cases. BMDM were transfected with 30 nmol/L siRNA (final concentration), using, as a transfection reagent, Lipofectamine RNAi Max (Invitrogen) in serum-free medium, according to the manufacturer's instructions. Mock transfection using Lipofectamine RNAi Max but no siRNA acted as a control. Cells were incubated at 37°C for at least 48 h prior to treatment. Transfection efficiency was 89 ⫾ 9%, as determined by counting the number of macrophages containing fluorescently tagged siRNA. The nucleotide sequences and concentration of siRNA used in these studies did not induce a detectable IFN-␣ response, as determined by cytokine secretion and STAT1 activation.

Cytokine quantification

RESULTS

M2 macrophages activated in vitro and in vivo selectively up-regulate SOCS1 but not SOCS3 expression SOCS1 exerts a negative-feedback control on macrophage proinflammatory responses in M1 macrophages, but its effect on M2 polarization has not been characterized [32]. Accordingly, we examined SOCS1 and SOCS3 expression in BMDM incubated with the canonical M2-activating cytokine, IL-4. SOCS1 mRNA was rapidly up-regulated, and this correlated with increased protein expression (Fig. 1A and B). By contrast,

BMDM were transfected with control, nontargeting siRNA or SOCS1 siRNA and stimulated with cytokines for 24 h, either 48 h or 72 h after transfection. The concentration of IL-6, TNF-␣, and IL-10 in the supernatant was determined by CBA (BD Biosciences) and IL-12p40 and IL-12p70 by ELISA (BioSource, Camarillo, CA, USA), according to the manufacturers’ instructions.

Western blot and flow cytometry analysis Protein lysates from BMDM (20 ␮g) were separated by SDS/PAGE for Western blot analysis with specific primary antibodies for SOCS1, SOCS3, and GAPDH (Abcam), ␤-actin (Sigma-Aldrich), and AKT and p-AKT (Cell Signaling Technology, Herts, UK). Immunolabeled proteins were detected by using appropriate HRP-conjugated secondary antibodies, followed by visualization with ECL (Amersham Pharmacia Biotech, Bucks, UK). Bands were normalized to ␤-actin or GAPDH. For flow cytometric analysis, cells were stained with FITC-conjugated anti-CD86, anti-MHC class II (BD PharMingen, Oxford, UK), or FITC-conjugated isotype control antibodies. MFI is shown to take into account any data that are not normally distributed.

Quantification of NO production and arginase I activity NO production was assessed by nitrite production in the culture supernatant using the Greiss reaction [28]. Arginase I activity was assessed by the production of urea generated by the arginase-dependent hydrolysis of larginine as described [31]. Briefly, 1–3 ⫻ 105 cells were lysed with 100 ␮l 0.1% Triton X-100. Following a 30-min incubation with shaking, 100 ␮l 25 mM Tris-HCl (pH 7.2) and 20 ␮l 10 mM MnCl2 were added, and the enzyme was activated by heating to 56°C for 10 min. Arginine hydrolysis was conducted by incubating 100 ␮l of this lysate with 100 ␮l 0.5 M l-arginine (pH 9.7) at 37°C for 60 min. The reaction was then stopped with 800 ␮l H2SO4 (96%)/H3PO4 (85%)/H2O (1/3/7, v/v/v), and 40 ␮l 9% isonitrosopropiophenone was added, followed by heating to 99°C for 30 min before reading on a microplate reader at 540 nm. A standard curve of urea solution was used to determine concentrations.

T cell proliferation assays BMDM (1⫻105/well) were pretreated with or without IL-4 for 24 h and pulsed with 5 ␮g/mL of a bacterial antigen (mycobacterial PPD), eliciting recall T cell responses indirectly via macrophages, which were washed several times and cocultured with CD4⫹ Th cells (ratio 1:3), isolated from splenocytes by a magnetic cell sorting isolation kit (Miltenyi Biotec, Surrey, UK). After 6 days, T cell proliferation was measured by [3H]-thymidine incorporation.

