Interleukin-25 fails to activate STAT6 and induce alternatively activated macrophages

Authors


G. Monteleone, Dipartimento di Medicina Interna, Università Tor Vergata, Via Montpellier, 1 – 00133, Rome, Italy.
Email: Gi.Monteleone@Med.uniroma2.it
Senior author: Giovanni Monteleone

Summary

Interleukin-25 (IL-25), a T helper type 2 (Th2) -related factor, inhibits the production of inflammatory cytokines by monocytes/macrophages. Since Th2 cytokines antagonize classically activated monocytes/macrophages by inducing alternatively activated macrophages (AAMs), we here assessed the effect of IL-25 on the alternative activation of human monocytes/macrophages. The interleukins IL-25, IL-4 and IL-13 were effective in reducing the expression of inflammatory chemokines in monocytes. This effect was paralleled by induction of AAMs in cultures added with IL-4 or IL-13 but not with IL-25, regardless of whether cells were stimulated with lipopolysaccharide or interferon-γ. Moreover, pre-incubation of cells with IL-25 did not alter the ability of both IL-4 and IL-13 to induce AAMs. Both IL-4 and IL-13 activated signal transducer and activator of transcription 6 (STAT6), and silencing of this transcription factor markedly reduced the IL-4/IL-13-driven induction of AAMs. In contrast, IL-25 failed to trigger STAT6 activation. Among Th2 cytokines, only IL-25 and IL-10 were able to activate p38 mitogen-activated protein kinase. These results collectively indicate that IL-25 fails to induce AAMs and that Th2-type cytokines suppress inflammatory responses in human monocytes by activating different intracellular signalling pathways.

Introduction

Monocytes/macrophages play an important role in both innate and acquired immune responses through their ability to recognize and respond to pathogens, and to produce a wide array of molecules, which participate in both the initiation and resolution of inflammatory processes.1 This reflects in part the plasticity and versatility of such cells, which are able to differentiate along different pathways in response to specific signals. Interferon-γ (IFN-γ), the signature cytokine of T helper (Th) type 1 cell responses, and microbial pathogen-associated molecular patterns induce classically activated monocytes/macrophages, which make the high amounts of inflammatory cytokines and reactive oxygen species necessary to eradicate invading organisms and tumour cells.2–4 However, persistent and uncontrolled activation of such cells can result in immunopathology.5–9 Monocytes/macrophages can also undergo a different activation programme, referred to as ‘alternative activation’.10 This phenotype is induced by interleukin-4 (IL-4) and IL-13, two cytokines produced in large quantities in Th2-associated immune responses.10–12 Alternatively activated monocytes/macrophages (AAMs) produce anti-inflammatory molecules and various components of extracellular matrix, and contribute to the host response to parasites, fibrosis in granulomatous diseases, angiogenesis, tissue repair and healing.13–15 Both IL-4 and IL-13 induce expression of typical markers of alternative activation in monocytes/macrophages (e.g. the mannose receptor CD206) and selective chemokines (e.g. CCL-17, CCL-18, CCL-22).16,17 These changes are distinct from those induced by IL-10, which enhances the expression of the haemoglobin scavenger receptor CD16318 and causes a ‘deactivation’ of macrophages.19 Notably, both alternatively activated and IL-10-treated macrophages can antagonize classically activated macrophages, hence contributing to the resolution of type I cytokine-associated inflammatory processes and tissue wound healing.10,20 Collectively these observations indicate that macrophage activation can be either pro-inflammatory or anti-inflammatory, depending upon the functional phenotypes that these cells acquire in response to distinct subsets of cytokines and other tissue-derived signals.

Interleukin-25 (also known as IL-17E) is a recently described cytokine produced by several cell types, including T lymphocytes, mast cells activated by IgE cross-linking, macrophages, activated eosinophils and basophils, and epithelial cells.21,22 Interleukin-25 has been shown to facilitate pathogenic Th2 cell responses.23 Indeed, studies in mice have shown that both transgenic expression of IL-25 and systemic administration of recombinant IL-25 increase the production of IL-4, IL-5 and IL-13, cause epithelial cell hyperplasia, and facilitate the recruitment of inflammatory cells (i.e. eosinophils, monocytes and T cells) into inflamed tissues.24,25 Interleukin-25 also sustains Th2 cytokine production and airway inflammation in an allergen-induced asthma model.26 Consistently, IL-25-knockout mice fail to expel the parasitic helminths Nippostrongylus brasiliensis and Trichuris muris from the gut, a defect that correlates with the inability of such mice to up-regulate Th2 cytokines.27 Administration of exogenous IL-25 to genetically susceptible mice confers resistance to helminths and accelerates expulsion of worms.27

