Protein tyrosine phosphatase 1B is a regulator of the interleukin-10-induced transcriptional program in macrophages.

RESEARCH ARTICLE IMMUNOLOGY

Protein Tyrosine Phosphatase 1B Is a Regulator of the Interleukin-10–Induced Transcriptional Program in Macrophages Kelly A. Pike,1* Andrew P. Hutchins,2,3* Valerie Vinette,1,4 Jean-François Théberge,1 Laurent Sabbagh,5 Michel L. Tremblay,1,4† Diego Miranda-Saavedra2,6†

INTRODUCTION

The cytokine interleukin-10 (IL-10) is one of the primary anti-inflammatory mediators required for resolution of the immune response. Binding of IL-10 to its cognate receptor leads to the activation of the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) signaling pathway. The JAK kinases JAK1 and TYK2 are associated with the IL-10 receptor (IL-10R), and once activated, they phosphorylate STAT3, which then shuttles into the nucleus, where it promotes the expression of a defined set of genes that encode anti-inflammatory mediators (1–3). Given that proinflammatory cytokine receptors also use JAK-STAT3 signaling, regulatory mechanisms are needed to ensure the specificity of gene targeting in response to such a diverse array of STAT3-activating cytokines. Both epigenetic differences and the differential abundance of cofactors contribute to cell type–specific transcriptional programs; however, alternative mechanisms are required to generate unique transcriptional programs in a single cell type capable of activating STAT3 in response to functionally distinct cytokines. For example, IL-6 activates STAT3 similarly 1 Goodman Cancer Research Centre, McGill University, 1160 Pine Avenue, Montreal, Quebec H3A 1A3, Canada. 2World Premier International (WPI) Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamadaoka, Suita, 5650871 Osaka, Japan. 3South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou 510663, China. 4Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. 5Department of Microbiology, Infectiology and Immunology, University of Montreal, CP 6128 Succursale Centre-Ville, Montreal, Quebec H3C 3J7, Canada. 6Fibrosis Laboratories, Institute of Cellular Medicine, Newcastle University Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK. *These authors contributed equally to this work. †Corresponding author. E-mail: [email protected] (M.L.T.); diego. [email protected] (D.M.-S.)

to IL-10; however, IL-6 promotes a proinflammatory response as compared to the anti-inflammatory response stimulated by IL-10 (4–6). This difference in target gene transcription has been ascribed to differential phosphorylation of STAT3. The IL-6R induces expression of the gene encoding suppressor of cytokine signaling 3 (SOCS3), which then inhibits IL-6R signaling, resulting in a transient increase in STAT3 phosphorylation (4–6). In comparison, the IL-10R is not susceptible to inhibition by SOCS3, and so, it stimulates prolonged STAT3 phosphorylation. The molecular mechanisms regulating the enhanced phosphorylation of STAT3 downstream of the IL-10R to ensure that only genes encoding anti-inflammatory factors are targeted remain undefined. Through computational analysis of genome-wide STAT3-binding sites in macrophages, CD4+ T cells, AtT-20 cells (a mouse pituitary epitheliallike tumor cell line), and embryonic stem cells, we previously showed that STAT3 has two main modes of binding: (i) a cell type–independent binding mode characterized by a set of 35 evolutionarily conserved STAT3-binding sites determines the outcome of STAT activity and cell growth, and (ii) a series of cell type–dependent binding modes that differ across these four cell types and that are responsible for the cell type–specific functions of STAT3. Among the targets in the cell type–independent binding mode is the gene Ptpn1, which encodes protein tyrosine phosphatase 1B (PTP1B) (7). PTP1B is a ubiquitous tyrosine phosphatase, which is localized to the endoplasmic reticulum. It is implicated in regulating multiple signaling pathways (8), and its list of substrates includes the insulin receptor and vascular endothelial growth factor receptor (9, 10). In addition, because JAK2, TYK2, STAT3, and STAT6 are substrates of PTP1B, PTP1B plays an important role in modulating JAK-STAT signaling (11–14). Dysfunction of JAK-STAT signaling is responsible for some of the immune dysfunctions identified in PTP1B-deficient mice. Such mice exhibit chronic, low-grade inflammation, which has, in part, been ascribed to perturbations in myeloid