Statistical analysis Results are presented as mean ⫾ sd, and differences between groups of cells or animals were determined by a two-tailed Student's t test using Prism 4 software (GraphPad Software, San Diego, CA, USA). P values of ⬍0.05 were designated as significant.

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Figure 1. M2 macrophages activated in vitro and in vivo selectively upregulate SOCS1 but not SOCS3 expression. SOCS1 mRNA (A) and protein (B) expression in IL-4-activated BMDM over time. Peritoneal macrophages isolated 6 days after sham-operated (sham) or B. malayiimplantated animals were FACS-purified, according to their F4/80 surface expression (F4/80hiSiglecF–) and subjected to RNA expression analyses for arginase I (Arg), RELM-␣, and YM1 (C) and SOCS1 and SOCS3 (D) by real-time PCR. Data show fold change in mRNA over sham-operated BALB/c WT control mice after normalization to GAPDH. Displayed are means ⫾ sem from at least five animals/group; one out of two independent experiments is shown. SOCS1 and SOCS3 immunofluorescent staining of peritoneal macrophages from shamoperated or B. malayi-implanted animals 6 days after surgery (E). SOCS1 expression was detected by a secondary antibody conjugated with Alexa fluor 488 (shown in green) and SOCS3 by a secondary antibody conjugated with Alexa fluor 594 (shown in red). Nuclei were identified by DAPI (blue). The nonspecific background is shown in this case by staining with secondary antibody alone (SOCS1 or SOCS3, ⫺ve); very similar results were observed with isotype control antibody. Images are representative of each group of at least five mice. Original magnification, ⫻400; original bars represents 50 ␮m.



SOCS3 mRNA and protein concentrations were decreased, demonstrating the strong polarization effect of IL-4 on SOCS protein expression, as anticipated from previous gene expression studies [33]. Macrophage activation in vivo is much more complex and does not necessarily reflect the in vitro models. We therefore analyzed SOCS1 and SOCS3 expression in peritoneal macrophages polarized to an M2 phenotype by implanting mice i.p. with the filarial nematode B. malayi. This produces a Th2-type immune response with little overt pathology and a highly enriched population of M2 macrophages [27]. At 6 days postimplantation, peritoneal macrophages expressed high levels of mRNA for the mouse M2 macrophage markers arginase I, RELM-␣, and YM1, as compared with shamoperated controls (Fig. 1C). However, nematode implantation did not induce the M2 markers in macrophages from BALB/c mice that were IL-4R␣⫺/⫺ [34]. This demonstrates the IL-4 dependence of the M2 macrophage polarization in this model. Peritoneal macrophages from sham-operated BALB/c control mice and IL-4R␣⫺/⫺ mice expressed low levels of SOCS1 mRNA, which was strongly induced in WT and IL-4R␣⫺/⫺ macrophages, 6 days after B. malayi implantation (Fig. 1D). The change in mRNA was reflected by an increase in SOCS1 protein expression in macrophages from BALB/c WT mice but not from the IL-4R␣⫺/⫺ animals (Fig. 1E), demonstrating the IL-4 dependence of SOCS1 protein expression in this context. Nematode implantation had the opposite effect on macrophage SOCS3 expression. After 6 days implantation in WT BALB/c mice, peritoneal macrophage SOCS3 mRNA expression had decreased substantially below baseline (Fig. 1D), consistent with results in IL-4-activated macrophages in vitro (Fig. 1A). In striking contrast, macrophages from IL-4R␣⫺/⫺ mice showed up-regulated SOCS3 protein levels (Fig. 1E), and SOCS3 gene expression was maintained (Fig. 1D). Thus, in vivo, M2 macrophage activation induced by nematode implantation in WT mice resulted in strongly polarized SOCS1-expressing macrophages, as observed in vitro, whereas in macrophages from IL-4R␣⫺/⫺ mice, this robust SOCS1 polarization was lost.