In addition to its Th2-inducing actions, IL-25 regulates other immune responses. For instance, we have recently shown that IL-25 inhibits the production of inflammatory cytokines by both human blood CD14+ monocytes and intestinal macrophages, and suppresses inflammatory processes induced by bacterial-activated Th1 cells.28,29 This inhibitory effect of IL-25 is associated with no change in IL-10 production, suggesting that IL-25 can regulate specific programmes of differentiation/activation of monocytes rather than acting as a general inhibitor of their functions.

In the present study, we assessed whether the anti-inflammatory action of IL-25 on monocytes/macrophages is associated with induction of AAMs.

Materials and methods

Cell isolation and culture

Human peripheral blood mononuclear cells were isolated from enriched buffy coats of healthy volunteer donors by Ficoll gradients. CD14+ cells were purified from peripheral blood mononuclear cells either by positive selection, using CD14 magnetic beads (Miltenyi Biotec, Bologna, Italy), or by negative selection using CD3, CD19 and CD56 magnetic beads (Miltenyi Biotec). Cell purity was routinely evaluated by flow cytometry and ranged between 93% and 99%. To evaluate the effect of Th2 cytokines on the synthesis of inflammatory chemokines, CD14+ cells were resuspended in RPMI-1640 medium, supplemented with 10% inactivated fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 μg/ml) (all from Lonza, Verviers, Belgium), seeded in 48-well culture dishes (1 × 106 cells/well) and incubated with human recombinant IL-25 (50 ng/ml; R&D Systems, Inc. Minneapolis, MN), human recombinant IL-13 (50 ng/ml; R&D Systems), human recombinant IL-4 (50 ng/ml; Peprotech EC LTD, London, UK) for 30 min and then stimulated with lipopolysaccharide (LPS; 100 ng/ml; Sigma-Aldrich, Milan, Italy) or IFN-γ (50 ng/ml; Peprotech) for 3 hr. CD14+ cells were incubated with human recombinant IL-10 (25 ng/ml; R&D Systems) for 30 min and then stimulated with LPS or IFN-γ for a further 3 hr. Cells were then used to extract RNA.

To examine whether IL-25 induced human AAMs, CD14+ cells were seeded in 48-well culture dishes and incubated with the above-indicated cytokines for 30 min and then stimulated or not with LPS (100 ng/ml) or IFN-γ (50 ng/ml) for 3–18 hr. Additionally, cell cultures were pre-incubated with IL-25 (50 ng/ml) for 0·5–6 hr and then stimulated or not with IL-4 (10 ng/ml) or IL-13 (10 ng/ml) for further 3–24 hr. To assess whether IL-10 promotes Th2 cytokine-driven AAM differentiation, CD14+ cells were pre-incubated with graded doses of IL-10 (12·5–50 ng/ml) for 30 min and then stimulated or not with IL-25, IL-4 or IL-13 (50 ng/ml) for further 3–24 hr. To determine whether IL-25 regulates the IL-10-mediated ‘deactivation’ of monocytes, CD14+ cells were pre-incubated with IL-25 for 0·5–6 hr and then stimulated with IL-10 (5 ng/ml). Cells were also pre-incubated with IL-25 or IL-10 for 30 min and then stimulated with LPS or tumour necrosis factor-α (TNF-α; 10 ng/ml; R&D Systems) for 12 hr. At the end, cells were used to extract RNA or analysed by flow-cytometry. We also evaluated the effect of IL-25, IL-4 and IL-13 on the induction of AAMs using human macrophages, which were obtained from circulating CD14+ cells as described elsewhere.30 Briefly, CD14+ cells were resuspended in Dulbecco’s modified Eagle’s medium (Lonza) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and seeded in 48-well culture dishes. Macrophage colony-stimulating factor and granulocyte–macrophage colony-stimulating factor (10 ng/ml and 1 ng/ml, respectively; R&D Systems) were daily added to the medium. On day 6, cells were washed with PBS and re-cultured with fresh medium. On day 8, fully differentiated monocyte-derived macrophages were stimulated with IL-25, IL-4 or IL-13 for 3 hr and then used to extract RNA. At the end of each experiment, cell recovery was assessed by evaluating the number of cells into each well.