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Both pro- and anti-inflammatory cytokines activate the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway; however, they elicit distinct transcriptional programs. Posttranslational modifications of STAT proteins, such as tyrosine phosphorylation, are critical to ensure the differential expression of STAT target genes. Although JAK-STAT signaling is dependent on reversible tyrosine phosphorylation, whether phosphatases contribute to the specificity of STAT-dependent gene expression is unclear. We examined the role of protein tyrosine phosphatase 1B (PTP1B) in regulating the interleukin-10 (IL-10)– dependent, STAT3-mediated anti-inflammatory response. We found that IL-10–dependent STAT3 phosphorylation and anti-inflammatory gene expression were enhanced in macrophages from PTP1B–/– mice compared to those in macrophages from wild-type mice. Consistent with this finding, the IL-10–dependent suppression of lipopolysaccharide-induced macrophage activation was increased in PTP1B–/– macrophages compared to that in wild-type macrophages, as was the IL-10–dependent increase in the cell surface expression of the anti-inflammatory cytokine receptor IL-4Ra. Furthermore, RNA sequencing revealed the expression of genes encoding proinflammatory factors in IL-10–treated PTP1B–/– macrophages, which correlated with increased phosphorylation of STAT1, which is not normally highly activated in response to IL-10. These findings identify PTP1B as a central regulator of IL-10R–STAT3 and IL-10R–STAT1 signaling, and demonstrate that phosphatases can tailor the quantitative and qualitative properties of cytokineinduced transcriptional responses.

RESEARCH ARTICLE comparable abundance of F4/80, major histocompatibility complex II (MHCII), CD11b, and CD80 was detected on the surfaces of PTP1B+/+, PTP1B+/–, and PTP1B–/– cells (fig. S2A). Similarly, a comparison of the transcriptional profiles of these cells by RNA sequencing (RNA-seq) confirmed their similarity with our previously published macrophage RNA-seq libraries (7) and their difference from those of other cell lineages (fig. S2B). The IL-10R associates with both JAK1 and TYK2. Because TYK2, but not JAK1, was identified as a PTP1B substrate, we assessed the phosphorylation of TYK2 in response to IL-10 (11). PTP1B–/– macrophages exhibited prolonged TYK2 phosphorylation compared to that in PTP1B+/+ and PTP1B+/– cells (Fig. 1A). The observed alteration in TYK2 phosphorylation kinetics influenced the downstream phosphorylation of STAT3. Whereas the IL-10–dependent stimulation of STAT3 tyrosine phosphorylation was comparable in PTP1B+/+ and PTP1B+/– macrophages, PTP1B–/– macrophages exhibited a 1.6- to 2-fold increase in the abundance of phosphorylated STAT3 (pSTAT3) (Fig. 1B). This increase in STAT3 phosphorylation was even more pronounced after 24 hours (Fig. 1C) and was evident in response to both low and high concentrations of IL-10 (Fig. 1D).

RESULTS

Deficiency in PTP1B results in the increased phosphorylation and activity of STAT3 in response to IL-10 Our previous study identified Ptpn1 as a core STAT3 target gene independent of cell type; however, whether the IL-10–STAT3 signaling axis induced Ptpn1 expression was unknown. Thus, we assessed the extent of Ptpn1 expression in purified, thioglycollateelicited peritoneal mouse macrophages that were stimulated with IL-10. Within 2 hours, we detected an increase in Ptpn1 expression and PTP1B protein abundance by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) on complementary DNA (cDNA) samples and Western blotting analysis of whole-cell lysates, respectively (fig. S1, A and B). To determine the contribution of PTP1B to the regulation of the phosphorylation and transcriptional activity of STAT3, we compared the responses of macrophages isolated from PTP1B wild-type (PTP1B+/+), PTP1B heterozygous (PTP1B +/–), and PTP1B knockout (PTP1B–/–) mice to IL-10 (19). Initially, purified macrophages were phenotypically characterized by flow cytometry. A

Fig. 1. Deficiency in PTP1B results in increased phosphorylation and transcriptional activity of STAT3 in response to IL-10, but not IL-6. (A to G) Thioglycollate-elicited peritoneal macrophages were harvested from PTP1B+/+, PTP1B+/–, and PTP1B–/– mice and were stimulated with either (A to E) IL-10 or (F and G) IL-6. (A) TYK2 was immunoprecipitated (IP) from whole-cell lysates of IL-10–stimulated macrophages with anti–TYK2-agarose. Immunoprecipitated samples were analyzed by Western blotting to detect phosphotyrosine (pTyr) and total TYK2 proteins. As a negative control, anti–glutathione S-transferase (GST)–agarose was used. The arrow indicates the upper band that corresponds to pTYK2. Western blots are representative of three independent experiments. (B to D and F) Whole-cell lysates of cells after the indicated treatments were subjected to Western blotting with antibodies against the indicated proteins. Blots are representative of at least three independent experiments. (B and F) Pixel intensities in the Western blots were quantified to determine the relative abundance of pSTAT3 relative to that of total STAT3. Data are means ± SEM of three independent experiments. **P < 0.01. (E and G) qRT-PCR analysis was performed on purified cDNA from the indicated cells to quantify the relative abundances of the indicated mRNAs in response to (E) IL-10 or (G) IL-6. Each experiment included triplicate technical samples, each normalized to the reference gene Gapdh. Data are means ± SEM of a single experiment and are representative of three independent experiments. ***P < 0.001. ns, not significant.