SOCS1 is necessary to maintain the arginase I:iNOS balance in M2 macrophages The next experiments used siRNA-mediated gene knockdown in BMDM to address the functional consequences of SOCS1 down-regulation in M2 macrophages. This technique avoids the risk of compensatory gene effects associated with congenital lack of SOCS1. SOCS1-specific siRNA, but not control siRNA or mock transfection, attenuated the IL-4-induced increase in SOCS1 mRNA and reduced protein expression, demonstrating the efficiency of our knockdown protocol (Fig. 2A and B). As expected, treatment with IL-4 in control siRNAtransfected BMDM enhanced expression of the M2 macrophage markers arginase I, mannose receptor (CD206), and indeed SOCS1 and decreased the basal expression of SOCS3 without influencing the M1 macrophage activation marker, iNOS (Fig. 2C). The characteristic IL-4-induced increase in the 848 Journal of Leukocyte Biology


Figure 2. SOCS1 knockdown deviates the relative arginase I:iNOS ratio of M2-polarized macrophages. Results from a representative experiment showing successful knockdown of SOCS1 mRNA as determined by PCR (A) and protein as determined by Western blotting (B). BMDM were treated with transfection reagent alone (mock transfection) or transfected in the presence of nontargeting control siRNA or SOCS1 siRNA and stimulated with IL-4 to induce SOCS1 expression. Knockdown of SOCS1 deviates the phenotype of IL-4-activated macrophages with enhanced mRNA for iNOS and SOCS3 and decreased expression of SOCS1 and arginase I, as analyzed by RT-PCR. MR, Mannose receptor (C). SOCS1 knockdown decreased the ratio of arginase I:iNOS mRNA expression, as determined by PCR and densitometric analyses of scanned bands from ethidium bromide-stained agarose gels. Arginase I and iNOS activity in SOCS1 knockdown BMDM (D). Mock-transfected, nontargeting siRNA-transfected, or SOCS1 siRNAtransfected macrophages were stimulated with IL-4 for 24 h and supernatants analyzed for nitrite production as a measure of iNOS activity (E). Cell lysates were analyzed for arginase I activity (urea production), as described in Materials and Methods. In all cases, values represent mean levels ⫾ sd from at least four separate BMDM preparations. *P ⬍ 0.05, compared with IL-4-stimulated, control siRNA-transfected macrophages, as determined by Student’s t test.

relative arginase I:iNOS expression ratio confirmed efficient M2 macrophage polarization (Fig. 2D). The arginase I:iNOS expression ratio was similar for mock-transfected macrophages (4.02⫾0.8). IL-4 treatment, however, induced a very different gene expression pattern in SOCS1 siRNA-transfected BMDM. SOCS1 knockdown inhibited the IL-4-induced increase in arginase I expression but strikingly, enhanced expression of iNOS (Fig. 2C), therefore decreasing the arginase I:iNOS expression ratio (Fig. 2D). There was, however, no detectable change in the IL-4-induced up-regulation of macrophage mannose receptor (Fig. 2C) or YM1 and RELM-␣ (data not included) with

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Whyte et al. SOCS1 directs macrophage activation and function

SOCS1 knockdown, showing specificity of SOCS1 in controlling expression of M2-activation genes. Thus, it appears that SOCS1 regulates specific aspects of M2 activation but not the overall IL-4R␣-dependent program. Importantly, on IL-4 activation, SOCS1 knockdown resulted in a reciprocal up-regulation of SOCS3 mRNA, previously associated with M1 macrophage activation [15]. To ensure that the changes in expression of iNOS and arginase I were reflected at a functional level, their activities were quantified in IL-4-activated BMDM following SOCS1 knockdown, which resulted in a significant reduction in IL-4-stimulated arginase I activity, reflecting gene expression results (Fig. 2E). Therefore, the enhanced SOCS1 expression observed in M2 macrophages could be critical in directing the arginase I-induced anti-inflammatory response of these cells [35]. The release of nitrite, as a measure of iNOS activity, was decreased below baseline levels with IL-4 treatment in control siRNAtransfected cells but not after SOCS1 knockdown. Indeed, in the absence of SOCS1, iNOS activity was equivalent to the baseline levels of nonstimulated cells, again consistent with the SOCS1 knockdown-induced increase in iNOS gene expression and verifying that SOCS1 controls the arginase I:iNOS activity balance.