RNA extraction, cDNA preparation and real-time PCR

The RNA was extracted using TRIzol reagent (Invitrogen, Milan, Italy), retro-transcribed into cDNA, and amplified using the following conditions: denaturation for 1 min at 95°, annealing for 30 seconds at 58° for IL-8 and CXCL9, at 60° for CCL18, MRC1 and β-actin, at 61° for CCL2 and CCL17, at 62° for CCL3 and GRP105, followed by 30 seconds of extension at 72°. Primers sequence was as follows: IL-8, forward: 5′-AGGAACCATCTCACTGTGTG-3′ and reverse: 5′-CCACTCTCAATCACTCTCAG-3′; CXCL9, forward: 5′- GGTGTTCTTTTCCTCTTGGG-3′ and reverse: 5′- GGATTGTAGGTGGATAGTCC-3′; CCL18, forward: 5′-CACTCTGACCACTTCTCTGC-3′ and reverse: 5′-TGCCAGGAGGTATAGACGAG-3′; MRC1, forward: 5′-TTTATGGAGCAGGTGGAAGATC-3′ and reverse: 5′-GTGTCATAGTCAGTAGTGGTTC-3′; CCL2, forward: 5′- TTCTGTGCCTGCTGCTCATAG-3′ and reverse: 5′-GACACTTGCTGCTGGTGATTC-3′; CCL17, forward: 5′-AGGGACCTGC-ACACAGAGAC-3′ and reverse: 5′-GCTTTCTAAGGGGAATGGCTC-3′; CCL3, forward: 5′-CACATTCCGTCACCTGCTCAG-3′ and reverse: 5′-CACTGGCTGCTCGTCTCAAAG-3′; GRP105, forward: 5′-CAGTGTTACCTTGGAGCCTAC-3′ and reverse: 5′-CAGTGAGCGTTTGTCGTCTTG-3′. β-actin (forward: 5′-AAGATGACCCAGATCATGTTTGAGACC-3′ and reverse: 5′-AGCCAGTCCAGACGCAGGAT-3′) was used as an internal control. Human Arginase1 (ARG1) was evaluated using a commercially available TaqMan probe (Applied Biosystems, Foster City, CA). Cytokine and chemokine RNA expression was calculated relative to the housekeeping β-actin gene on the base of the ΔΔCt algorithm.

Flow cytometry

The following monoclonal anti-human antibodies were used: CD86-FITC (cat. 555657; Becton Dickinson, Milan, Italy), CD163-phycoerythrin (cat. 12-1639-73; eBioscience, San Diego, CA), and CD23-peridinin chlorophyll protein (cat. 25-0238-73; eBioscience). Phosphorylated signal transducer and activator of transcription (pSTAT6) and p-p38 were evaluated using monoclonal anti-human antibodies recognizing the phosphorylated-Y641 residue of STAT6 (cat. 612600; Becton Dickinson) and the phosphorylated-T180/Y182 residues of p38 (cat. 612565; Becton Dickinson), respectively, according to the manufacturer’s instruction.

STAT6 knockdown by small interfering RNA

CD14+ cells were transfected with STAT6 or control small interfering (si) RNA (all from Santa Cruz Biotechnology, Santa Cruz, CA) using lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours after the transfection, an aliquot of cells was used to assess their viability by staining with Annexin V/propidium iodide. The remaining cells were either left untreated or stimulated with IL-4 or IL-13. The efficiency of the siRNA transfection was assessed using fluorescein-conjugated control siRNA (Santa Cruz Biotechnology).

Data analysis

Values are expressed as means ± SD. Differences between groups were compared using the Student’s t-test. Significance was defined as P-values < 0·05.

Results

Th2-cytokines inhibit the expression of inflammatory chemokines in human blood monocytes