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cell development and activation. Notably, deficiency in PTP1B results in enhanced colony-stimulating factor 1 receptor signaling and increased sensitivity to lipopolysaccharide (LPS) in spleen-derived macrophages (15–17). However, mice with a myeloid cell–specific deletion of PTP1B are protected against inflammation induced by high-fat diet or LPS. Protection was ascribed to increased amounts of systemic IL-10 and an associated increase in STAT3 phosphorylation in these mice (18). Whether the loss of PTP1B altered the functional properties of the IL-10–mediated transcriptional profile was not investigated. Here, we provide evidence that PTP1B is a required inhibitor of the IL10R–TYK2–STAT1/3 signaling axis. Through mouse genetics and experiments with a PTP1B-specific inhibitor, we demonstrated that, in the absence of PTP1B, both quantitative and qualitative changes in IL10–induced transcription occurred. Such changes can be ascribed to enhanced activation of STAT3 and STAT1. Although enhanced phosphorylation of STAT3 corresponded with the increased expression of genes encoding anti-inflammatory factors, the unexpected enhancement in STAT1 phosphorylation downstream of the IL-10R was associated with the induction of a set of genes encoding proinflammatory factors. Therefore, these findings identify PTP1B as a nonredundant inhibitor of IL-10R–STAT3 signaling. The requirement for PTP1B to ensure that only a STAT3-dependent anti-inflammatory gene program is expressed in response IL-10 is an example of how protein tyrosine phosphatases (PTPs) can contribute to targeting STAT-dependent gene expression.

RESEARCH ARTICLE To determine whether the increase in STAT3 phosphorylation in PTP1B–/– cells was sufficient to affect gene expression, we performed qRT-PCR analysis to quantify the extent of expression of known IL10–induced, STAT3 target genes. These include genes whose products are implicated in directing the anti-inflammatory response by inhibiting proinflammatory cytokine receptor signaling (Socs3), compromising tumor necrosis factor–a (TNF-a) mRNA stability (Zfp36), or acting as transcriptional co-repressors (Sbno2 and Bcl3) (Fig. 1E). The mRNAs of all of these genes were increased in abundance in PTP1B–/– peritoneal macrophages compared to those in wild-type macrophages between 2 and 8 hours after stimulation with IL-10. Moreover, whereas expression of these genes reached a plateau after 4 hours of treatment in PTP1B+/+ and PTP1B+/– cells, we observed a continual increase in transcription in PTP1B–/– cells until 8 hours after stimulation with IL-10 (Fig. 1E).

PTP1B is not required for the inhibition of STAT3 phosphorylation downstream of the IL-6R

Loss of PTP1B results in the increased cell surface abundance of IL-4Ra in response to IL-10 In addition to promoting the expression of genes associated with an antiinflammatory response, IL-10 primes macrophages for polarization to become M2 macrophages by increasing the cell surface abundance of the IL-4Ra chain. In combination with IL-13, IL-4 directs M2 polarization (22). qRT-PCR analysis demonstrated about a threefold increase in the abundance of Il4ra mRNA in PTP1B–/– cells compared to that in PTP1B+/+ and PTP1B+/– cells in response to IL-10 (Fig. 3A). This increase in Il4ra mRNA abundance coincided with an increase in the amount of IL-4Ra protein on the cell surface. Specifically, in the absence of IL-10, less than 5% of macrophages were IL-4RaHIGH independent of the presence or absence of PTP1B (Fig. 3B); however, whereas overnight exposure to IL-10 resulted in about 18% of PTP1B+/+ cells exhibiting increased cell surface IL-4Ra abundance, 54% of PTP1B–/– cells became IL-4RaHIGH (Fig. 3B). Therefore, the proportion of IL-4RaHIGH cells was about threefold higher in cultures of PTP1B–/–

The loss of PTP1B increases the IL-10–mediated suppression of LPS-induced TNF-a production We next sought to determine whether the increased phosphorylation and activation of STAT3 observed in PTP1B–/– peritoneal macrophages in response to IL-10 affected the functional properties of the cell. As described earlier, we observed an increase in the abundance of mRNAs of Zfp63 and Bcl3, whose products are implicated in inhibiting TNF-a abundance, ZFP36 by directly binding to TNF-a mRNA, leading to its degradation (20), and B cell leukemia 3 (BCL3) by inhibiting the transcriptional activity of nuclear factor kB (NF-kB) (21). We therefore assessed the capacity of IL-10 to suppress the production of TNF-a in response to LPS in PTP1B+/+, PTP1B+/–, and PTP1B–/– peritoneal macrophages. We used a high concentration of LPS because of our previous findings that showed that although PTP1B–/– macrophages are more sensitive than are PTP1B+/+ macrophages to low concentrations of LPS, the responses of both cell types to high doses of LPS are comparable (15). Indeed, about 85% of PTP1B+/+, PTP1B+/–, and PTP1B–/– macrophages produced high amounts of TNF-a in response to LPS alone

Fig. 2. PTP1B –/– macrophages exhibit increased IL-10–mediated suppression of LPS-induced TNF-a production. (A to C) Peritoneal macrophages were isolated from PTP1B+/+, PTP1B+/–, and PTP1B–/– mice and were either left untreated or treated with IL-10 overnight. The following day, cells were stimulated with LPS for 10 hours in the presence or absence of IL-10. TNF-a production was detected by intracellular flow cytometric analysis. (A) Histograms are representative of three independent experiments. (B) Data are means ± SEM of the percentage reduction in the number of TNF-a+ cells of the indicated genotypes from three independent experiments, one of which was shown in (A). **P < 0.01, ***P < 0.001. (C) Wholecell lysates from the indicated untreated and treated macrophages were analyzed by Western blotting with antibodies against the indicated proteins. Blots are representative of three independent experiments.