SOCS1 regulates the capacity of M2 macrophages to suppress T cell proliferation M2 macrophages acquire an inhibitory activity against T cells, and one way they achieve this is through induction of high arginase I activity, which depletes the arginine required for T cell proliferation [35]. In line with this, we show that the decrease in T cell proliferation by IL-4 pretreated, antigen-pulsed macrophages can be restored by addition of l-arginine in our model system (Supplemental Fig. 1). SOCS1 knockdown caused a reduction in M2-induced arginase I activity, so we

determined whether this would also alter the impaired ability of M2 macrophages to support T cell expansion. T cells proliferated robustly when incubated with antigen-pulsed, nonstimulated, normal macrophages or SOCS1 knockdown macrophages, and as expected, this response was reduced when normal macrophages were pretreated with IL-4 (Fig. 3A). However, IL-4 pretreatment had no such effect on proliferation induced by SOCS1 knockdown macrophages, consistent with the idea that decreased arginase I activity after SOCS1 knockdown restores the ability of M2 macrophages to support T cell responses.

Effect of SOCS1 on IL-4-induced unresponsiveness of macrophages to IFN-␥/LPS Pretreatment of macrophages with IL-4 induces unresponsiveness to subsequent challenge with IFN-␥/LPS [7], and so, we tested whether this inhibition was likewise mediated by SOCS1. Preincubation with IL-4 attenuated the IFN-␥/LPS-induced NO generation by 27 ⫾ 6% in control siRNA-transfected macrophages (Fig. 3B), most likely a result of up-regulated arginase I activity and competition between iNOS and arginase I for the common substrate l-arginine. This effect was prevented by SOCS1 knockdown (4⫾4%). SOCS1 had a similar role in IFN-␥/LPS-induced IL-6 secretion (Fig. 3C) but not TNF-␣ secretion (data not included). In control siRNA-transfected BMDM, prior exposure to IL-4 decreased the IFN-␥/LPS-induced IL-6 by 38 ⫾ 8%, whereas in SOCS1 siRNA-transfected cells, IL-4 pretreatment did not decrease IL-6 secretion, and indeed, levels were increased significantly by 39 ⫾ 12% (P⬍0.05) over macrophages without IL-4 pretreatment. Thus, SOCS1 contributes to another aspect of M2 macrophage antiinflammatory activity, the hyporesponsiveness to IFN-␥/LPS following IL-4 treatment.

Figure 3. SOCS1 knockdown inhibits the functional properties of IL-4-treated macrophages. SOCS1 expression is required by IL-4-activated macrophages to inhibit antigen-specific T cell proliferation (A). SOCS1 knockdown or control siRNA-treated macrophages were pretreated with or without IL-4 and loaded with activating antigen (PPD). CD4⫹ cells were added to macrophage cultures and proliferation measured after 6 days by [3H]-thymidine incorporation. Results are expressed as mean percentage proliferation ⫾ sd as compared with nonactivated T cells. *P ⬍ 0.05 represents a significant decrease in proliferation as compared with nonstimulated, control siRNA-transfected macrophages, as determined by Student’s t test. The experiment was conducted twice with similar results. Knockdown of SOCS1 abrogates the IL-4-mediated suppression of IFN-␥/ LPS-induced nitrite (B) and IL-6 (C) production in BMDM, which were stimulated with IFN-␥/LPS without IL-4 pretreatment or incubated for 4 h with IL-4 followed by 18 h with IFN-␥/LPS. NO and IL-6 were assessed in culture supernatant, with or without SOCS1 knockdown. Values represent mean percentages ⫾ sd as compared with BMDM stimulated with IFN-␥/LPS alone. *P ⬍ 0.05 represents a statistically significant decrease in nitrite or IL-6 as compared with IFN-␥/LPS-activated BMDM and determined by Student’s t test. Results are representative of four independent experiments.