Blood CD14+ cells were isolated by positive selection using CD14 magnetic beads unless specified. Following stimulation with inflammatory cytokines and bacterial components/products, monocytes produce a wide panel of chemokines (e.g. CCL2, CCL3, CXCL9, IL-8), which participate in the development of Th1 immunity and inflammation.31 Induction of such chemokines is inhibited by Th2 cytokines and IL-1032,33 so we evaluated whether IL-25 was effective in inhibiting the RNA expression of inflammatory chemokines induced by IFN-γ or LPS. In initial time–course studies, we selected the doses of LPS/IFN-γ and time of stimulation to obtain maximal monocyte response. Similarly, we selected the optimal doses of each cytokine to get the maximal inhibition of the LPS-driven chemokine expression (not shown). CD14+ cells were pre-incubated with IL-25, IL-4, IL-13 or IL-10 for 30 min and then stimulated with LPS/IFN-γ for a further 3 hr. Real-time PCR analysis revealed that induction of CCL2, CCL3, IL-8 and CXCL9 by IFN-γ (Fig. 1a, c, e, g) or LPS (Fig. 1b, d, f, h) was significantly inhibited by IL-25, IL-4, IL-13 and IL-10. These data indicate that IL-25, like other Th2-related cytokines, is effective in reducing the expression of inflammatory chemokines in human monocytes. To confirm these observations, we next tested the effect of Th2 cytokines and IL-10 on inflammatory chemokines induced in CD14+ cells purified by negative selection. Data shown in Fig. S1 show that IL-25, IL-4, IL-13 and IL-10 significantly inhibited CCL3 and CXCL9 induced by IFN-γ and LPS.

Figure 1.

 T helper type 2 (Th2) -related cytokines inhibit the expression of inflammatory chemokines induced by interferon-γ (IFN-γ) and lipopolysaccharide (LPS) in human blood CD14+ cells. CD14+ cells were isolated from three healthy volunteers, pre-incubated with interleukin-25 (IL-25; 50 ng/ml), IL-4 (50 ng/ml), IL-13 (50 ng/ml) or IL-10 (25 ng/ml), for 30 min, then either left unstimulated or stimulated with IFN-γ (50 ng/ml; a, c, e, g) or LPS (100 ng/ml; b, d, f, h) for a further 3 hr. RNA was extracted and amplified by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of all experiments. IFN-γ/LPS-treated cells versus IFN-γ/LPS + IL-25 or Th2-cytokine-treated cells: *P < 0·001; **P < 0·01.

IL-25 fails to induce the differentiation of AAMs

We next performed a flow-cytometry analysis of cell-surface molecules and assessed whether the above inhibitory effect of IL-25 was associated with induction of AAMs. As compared with unstimulated monocytes, the expression of CD23 was up-regulated in cells stimulated with IL-4 or IL-13, but not with IL-25 (Fig. 2). Interleukin-4 and IL-13 enhanced the percentage of cells co-expressing CD23 and CD86, even when cells were treated with LPS or TNF-α (Fig. 2). No change in cell recovery was seen in cultures stimulated with IL-25, IL-4 or IL-13 (not shown), arguing against the possibility that the observed differences in CD23 and CD86 expression following Th2 cytokine stimulation are the result of differential cell loss.

Figure 2.

 Interleukin-4 (IL-4) and IL-13, but not IL-25, enhance the fraction of CD23-expressing cells. Representative dot-plots showing the expression of CD23 and CD86 in CD14+ cells pre-incubated with medium, IL-25, IL-4 or IL-13 (50 ng/ml) for 30 min and then stimulated or not with lipopolysaccharide (LPS; 100 ng/ml) or tumour necrosis factor-α (TNF-α; 10 ng/ml) for a further 12 hr. CD23 and CD86 expression was assessed by flow-cytometry. Numbers indicate the percentage of positive cells within the designated quadrants. One of three representative experiments in which similar results were obtained is shown.

These findings suggest that IL-25, unlike other Th2-related molecules, is unable to induce the alternative activation of monocytes. To confirm this hypothesis, we evaluated the RNA expression of additional markers of AAMs. Stimulation of blood monocytes with graded doses of IL-25 for different time-points did not increase the RNA content for CCL17, CCL18 and mannose receptor (MRC1) (Fig. S2a–c). In the same experiments IL-4, used as a positive control for the induction of AAMs, increased the RNA expression of CCL17, CCL18 and MRC1; this increase was evident as early as 3 hr after cell stimulation (Fig. S2a–c). Consistently, IL-4 and IL-13, but not IL-25, induced CCL17, CCL18 and MRC1 RNA expression in cells stimulated with IFN-γ (Fig. S3a, c, e) or LPS (Fig. S3b, d, f). No change in the RNA content of ARG1 and GRP105, other AAM-related markers, was seen in cytokine-stimulated cells (data not shown), confirming previous studies that showed that IL-4 and IL-13 do not enhance ARG1 or GPR105 expression in human monocytes.17

Consistent with the above data, IL-4 and IL-13, but not IL-25, induced CCL17, CCL18 and MRC1 in monocyte-derived macrophages (Fig. S4).