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The requirement for PTP1B to inhibit IL-10–induced STAT3 phosphorylation and transcriptional activity raised the question of whether PTP1B was also required to inhibit the parallel IL-6–JAK–STAT3 axis. This is of particular interest given that IL-6 induced Ptpn1 expression in macrophages (fig. S3). We found that the phosphorylation status of STAT3 was similar in PTP1B+/+, PTP1B+/–, and PTP1B–/– macrophages in response to IL-6 (Fig. 1F). Furthermore, the abundances of mRNAs of the STAT3 targets Stat3 and Socs3 were not increased in IL-6–stimulated PTP1B–/– macrophages compared to wild-type macrophages (Fig. 1G). This finding suggests that PTP1B is not a critical inhibitor of IL-6R–STAT3 signaling and identifies differential use of PTP1B by the IL-10R– STAT3 and IL-6R–STAT3 pathways.

(Fig. 2A). Whereas IL-10 suppressed TNF-a production by all cells, the extent of its suppression was increased in PTP1B–/– cells (Fig. 2B). In parallel, we assessed the activation of the NF-kB pathway given that NF-kB is responsible for the induction of TNF-a production in response to LPS. After 10 hours of stimulation with LPS, PTP1B–/– macrophages exhibited robust phosphorylation of inhibitor of kBa (IkBa) (Fig. 2C). Therefore, we do not attribute the reduction in TNF-a production by PTP1B–/– macrophages to impaired NF-kB activation. Furthermore, the LPS-dependent phosphorylation of STAT1 and STAT3 in PTP1B–/– macrophages was intact (Fig. 2C).

RESEARCH ARTICLE macrophages than in cultures of PTP1B+/+ cells in response to IL-10 (Fig. 3B). Changes in the cell surface abundance of the IL-15Ra were not observed, demonstrating the specific effect of loss of PTP1B on the abundance of the IL-4Ra chain (Fig. 3B).

The phenotype of PTP1B–/– macrophages can be recapitulated with a PTP1B-specific inhibitor

Critical changes to the IL-10–induced transcriptome are detected in PTP1B–/– macrophages

Fig. 3. PTP1B deficiency results in the increased cell surface abundance of IL-4Ra in response to IL-10. (A) Total RNA was purified from IL-10–stimulated PTP1B+/+, PTP1B+/–, and PTP1B–/– peritoneal macrophages and was analyzed by qRT-PCR to determine the relative abundances of Il4ra mRNA. Each experiment included triplicate technical samples, each normalized to the reference gene Gapdh. Data are means ± SEM of a single experiment, which is representative of three independent experiments. ***P < 0.001. (B) Macrophages from PTP1B+/+ and PTP1B–/– mice were left untreated or were treated with IL-10 overnight before being analyzed by flow cytometry to determine the cell surface abundances of IL-4Ra and IL-15R. Histograms are representative of three independent experiments. A bar graph shows means ± SEM of the percentage of IL-4RaHIGH cells from three independent experiments. **P < 0.001.

Although our findings indicated that PTP1B played a critical role in inhibiting STAT3 activation downstream of the IL-10R, it remained unclear how the loss of PTP1B would affect the entire transcriptional landscape of macrophages in response to IL-10. We therefore used RNA-seq to identify all of the quantitative and qualitative changes to the IL-10–induced transcriptional program. We performed RNA-seq on unstimulated and IL-10– stimulated peritoneal macrophages purified from PTP1B+/+ and PTP1B–/– mice. Gene expression is described in the units of “counts per million” (cpm), which represents the number of sequence tags aligned to a specific gene per million total sequence tags in the library. Initially, we observed an increase in the abundance of transcripts of anti-inflammatory genes in IL-10– stimulated PTP1B–/– cells, including Etv3, Bcl3, Zfp36, Sbno2, and Nfil3 (table S1). Because sequencing was only performed on cells stimulated for 4 hours, the extent of the increase varied between genes, most likely reflecting their distinct transcriptional kinetics in response to IL-10. Multiple molecules have been implicated in the inhibition of JAK-STAT signaling; thus, we next verified that the loss of PTP1B did not result in the unexpected loss of gene expression of any such regulators (table S1). Indeed, we found that PTP1B+/+ and PTP1B–/– peritoneal macrophages exhibited similar expression of genes encoding relevant PTPs, SOCS, or PIAS family members implicated in JAK-STAT signaling (table S1). We then sought to compare the fold induction in gene expression in PTP1B+/+ versus PTP1B–/– macrophages in response to IL-10 globally. Analysis of overlapping gene sets from PTP1B+/+ and PTP1B–/– peritoneal macrophages identified 78 genes whose expression were increased in both cell types. Of these 78 genes, 75% (59 of 78) were more highly expressed in PTP1B–/– cells than in PTP1B+/+ cells (Fig. 5A). Thus, loss of PTP1B resulted in quantitative changes in IL-10–dependent transcription.