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SOCS1 preserves the IL-4-induced PI3K activity that drives M2 activation IL-4-induced PI3K activity is considered to be highly important for M2 macrophage polarization and especially, arginase I expression [36 –38]. To define a possible mechanism by which SOCS1 directs M2 macrophage functions, we therefore addressed the effects of SOCS1 knockdown on this signaling pathway. PI3K activity was increased 30 min after stimulation with IL-4, as shown by increased phosphorylation of the downstream signaling molecule AKT. Critically, knockdown of SOCS1 attenuated IL-4-induced p-AKT for up to 60 min poststimulation, without an apparent change in total AKT (Fig. 4). The attenuated PI3K activity correlated with an increase in SOCS3 protein expression, and therefore, the result is consistent with the previously reported ability of SOCS3 to inhibit PI3K [22]. In this set of experiments, the characteristic decrease in SOCS3 protein expression with IL-4 activation was observed slightly later than in Fig. 1B at 90 min poststimulation (data not included).

SOCS1 regulates the phenotype of M1 macrophages As our data suggested that exclusive up-regulation of SOCS1 with concomitant down-regulation of SOCS3 is important for the regulation of key aspects of M2 macrophage polarization,

we next examined their influence on M1 polarization. First, we confirmed our previous observation that incubation of BMDM with IFN-␥ and LPS greatly increased SOCS3 expression without a change in SOCS1, whereas incubation with LPS alone increased mRNA for SOCS1 and SOCS3 (Fig. 5A) [15]. IFN-␥ alone enhanced SOCS1 expression and only marginally upregulated SOCS3. Stimulation of control BMDM with IFN-␥ and LPS, individually or in combination, caused a substantial increase in iNOS expression, which was greatly attenuated when SOCS1 was knocked down (Fig. 5A). Enhanced arginase I expression is linked to M2 macrophages but is also induced in M1 macrophages through a MyD88-dependent pathway [39]. In contrast to iNOS, arginase I mRNA was robustly upregulated in SOCS1 knockdown macrophages activated with IFN-␥, LPS, or IFN-␥/LPS. Accordingly, the relative arginase I:iNOS mRNA ratio, commonly taken as an indicator of macrophage-inflammatory status [5–7], was significantly increased (Fig. 5B). No change in expression of the M2 marker, mannose receptor (CD206), with SOCS1 knockdown confirmed specificity of effects. To corroborate whether changes in gene expression were reproduced at a functional level, iNOS and arginase I activities were quantified in M1-activated BMDM following SOCS1 knockdown, which markedly limited the IFN-␥/LPS-induced increase in iNOS activity (Fig. 5C), consistent with the reduction of iNOS gene expression. No significant decrease in iNOS activity was observed in LPS-activated SOCS1 knockdown macrophages, suggesting that additional post-translational regulatory mechanisms are important in this case. By contrast, in LPS- and IFN-␥/LPS-activated macrophages, SOCS1 knockdown significantly enhanced arginase I activity (Fig. 5D) and as with gene expression data, substantially changed the relative iNOS:arginase I activity balance in these cells. Thus, SOCS1 regulates the iNOS:arginase I balance in M1 and M2 macrophages but in opposite directions.

SOCS1 limits the proinflammatory characteristics of M1 macrophages but also anti-inflammatory IL-10 production

Figure 4. SOCS1 knockdown inhibits PI3K activity by enhancing SOCS3 expression. Immunoblot analysis of p-AKT in a lysate of BMDM transfected with control siRNA or SOCS1 siRNA, treated with IL-4 for 0, 30, and 60 min. SOCS1 knockdown decreased SOCS1 protein expression, increased SOCS3 expression, and limited p-AKT with no apparent effect on total AKT. Figure shows expression of proteins as determined by an immunoblot, representative of three individual experiments (A) and normalized densitometric analysis of scanned bands in arbitary units (a.u.) from three separate immunoblots (B).