As Th2 cytokines are known to reciprocally potentiate their regulatory effect on various cell types,24,34,35 we first determined whether IL-25 regulates the expression of IL-4/IL-13-induced AAM markers. Pre-incubation of CD14+ cells with IL-25 for 6 hr did not modify the percentages of CD23+ cells induced by IL-4/IL-13 (Fig. 3a). Similar findings were seen when cells were stimulated simultaneously with IL-25 and IL-4 or IL-13 (not shown). Moreover, IL-25 did not affect the IL-4/IL-13-induced RNA expression of CCL17, CCL18 and MRC1 (Fig. 3b–d). As IL-10 sensitizes macrophages to the IL-4/IL-13-induced alternative activation,36 we next examined whether pre-incubation of CD14+ cells with increasing doses of IL-10 (12·5–50 ng/ml) promoted IL-25-driven AAM differentiation. Pre-incubation of CD14+ cells with IL-10 enhanced the percentage of CD23+ cells and RNA expression for CCL18 following treatment with IL-4/IL-13 but not with IL-25 (Fig. 4a, b). No increase in the RNA content of CCL17 and MRC1 was observed in cytokine-stimulated CD14+ cells pre-incubated with IL-10 (Fig. 4b).

Figure 3.

 Interleukin-25 (IL-25) does not affect the IL-4- or IL-13-induced expression of alternatively activated macrophage-associated markers. (a) Representative histograms showing the percentage of CD14+ cells positive for CD23. Cells were pre-incubated or not with IL-25 for 6 hr and then stimulated with IL-4 or IL-13 for a further 24 hr. Data indicate the mean ± SD of three experiments (b–d). Representative histograms showing CCL17, CCL18 and MRC1 mRNA expression in CD14+ cells, pre-incubated or not with IL-25 for 30 min and then stimulated with IL-4 or IL-13 for a further 3 hr. RNA was extracted and amplified by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of three experiments. nd = not detectable.

Figure 4.

 Interleukin-25 (IL-25) fails to induce alternatively activated macrophage-associated markers in human monocytes pre-incubated with IL-10. (a) Representative dot-plots showing the expression of CD23 in CD14+ cells pre-incubated with medium or IL-10 (25 ng/ml) for 30 min and then either left unstimulated (Uns) or stimulated with IL-25, IL-4 or IL-13 (50 ng/ml). After 12 hr, CD23 expression was assessed by flow-cytometry. Numbers indicate the percentage of positive cells within the designated areas. One of three representative experiments in which similar results were obtained is shown. Right inset. Representative histograms showing the percentage of CD14+ cells expressing CD23 and cultured as indicated above. Data indicate the mean ± SD of three experiments (b) Representative histograms showing CCL17, CCL18 and MRC1 mRNA expression in CD14+ cells, pre-incubated or not with IL-10 for 30 min and then stimulated with IL-25, IL-4 or IL-13 for a further 3 hr. RNA was extracted and amplified by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of three experiments. nd = not detectable.

IL-25 does not regulate the expression of CD163 and CD86 on human monocytes

In subsequent experiments we assessed whether IL-25-treated monocytes express surface markers that are similar to those seen in cells stimulated with IL-10. Interleukin-10, but not IL-25, reduced the fraction of CD86+ cells. This effect was seen in cells cultured with medium alone and in cells stimulated with LPS or TNF-α (Fig. 5a). Moreover, IL-10 but not IL-25 increased the fraction of CD163-expressing cells, regardless of whether cells were treated or not with LPS or TNF-α (Fig. 5a). Finally, we showed that pre-incubation of monocytes with IL-25 did not modify the percentage of IL-10-induced CD163+ cells (Fig. 5b).

Figure 5.

 Interleukin-25 (IL-25) does not alter the expression of CD163 and CD86 in human CD14+ cells. (a) Representative dot-plots showing the expression of CD163 and CD86 in CD14+ cells pre-incubated with medium, IL-25 (50 ng/mL) or IL-10 (25 ng/ml) for 30 min and then either left unstimulated (Uns) or stimulated with lipopolysaccharide (LPS; 100 ng/ml) or tumour necrosis factor-α (TNF-α; 10 ng/ml). After 12 hr, CD23 and CD163 expression was assessed by flow cytometry. Numbers indicate the percentage of positive cells within the designated quadrants. One of three representative experiments in which similar results were obtained is shown. (b) Induction of CD163 by IL-10 is not influenced by IL-25. Representative histograms showing the percentage of CD14+ cells positive for CD163. Cells were pre-incubated with IL-25 (50 ng/ml) for 6 hr and then stimulated with IL-10 (25 ng/ml) for a further 24 hr. Data indicate the mean ± SD of three experiments.