IL-10 induces expression of a proinflammatory gene set in PTP1B-deficient macrophages The IL-10–dependent expression of an additional 42 genes was induced in PTP1B–/– cells only (Fig. 5, A and B). Gene ontology (GO) enrichment


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Given that peritoneal macrophages were isolated from mice in which PTP1B was systemically knocked out, we used a pharmacological approach to confirm the cell-intrinsic effect of PTP1B deficiency. We used the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-phosphono(difluoro-methyl)3-napthalenonitrile (PTP1Bi) to inhibit PTP1B (23, 24). PTP1Bi is an order of magnitude more selective for PTP1B than for the highly similar phosphatase T cell–PTP (TC-PTP) (25). We treated peritoneal macrophages from PTP1B+/+ mice with PTP1Bi before stimulating them with IL-10. First, we treated peritoneal macrophages with increasing concentrations of the inhibitor. Western blotting analysis of whole-cell lysates demonstrated the increased abundance of pSTAT3 in IL-10–treated cells in the presence of increasing concentrations of PTP1Bi (Fig. 4A). Second, we pretreated macrophages with PTP1Bi before stimulating them with IL-10 for 6 hours. As we observed in PTP1B–/– peritoneal macrophages, we saw an increase in pSTAT3 abundance within 2 hours of stimulation with IL-10 (Fig. 4B). PTP1Bi also caused an increase in expression of the STAT3 target Socs3 (Fig. 4C), enhanced the IL-10–dependent suppression of LPS-induced TNF-a production (Fig. 4D), and increased the cell surface abundance of IL-4Ra in response to IL-10 (Fig. 4E), as we described earlier for PTP1B–/– peritoneal macrophages. Thus, treatment of PTP1B+/+ macrophages with PTP1Bi completely recapitulated the phenotype of PTP1B–/– macrophages.

RESEARCH ARTICLE analysis was performed to identify potential functional trends among this gene set that were specific to PTP1B–/– macrophages. The top terms [response to interferon-g (IFN-g), immune response] identified most of these PTP1B–/– cell–specific genes as proinflammatory in nature (Fig. 6A). A comparison of the average cpm of a selection of transcripts encoding IFNinducible (IFI) proteins, IFN-response factors (IRFs), and small IFN-inducible guanylate-binding proteins (GBPs) identified an overall increase in their cpm in PTP1B–/– macrophages compared to those in PTP1B+/+ macrophages,

both under basal and IL-10–stimulated conditions (Table 1). This increase was not indiscriminate. For example, among genes encoding IRFs, Irf7 was particularly affected. In contrast, among almost all IFI genes verified, we observed an increase in cpm. Similarly, we detected an increase in the abundance of almost all Gbp gene transcripts by RNA-seq. qRT-PCR analysis of purified RNA extracted from IL-10–stimulated PTP1B+/+, PTP1B+/–, and PTP1B–/– macrophages was performed to confirm the increased expression of select genes, including Ifi47, Oas2, Gbp2, and Gbp6, in the PTP1B–/– macrophages (Fig. 6B). Together, these results suggest that a pro-inflammatory gene program is erroneously activated in IL-10– stimulated PTP1B–/– peritoneal macrophages.