Efficiently polarized M1 macrophages demonstrate an increase in the surface expression of MHC class II and costimulatory molecule CD86 and up-regulate secretion of proinflammatory IL-6, IL-12, and TNF-␣ and their feedback regulator, IL-10. Next, we defined whether SOCS1 played a role in regulating these functional effects. Flow cytometry with statistical analyses of MFI values showed that the M1-induced increase in MHC class II surface expression was enhanced significantly with SOCS1 knockdown with all stimuli, and an increase in CD86 was evident in LPS-activated macrophages (Fig. 6A and B). SOCS1 knockdown resulted in a significant up-regulation of LPS-induced IL-6 and IFN-␥/LPS-induced IL-12p40 in line with SOCS1 overexpression and knockout studies [25, 40 – 43], whereas there was no significant change in TNF-␣ secretion (Fig. 6C–F). Production of IL-12p70 was substantially less than that of IL-12p40, and despite an increase in IFN-␥/LPS-induced IL-12p70 with SOCS1 knockdown, this did not reach statistical significance (control siRNA 31.4⫾2.5 pg/ml; SOCS1 siRNA 38.9⫾6.6 pg/ml; P⫽0.134).

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Whyte et al. SOCS1 directs macrophage activation and function Figure 5. Effect of SOCS1 knockdown on gene expression in M1activated macrophages. Control siRNA-transfected or SOCS1 siRNAtransfected BMDM were treated with medium (no stimulation; control) or activating stimuli for 2 h, cells harvested, and the levels of SOCS1, iNOS, arginase I, mannose receptor, and SOCS3 mRNA analyzed by RT-RCR (A). SOCS1 knockdown decreases the ratio of arginase I:iNOS mRNA expression, as determined by PCR and densitometric analyses of scanned bands from ethidium bromide-stained agarose gels (B). Control siRNA-transfected or SOCS1 siRNA-transfected macrophages were stimulated with IFN-␥, LPS, or a combination of IFN-␥/LPS for 18 h and supernatants analyzed for nitrite (C) or arginase I activity, as determined by urea production (D). *P ⬍ 0.05 as compared with control siRNA-transfected macrophages and determined by Student’s t test. In all cases, the data are representative of four independent experiments.

Remarkably, the LPS- and IFN-␥/LPS-induced up-regulation of the anti-inflammatory cytokine IL-10 was significantly greater in SOCS1-depleted macrophages (Fig. 6F), supporting a novel and crucial role for SOCS1 in limiting IL-10-induced anti-inflammatory-feedback responses and in driving efficient polarization of M1 cells. Therefore, taken together, results show that on M1 activation, SOCS1 regulates both sides of the inflammatory balance (IL-6, IL-12, iNOS, as well as arginase I and IL-10).

DISCUSSION Here, we define a significant role for SOCS1 in controlling M1 and M2 macrophage activation and identify key differences in the way SOCS proteins control effective polarization of macrophage subsets. We show that IL-4-activated M2 macrophages exclusively expressed SOCS1 in vivo and in vitro and provide the first evidence that this up-regulated SOCS1 has important and nonredundant roles in acquisition of the M2 characteristics. By contrast, for M1 macrophages, SOCS1 attenuates some but not all proinflammatory properties of M1 macrophages, but crucially, it also attenuates their anti-inflammatory IL-10 response. Thus, we demonstrate that SOCS1 is not simply part of a negative-feedback loop that protects against inflammatory consequences of M1 activation but that it may also have a role in sustaining their inflammatory properties, albeit at a lower, less-destructive level. A key finding of our study was the highly polarized up-regulation of SOCS1 but not SOCS3 expression in macrophages infiltrating an M2-activating environment in vivo, an observation consistent with in vitro IL-4-activated macrophages [15, 33]. This in vivo polarization was at least partly dependent on IL-4, as under the same activating conditions, macrophages from IL-4R␣⫺/⫺ mice did not support M2 activation and show