IL-25 does not activate STAT6, a key regulator of AAM induction

Studies in mice have convincingly shown that STAT6 plays a major role in the induction of AAMs.37,38 We next asked whether STAT6 was required for the alternative activation of human monocytes following stimulation with Th2 cytokines. Flow cytometry analysis showed that IL-4 and IL-13, but not IL-25 and IL-10, enhanced STAT6 activation in CD14+ cells cultured with these cytokines for different periods (i.e. 15–240 min) (Fig. 6a, and not shown). Similar results were seen when experiments were performed using CD14+ cells purified by negative selection (Fig. S5).

Figure 6.

 Interleukin-4 (IL-4) and IL-13 promote the induction of alternatively activated macrophages through a signal transducer and activator of transcription 6 (STAT6) -dependent mechanism. (a) IL-4, IL-13, but not IL-25 and IL-10, activate STAT6. Representative dot-plots showing the expression of p-STAT6 (Y641) in CD14+ cells either left unstimulated (Uns) or stimulated with IL-25, IL-4, IL-13 (50 ng/ml) or IL-10 (25 ng/ml) for 1 hr. Numbers in the selected areas indicate the percentage of p-STAT6 (Y641)-positive cells. Right inset. Representative histograms showing the percentage of CD14+ cells expressing p-STAT6 (Y641). Data indicate the mean ± SD of three experiments. (b) Representative dot-plots showing the percentages of propidium iodide (PI) -positive and fluorescent (FITC) labelled small interfering (si) RNA-transfected CD14+ cells. One of four representative experiments is shown. (c) Representative dot-plots showing the fraction of p-STAT6 (Y641)-positive CD14+ cells following transfection with control or STAT6 siRNA and then either left unstimulated (Uns) or stimulated with IL-4 or IL-13 (50 ng/ml) for 1 hr. Numbers in the selected areas indicate the percentage of p-STAT6 (Y641)-positive cells. One of four separate experiments in which similar results were obtained is shown. (d) Treatment of CD14+ cells with STAT6 siRNA does not affect cell viability. Representative dot-plots showing the percentages of Annexin V and/or PI-positive CD14+ cells transfected with control (CTR) or STAT6 siRNA. One of four separate experiments is shown. (e) Representative histograms showing the percentages of CD14+ cells expressing CD23. Cells were treated with control or STAT6 siRNA then stimulated with IL-4 or IL-13 for 12 hr. Data indicate the mean ± SD of four experiments. CTR siRNA-transfected cells stimulated with IL-4/IL-13 versus STAT6 siRNA-transfected cells stimulated with IL-4/IL13: *P < 0·05. (f, g) Silencing of STAT6 significantly reduces the IL-4/IL-13-induced MRC1 and CCL18 RNA expression. CD14+ cells were transfected with control or STAT6 siRNA and then stimulated with IL-4 or IL-13 for 3 hr. RNA was extracted and amplified by real-time PCR. Levels are normalized to β-actin and indicate the mean ± SD of four experiments. CTR siRNA-transfected cells stimulated with IL-4/IL-13 versus STAT6 siRNA-transfected cells stimulated with IL-4/IL13: *P < 0.05.

To examine the functional role of STAT6 in regulating AAM induction, we treated cells with STAT6 siRNA or control siRNA, and then stimulated them with IL-4/IL-13. Using a fluorescein-labelled siRNA, we initially showed that > 50% of human CD14+ cells were efficiently transfected (Fig 6b). As expected, transfection of cells with STAT6 siRNA markedly decreased the fraction of phospho-STAT6-positive cells (Fig. 6c), without altering the rate of cell death (Fig. 6d). Notably, knockdown of STAT6 was accompanied by a significant reduction of the percentage of CD23+ cells (Fig. 6e) and diminished expression of CCL18 and MRC1 (Fig. 6f–g) in response to IL-4/IL-13.

IL-25 and IL-10, but not IL-4 or IL-13, activate p38 mitogen-activated protein kinase

We have recently shown that the IL-25-mediated inhibition of CD14+ cell response to inflammatory stimuli occurs through a p38 mitogen-activated protein kinase (MAPK)-driven suppressor of cytokine signalling (SOCS)-3-dependent mechanism.28We therefore evaluated the involvement of p38 in the Th2 cytokine-mediated negative regulation of LPS-stimulated monocyte response. As the positive selection of monocytes by engagement of CD14 receptor may regulate the activation of p38,39 we first evaluated basal p38 activation using in parallel both positively and negatively isolated CD14+ cells. The p38 level phosphorylation was not affected by the purification procedure (Fig. S6a). As shown in Fig. 7, IL-25 and IL-10, but not IL-4 or IL-13, enhanced the percentages of cells expressing active p38. Moreover, IL-4 and IL-13 were unable to activate p38 in cells purified by negative selection (Fig. S6b).