IL-10 leads to an increase in STAT1 phosphorylation in PTP1B–/– macrophages


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Fig. 4. Inhibition of PTP1B increases IL-10R–dependent STAT3 activation and target gene transcription. (A) Macrophages isolated from PTP1B+/+ mice were treated with increasing concentrations of the PTP1B inhibitor PTP1Bi before being stimulated with IL-10 for 4 hours. Whole-cell lysates were then analyzed by Western blotting with antibodies against the indicated proteins. Blots are representative of two independent experiments. (B) PTP1B+/+ macrophages pretreated with 30 mM PTP1Bi or vehicle control were then stimulated with IL-10 for the indicated times. Whole-cell lysates were then analyzed by Western blotting with antibodies against the indicated proteins. Blots are representative of four independent experiments. Bar graph shows the relative abundances of pSTAT3 (normalized to that of total STAT3) in nonpretreated (gray) and PTP1Bi-pretreated (black) cells. Data are means ± SEM from four independent experiments. *P < 0.05. (C) Total RNA was purified from unstimulated and IL-10–stimulated PTP1B+/+ peritoneal macrophages. Macrophages were incubated with either vehicle or 30 mM PTP1Bi before being stimulated. Isolated RNA was analyzed by qRT-PCR to determine the relative abundance of Socs3 mRNA. Each experiment included triplicate technical samples, each normalized to the reference gene Gapdh. Data are means ± SEM of a single experiment, which is representative of three independent experiments. ***P < 0.001. (D) Isolated macrophages from PTP1B+/+ were left unstimulated or were stimulated with IL-10 overnight in the presence or absence of PTP1Bi. Cells were then exposed to LPS for 10 hours before being analyzed by flow cytometry to detect TNF-a by intracellular staining. Histograms are representative of four independent experiments, and values indicate the percentage of TNF-a+ cells. Bar graph shows pooled data from all experiments demonstrating the percentage reduction in the number of TNF-a+ cells. *P < 0.05. (E) Macrophages isolated from PTP1B+/+ mice were treated overnight with the indicated concentrations of PTP1Bi in the presence or absence of IL-10 before being analyzed by flow cytometry to determine the percentage of cells with cell surface IL-4Ra. Histograms are representative of three independent experiments. Bar graph shows the percentage of IL-4RaHIGH cells treated with the indicated concentrations of IL-10 and PTP1Bi. Data are means ± SEM from three independent experiments. ***P < 0.001.

STAT1 is primarily responsible for the transcription of Ifi and Gbp genes in response to IFN-g. Given that PTP1B –/– macrophages did not produce IFN-g in the presence or absence of IL-10 (fig. S4), we investigated the possibility that in the absence of PTP1B, enhanced IL-10R signaling resulted in sufficient STAT1 activation to stimulate the transcription of STAT1 target genes. Indeed, an enhanced and sustained phosphorylation of STAT1 was readily detectable in PTP1B–/–, but not in PTP1B+/+, peritoneal macrophages in response to IL-10 (Fig. 7A). When the concentration of IL-10 used to stimulate the cells was reduced, we still observed STAT1 phosphorylation in PTP1B–/– macrophages (Fig. 7B). In contrast, STAT5 phosphorylation was not increased in IL-10–stimulated PTP1B–/– peritoneal macrophages, despite a report that STAT5 is a PTP1B substrate (Fig. 7B) (26). We also detected pSTAT1 in purified PTP1B+/+ peritoneal macrophages that were treated with PTP1Bi in vitro before being stimulated with IL-10 (Fig. 7C). Increasing STAT1 phosphorylation correlated with increasing concentrations of PTP1Bi and was maintained for 8 hours (Fig. 7C). In addition, qRT-PCR analysis indicated the increased transcription of proinflammatory genes in PTP1B+/+ macrophages incubated with PTP1Bi before being stimulated with IL-10 (Fig. 7D). To explore the relative contributions of STAT1 and STAT3 to the quantitative and qualitative changes to the IL-10–dependent transcriptome in PTP1B–/– macrophages, we examined genome-wide, chromatin immunoprecipitation–sequencing (ChIP-seq) libraries of peritoneal macrophage activated through the IL-10R–STAT3 pathway (7, 27) and of bone marrow–derived macrophages stimulated through the IFN-g–STAT1 pathway (28). We then determined the cumulative occurrence of STAT3- and STAT1-binding

RESEARCH ARTICLE sites in the vicinity of genes whose expression was increased exclusively in PTP1B–/– macrophages compared to those whose expression was increased in both PTP1B+/+ and PTP1B–/– macrophages. This analysis indicated that STAT3 induced genes that were commonly expressed by PTP1B+/+ and PTP1B–/– cells in response to IL-10. In contrast, STAT1 had a greater tendency than STAT3 to regulate those genes whose expression was increased only in PTP1B–/– cells, of which ~70% have a STAT1binding site within 20 kb of their transcription start site (Fig. 7E). This suggests that most of the quantitative changes to the IL-10 transcriptome in PTP1B–/– macrophages can be attributed to increased STAT3 activity, whereas qualitative changes are likely a result of the increased activation of STAT1.

Fig. 5. Loss of PTP1B results in quantitative and qualitative changes to the IL-10–induced transcriptome. (A and B) PTP1B+/+ and PTP1B–/– macrophages were treated with IL-10 and then were analyzed by RNA-seq to assess the effect of loss of PTP1B on the IL-10–induced transcriptome. (A) Representation of the number of genes whose expression is increased by IL-10 in PTP1B+/+ and PTP1B–/– macrophages

showing the extent of overlap. (B) Graphical representation of the fold changes in expression of genes whose expression was increased in PTP1B+/+ and PTP1B–/– macrophages in response to IL-10. Inset graphs show overall increase in expression of all IL-10–stimulated genes in PTP1B+/+ and PTP1B–/– cells. Genes with a known STAT3-binding site are identified by a gray bar.