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increased expression of SOCS3 and a substantially reduced SOCS1/SOCS3 ratio. A similar increase in SOCS1 but not SOCS3 expression during parasitic infections is observed in human monocytes derived from B. malayi-infected patients after antigen-specific restimulation [44]. This suggests that exclusive up-regulation of SOCS1 or indeed, a high SOCS1/ SOCS3 expression ratio has potential as a useful in vivo biomarker for M2 macrophages. Strikingly, this contrasts with macrophages infiltrating an in vivo-inflamed, M1-activating environment, where macrophages with enhanced SOCS3 but not SOCS1 are prominent [15]. Indeed, in our B. malayi-implant model, high SOCS1 protein levels were maintained in a later, more chronic stage of infection (Day 27) but were accompanied by increased SOCS3 expression, which was associated with the presence of M1-activating factors (unpublished results). This transition or “hybrid” phenotype [45] may be similar to the population, which we have shown to be augmented when M1 macrophages enter the resolution phase of inflammation [15]. The IL-4-induced expression of SOCS1 in macrophages was functionally important in preserving their M2-programmed anti-inflammatory phenotype. Knockdown of SOCS1 not only decreased M2-induced arginase I expression and activity but also reciprocally enhanced that of iNOS and up-regulated the SOCS3 expression associated with M1 activation. This was surprising given that SOCS1 overexpression has previously been reported to inhibit IL-4-induced STAT6 in Hela and Th cells [30, 33, 46], and IL-4-induced arginase I gene expression is shown to be somewhat enhanced in macrophages from SOCS1- and IFN-␥-deficient mice via a STAT6-dependent mechanism [33]. However, IL-4 also induces arginase I expression through non-STAT6 pathways, and indeed, PI3K activation is essential for driving macrophage arginase I activity [37, 38]. Our demonstration that SOCS1 knockdown attenuated Volume 90, November 2011


Figure 6. Knockdown of SOCS1 decreases cell-surface expression of MHC class II and CD86 and deviates the production of pro- and anti-inflammatory mediators by M1 macrophages. Representative results from flow cytometric analysis of BMDM after stimulation with IFN-␥, LPS, or IFN-␥/LPS for 24 h, as described in Materials and Methods (A). Black lines represent control siRNA-transfected macrophages, gray lines represent SOCS1 siRNA-transfected macrophages, and filled gray area represents labeled, isotype-matched control antibody. Mean MFI ⫾ sd of triplicate experiments for MHC class II and CD86 surface expression on control siRNA- and SOCS1 siRNA-transfected BMDM (B). Control siRNA-transfected or SOCS1 siRNA-transfected macrophages were stimulated with IFN-␥, LPS, or a combination of IFN-␥/ LPS for 18 h and supernatants analyzed for IL-6 (C), IL-12p40 (D), TNF-␣ (E), and IL-10 (F), as described in Materials and Methods. Values represent mean values ⫾ sd from five different BMDM preparations. *P ⬍ 0.05 as compared with control siRNA-transfected macrophages, as determined by Student’s t test.

IL-4-induced PI3K activity provides an important and novel mechanism for the abrogation in arginase I activity and associated effects on T cell proliferation and IL-4-induced IFN-␥/ LPS unresponsiveness observed in these cells. Notably, the lack of a detectable change in expression of macrophage mannose receptor (CD206), YM1, or RELM-␣ in the same cells indicates that SOCS1 regulates a specific module of macrophage signaling, rather than the whole M2 activation pathway. SOCS3 impairs IL-4-induced gene expression in macrophages by inhibitory effects on PI3K activity [22], and here, we propose that the increase in SOCS3 expression observed with SOCS1 knockdown is restraining PI3K-induced arginase I expression. SHIP, 852 Journal of Leukocyte Biology


induced by M1-stimulating agents such as LPS, also effectively prevents M2 activation by inhibiting PI3K [38], although it remains to be determined whether effects are mediated through similar or different mechanisms to that observed with SOCS1 knockdown. In stark contrast to M2 macrophages, our data suggest that for M1 macrophages, SOCS1 acts to fine-tune expression of proinflammatory mediators, while actively repressing anti-inflammatory responses. This distinguished it from SOCS3, which uniquely controls M1 macrophage proinflammatory properties [15]. This key and divergent role of SOCS1 in regulating M1 and M2 activation is analogous to its role in polariz-