Figure 7.

 Interleukin-25 (IL-25) and IL-10 but not IL-4 or IL-13 activate p38 in human monocytes. (a) Representative dot-plots showing the expression of p-p38 in CD14+ cells either left unstimulated (Uns) or stimulated with IL-25, IL-4, IL-13 (50 ng/ml) or IL-10 (25 ng/ml) for 15 min. Moreover cells were pre-incubated with medium or the above cytokines for 15 min and then stimulated with lipopolysaccharide (LPS) for a further 15 min. Numbers in the selected areas indicate the percentage of p-p38-positive cells. (b) Histograms showing the percentage of CD14+ cells expressing p-p38 in cultures stimulated as indicated above. Data indicate the mean ± SD of three experiments.

Discussion

Since its discovery, IL-25 has been considered an important regulator of Th2 cell responses,24 even though the basic mechanism by which IL-25 expands Th2 cell-driven inflammatory reactions is not fully understood. However, it has emerged that IL-25 can regulate the function of cells other than Th2 lymphocytes. For instance, IL-25 can target antigen-presenting cells (APC) and inhibit the production of inflammatory cytokines induced by bacterial components/products.28,29,40 In this study, we confirmed and expanded on these data by showing that pre-incubation of human blood monocytes with IL-25 results in a significant inhibition of inflammatory chemokines induced by LPS or IFN-γ. This inhibitory effect of IL-25 mirrors that mediated by other Th2-type cytokines, such as IL-4 and IL-13. Previous studies have shown that the anti-inflammatory actions of IL-4 and IL-13 on monocytes/macrophages is strictly dependent on the ability of these cytokines to alter the APC differentiation state from one that is classically activated to one that is alternatively activated.1,13 The AAMs are induced in various inflammatory diseases, where they contribute to restrain macrophage activation and favour the resolution of detrimental immune reactions.41 These observations together with the demonstration that, in vivo in mice, administration of IL-25 attenuates APC-driven pathology in the gut,29,40 led us to determine if IL-25 was able to orchestrate the differentiation of human AAMs. By analysing a panel of cell-surface molecules and various chemokines, which are expressed by AAMs, we show that IL-25, unlike IL-4/IL-13, does not promote the induction of AAMs. Similarly, IL-25 is unable to potentiate the effect of IL-4 and IL-13 on the differentiation of AAMs and fails to induce AAMs following pre-incubation of monocytes with IL-10.

The above findings collectively suggest that induction of AAMs in cultures of human monocytes/macrophages probably relies on the activation of distinct signalling programmes, which are triggered by IL-4/IL-13, but not IL-25. Further experimentation was conducted to address this issue. Both IL-4 and IL-13 use the IL-4 receptor α as a receptor component, and this receptor activates STAT6, a transcription factor that translocates into the nucleus where it binds to the promoter region of target genes, thereby regulating the expression.42 In this context, studies in mice have shown that STAT6 plays a major role in the control of AAM-related genes. For example, infection of mice with a live vaccine strain of Francisella tularensis, the causative agent of tularaemia, results in induction of AAMs; the same strain does not induce AAMs in STAT6-null macrophages.37 Moreover, Heligmosomoides polygyrus (a gastrointestinal helminth parasite) infection fails to promote the development and accumulation of AAMs in the colon of STAT6-deficient mice.38 Interestingly up-regulation of AAM-associated markers occurs in wild-type but not STAT6-null recipients following transfer of CD4+ T cells from Heligmosomoides polygyrus-cured wild-type mice.43 It has also been shown that AAM-associated marker genes are epigenetically regulated by reciprocal changes in histone H3 lysine-4 and histone H3 lysine-27 (H3K27) methylation. Notably, IL-4 reduces H3K27 methylation by increasing the expression of the H3K27 demethylase Jumonji domain containing 3 through a STAT6-dependent mechanism.44 In line with these findings, we here show that IL-4 and IL-13 activate STAT6 in human monocytes, and that knockdown of STAT6 significantly reduces the IL-4/IL-13-driven AAM induction. We would like to point out, however, that AAM-associated markers were still detectable in STAT6 siRNA-transfected monocytes following IL-4/IL-13 stimulation. Since we were not able to fully silence STAT6, it is likely that the remaining STAT6 activity was sufficient to sustain the IL-4/IL-13-induced expression of AAM-related markers. Another possibility is that induction of AAMs may rely on the activation of additional signalling pathways other than STAT6. Indeed, it was recently shown that the p50 subunit of nuclear factor-κB (NF-κB) plays a key role in the orientation of macrophages towards the alternatively activated phenotype.45 We cannot also exclude the possibility that induction of human AAMs requires co-operation between STAT6 and NF-κB/p50. Indeed, it is well known that STAT6 and NF-κB family members can directly bind each other in vitro and in vivo to synergistically activate transcription of specific genes.46 No effect of IL-25 on STAT6 activation was found in human monocytes so it is highly likely that the lack of effect of IL-25 on AAM induction is the result of the inability of this cytokine to activate STAT6.