DISCUSSION


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IL-10 has a critical, nonredundant role in controlling inflammation (29–34). Although the mechanisms by which the IL-10R activates STAT3 have been studied extensively, the regulatory machinery controlling the amplitude of STAT3 phosphorylation and the specificity of STAT3 activity in response to IL-10 is only partially understood. For example, the availability of IL-10 is a contributor to the regulation of IL-10R signaling, and degradation of cell surface IL-10R1 after stimulation with IL-10 leads to reduced STAT3 activation (35, 36). Here, we described a previously uncharacterized regulatory mechanism controlling STAT3 activity in response to IL-10. We identified PTP1B as an inducible inhibitor of the IL-10R–TYK2–STAT3 anti-inflammatory signaling response. PTP1B-deficient macrophages

exhibited prolonged TYK2 phosphorylation and enhanced STAT3 phosphorylation in response to IL-10. These perturbations resulted in increased transcription of STAT3 target genes and more efficient suppression of macrophage activation. Multiple PTPs, including TC-PTP (37), SHP1 (38, 39), SHP2 (40), receptor PTPe (RPTPe) (41), and PTP receptor T (PTPRT) (42), regulate the JAK-STAT3 pathway. However, our findings indicate that the loss of PTP1B cannot be compensated for by such alternative PTPs. The reason for a lack of redundancy may be that PTP specificity and activity are highly dependent on intracellular localization. Hence, one might predict that there exists a hierarchy of PTPs, each acting at different subcellular locations with different kinetics in response to cytokine stimulation (43). For example, PTP1B may be more critical in regulating early, membraneproximal events, such as TYK2 activation, whereas TC-PTP may play a greater role downstream on STAT3 in the nucleus. In addition, the effect of cellular context should not be underestimated. Thus, although the targeting of STAT3 by PTPRT is implicated in colorectal cancer (42), PTPRT is dispensable for the dephosphorylation of STAT3 in keratinocytes in response to ultraviolet B irradiation (44). Further work will be needed to determine how such differential use of PTPs is orchestrated within one cell and why particular PTPs are more or less critical in distinct cell lineages. Nevertheless, PTP1B was required to inhibit IL-10–JAK– STAT3 signaling in macrophages in response to IL-10. Because inflammation increases PTP1B protein abundance, increasing the amount of PTP1B may curtail any IL-10–dependent anti-inflammatory responses during the immune response by inhibiting IL-10R signaling (45–47).

RESEARCH ARTICLE Fig. 6. In the absence of PTP1B, IL-10 aberrantly induces the transcription of proinflammatory genes. (A) GO terms describing genes of the category “Biological Process” whose expression in IL-10–stimulated PTP1B–/– macrophages was increased compared to that in PTP1B+/+ macrophages. (B) PTP1B+/+, PTP1B+/–, and PTP1B–/– macrophages were left unstimulated or were stimulated with IL-10 for 4 hours. Samples were then analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. Each experiment included triplicate technical samples, each normalized to the reference gene Gapdh. Data are means ± SEM of a single experiment, which is representative of three independent experiments. **P < 0.01, ***P < 0.001.

Functional group

Gene

IFI

Ifi205 Ifi204 Ifit3 Ifi47 Ifitm1 Ifitm3 Ifit1 Ifi203 Ifit2 Oas1a Oas1b Oas1c Oas1g Oas2 Oas3 Irf1 Irf2 Irf3 Irf4 Irf5 Irf7 Irf8 Irf9 Gbp2 Gbp3 Gbp5 Gbp6 Gbp7 Gbp9

IRF

GBP

Decreased PTP1B abundance leads to an increase in the number of myeloid suppressor cells derived from bone marrow cells, providing protection against chronic inflammation and colitis (48).

PTP1B+/+ (cpm)

PTP1B−/− (cpm)

−IL-10

+IL-10

−IL-10

+IL-10

80.5 1746 27 163.5 34 4589 40.5 614 70 1200 29.5 102 126 256.5 678 986 1549 976 77 6777 633 3124 740 507 164 21 35 230 55

86.5 3037 36 295 72 5056.5 50.5 880 70.5 1412 32 112.5 139 369 740 1350 1874.5 1032 70 6115.5 728 2926.5 865.5 878.5 348 62 51 456.5 77.5

83 2759 166 273.5 39 5676.5 172.5 973 211.5 2590 51.5 155.5 461.5 434 1262.5 1267 1535 1063 84 6610 2146 3304.5 983.5 828 205 25 109.5 281.5 95.5

230 6454.5 224 1308 123 6468.5 342 2150 677.5 3088.5 60 150.5 597 1006 1807 2002.5 2236.5 1113.5 74.5 5939 2555 3021 1367.5 2026.5 853.5 235 308.5 849 89.8

The function of PTP1B in the immune system is, however, not restricted to controlling the anti-inflammatory response. As evidence, exacerbated inflammation and increased leukocyte trafficking are observed in PTP1B-deficient mice during allergic responses (17). Similarly, peripheral T cells from PTP1B–/– mice produce increased quantities of IFN-g in response to mitogenic stimulants (16). Such an enhanced inflammatory response may be in part attributed to regulation of the IL-4R,


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Table 1. Proinflammatory genes are more highly expressed in PTP1B–/– macrophages than in PTP1B+/+ macrophages. Average cpm values from 2 biological replicates as detected by RNA-Seq.