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Whyte et al. SOCS1 directs macrophage activation and function

ing Th1 and Th2 cells by IL-12 and IL-4, respectively [46 – 48]. Therefore, as with T cells, the role of SOCS1 in directing macrophage polarization is dependent on the activating factors present in the extracellular environment and the signaling pathways they initiate. The role of SOCS1 in controlling macrophage proinflammatory cytokines and TLR signaling pathways has been addressed previously by several overexpression and knockout studies. It is proposed that SOCS1 inhibits LPS-induced production of IL12, TNF-␣, and NO via down-regulating NF-␬B activity [25, 40 – 43], but this remains controversial [25, 43]. Gingras et al. [42], for example, found no evidence that SOCS1 blocks LPSinduced NF-кB signaling or TNF-␣ and NO production in macrophages from SOCS1-deficient mice and indeed, as with our SOCS1 knockdown studies, found a decrease in IFN-␥/ LPS-induced NO. Critically, in the current study, we examined the effects of SOCS1 on the arginase I:iNOS activity ratio and subsequent M1 polarization. We propose that with SOCS1 knockdown, the significant increase in arginase I activity will compete with iNOS for the common substrate l-arginine [39, 48], accounting for the observed decrease in NO production in these cells. A novel and surprising finding of our studies was the striking up-regulation of the potent anti-inflammatory cytokine IL-10 by M1-activated SOCS1 knockdown cells. IL-10 is up-regulated by TLR agonists to prevent overshooting of TLR-mediated proinflammatory responses. SOCS1 thus limits this feedback mechanism to sufficiently maintain the natural cytotoxicity of M1-polarized macrophages. Interestingly, macrophages transduced with an inhibitor of NF-␬B demonstrated a similar phenotype with reduced iNOS and high secretion of IL-10, which significantly decreased the severity of experimental nephritis [49]. Our findings with SOCS1 knockdown in macrophages have parallels with those in DCs. Splenic DCs from SOCS1-deficient mice showed higher expression of MHC class II and CD86, whereas SOCS1 knockdown and SOCS1-deficient DCs were more responsive than controls to LPS stimulation, as indicated by enhanced IL-6 production [24, 50]. However, regulation of IL-10 production by SOCS1 was not investigated in these previous studies. In summary, we show that specific discriminating properties of M2 macrophages are dependent on the IL-4-induced upregulation of SOCS1 expression and propose that selective SOCS1 protein expression could be a useful biomarker for assessing macrophage activity in vivo. Moreover, we confirm that basal SOCS1 expression directs properties of M1 macrophages, where it acts to limit excessive signaling and coordinate proinflammatory responses. We therefore conclude that SOCS1 is a critical molecular switch that tunes key signaling pathways to effectively program M1 and M2 macrophage polarization.

AUTHORSHIP C.S.W. provided biochemistry, molecular biology, and data interpretation. E.T.B. performed immunohistochemistry. D.R. provided in vivo experiments, data interpretation, and manu-

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script preparation. S.G-P. performed biochemistry. R.N.B. helped with the manuscript preparation. J.E.A. supplied the study design, data interpretation, and manuscript preparation. A.J.R. provided study design and manuscript preparation. H.M.W. designed experiments, provided supervision, analyzed data and wrote the manuscript.

ACKNOWLEDGMENTS This work was supported by the Medical Research Council (grant nos. 74804 and 78896), NHS Grampian Endowments Research Trust (grant no. 09/02), and Cunningham Trust (grant no. ACC/KWF/CT08/03). REFERENCES

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KEY WORDS: inflammation 䡠 M1 activation 䡠 M2 activation 䡠 inducible nitric oxide synthase 䡠 arginase I 䡠 Brugia malayi

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