In monocytes/macrophages, IL-10 inhibits the production of inflammatory cytokines and chemokines and antigen presentation of such cells via down-regulation of MHC II and co-stimulatory molecules.47 These inhibitory effects are paralleled by a marked up-regulation of CD163. Cells exposed to IL-10 are therefore regarded as deactivated monocytes/macrophages. Our data show that IL-25 did not alter the expression of both CD163 and CD86. Therefore we feel that the IL-25-mediated negative regulation of monocyte-derived inflammatory chemokines is not the result of a cell deactivation. Another subset of macrophages, termed ‘type II macrophages’ or ‘M2b cells’, has immunoregulatory functions and is inducible by immune complexes, Toll-like receptors, and the IL-1 receptor antagonist. These cells are distinct from classical AAMs because they produce high levels of IL-10, IL-6 and TNF-α.1 We previously showed that IL-25 markedly reduces the production of IL-6 and TNF-α by human CD14+ cells following stimulation with LPS28 so it is fair to conclude that IL-25 does not favour the development of type II macrophages.

We have recently shown that IL-25 inhibits the cytokine response in CD14+ cells treated with inflammatory stimuli by activating a p38 MAPK-mediated SOCS-3-dependent mechanism.28 These findings support data of previous studies showing that SOCS-3 negatively regulates cytokine production by monocytes/macrophages after inflammatory stimuli.48–50 The data presented here confirm that IL-25 enhances p38 activation in human monocytes. Activation of p38 was also seen in CD14+ cells treated with IL-10. It is widely known that IL-10 induces SOCS-3 in various cell types via the activation of different intracellular pathways, including p38 MAPK,49 so it is conceivable that the anti-inflammatory properties of IL-10 and IL-25 rely, at least in part, on the p38-driven SOCS-3 induction.28,48 Nonetheless, it has recently been shown that, in rat bone marrow-derived macrophages, SOCS-3 deficiency associates with impaired ability to develop pro-inflammatory properties and enhanced AAM-related anti-inflammatory characteristics.51 The reason why these studies provided us with different results is unknown, even though it is plausible that the different cell populations used for assessing the role of SOCS-3 in the macrophage activation can account for this discrepancy.

Unlike IL-25 and IL-10, neither IL-4 nor IL-13 enhanced the fraction of active p38-expressing cells. Previous studies have shown that IL-4-dependent regulation of p38 signalling relies on the cell type. Indeed, IL-4 is unable to activate p38 in murine macrophages and human monocytes,52 whereas IL-4 activated p38 in epithelial cells.53 In contrast, it has been reported that pre-treatment of human monocytes with IL-4 for 24 hr augments the LPS-induced activation of p38,52 suggesting that activation of p38 in monocytes is a secondary effect rather than a direct effect of IL-4. Our results conflict with those published by Cathcart’s group showing that IL-13 activates p38 in human monocytes.54,55 The reason for this apparent discrepancy remain unknown, even though the different techniques adopted to purify monocytes and analyse p38 activation could explain such a difference.

In conclusion, our data indicate that Th2-related cytokines share anti-inflammatory effects on human monocytes, even though this function is mediated by distinct signalling pathways.

Acknowledgements

This work received support from the ‘Fondazione Umberto di Mario’, Rome, the Broad Medical Research Program Foundation (Nr: IBD-0242), and Giuliani SpA, Milan, Italy.

Disclosures

G.M. has filed a patent entitled ‘A treatment for inflammatory diseases.’ The other authors declare no competing financial interests.

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