RESEARCH ARTICLE IFN-a/bR, and Toll-like receptors by PTP1B (13, 14, 49, 50). Therefore, PTP1B plays an inhibitory role in both pro- and anti-inflammatory signaling pathways. In relation to JAK-STAT signaling, we propose that the loss of PTP1B alters the quantitative and qualitative properties of STAT-mediated target gene transcription downstream of various receptors in a manner dependent on cellular context and the nature of the stimulating cytokine. This is of particular importance given the development of PTP1B inhibitors for the treatment of obesity and type II diabetes. Such metabolic disorders are associated with chronic inflammation, with macrophages being the

primary source of inflammatory mediators in adipose tissue. Therefore, an understanding of the role of PTP1B in controlling the pro- versus antiinflammatory responses of macrophages is needed to predict any secondary effects associated with the administration of a PTP1B inhibitor. A report demonstrated that when mice with a macrophage-specific deletion of PTP1B were maintained on a high-fat diet, they were partially protected from LPS-induced inflammation, hyperinsulinemia, and endotoxemia. It was therefore concluded that the inhibition of PTP1B is in fact a possible anti-inflammatory therapy in obesity (18). Our findings suggest that whereas inhibition of PTP1B can enhance the anti-inflammatory response,


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Fig. 7. The expression of Ifi genes is associated with aberrant IL-10–dependent activation of STAT1. (A and B) Macrophages isolated from PTP1B+/+ and PTP1B–/– mice were stimulated with (A) IL-10 for the indicated times or (B) the indicated concentrations of IL-10 for 4 hours. Whole-cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three independent experiments. IFN-g–stimulated macrophages were used as a positive control (+) for pSTAT1, whereas IL-7–stimulated T cell progenitors were used as a positive control (+) for pSTAT5. (C) Top: Macrophages isolated from PTP1B+/+ mice were left untreated or were pretreated with the indicated concentrations of PTP1Bi before being stimulated with IL-10. Bottom: PTP1B+/+ macrophages were left untreated or were treated with 30 mM PTP1Bi before being treated with IL-10 for the indicated times. Whole-cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three independent experiments. (D) Macrophages from PTP1B+/+ mice were left untreated or were treated with IL-10 in the presence or absence of PTP1Bi. Samples were analyzed by qRT-PCR to determine the relative abundances of the indicated mRNAs. Each experiment included triplicate technical samples, each normalized to the reference gene Gapdh. Data are means ± SEM of a single experiment, which is representative of three independent experiments. **P < 0.01, ***P < 0.001. (E) Genome-wide analyses of STAT3- and STAT1-binding sites were extracted from the studies of Hutchins et al. (27) and Ostuni et al. (28), respectively. The binding sites for STAT3 and STAT1 were mapped to the gene set common to IL-10–stimulated PTP1B+/+ and PTP1B–/– macrophages, as well as to the distinct gene set expressed in IL-10–stimulated PTP1B–/– macrophages. For comparison, 30 lists of randomly distributed, genome-wide coordinates are displayed in gray to indicate the number of genes expected to be bound by chance alone. WT, wild type; KO, knockout.

RESEARCH ARTICLE

MATERIALS AND METHODS

Mice

The generation of Ptpn1–/– (PTP1B–/–) gene–targeted mice has been previously described (19). Heterozygous breeding pairs were used to ensure that littermate wild-type and heterozygous controls were used. Mice were housed in a specific pathogen–free facility, in sterile microisolator caging. Animal protocols were in accordance with the regulations of the Canadian

Council on Animal Care, and were approved by the McGill University animal care committee.

Isolation and stimulation of thioglycollate-elicited peritoneal macrophages Mice were subjected to intraperitoneal injection of 1 ml of a 3% thioglycollate solution. Three to 5 days later, peritoneal macrophages were harvested by peritoneal lavage with 10 ml of phosphate-buffered saline (PBS). Macrophages were allowed to adhere to cell culture plates overnight, after which nonadherent cells were removed by extensive washing. Recombinant IL-10 (PeproTech) was added at a concentration of 10 or 100 ng/ml, IL-6 (PeproTech) was used at 100 ng/ml, and LPS (Sigma-Aldrich) was used at a concentration of 100 ng/ml. When PTP1Bi was used, cells were pretreated with the inhibitor overnight before the addition of IL-10.

RNA isolation, reverse transcription, and quantitative real-time PCR Total RNAwas extracted from peritoneal mouse macrophages with the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Any potential contaminating genomic DNA was degraded with the DNase I RiboPure kit (Life Technologies). RNA was transcribed to cDNA with the SuperScript III Reverse Transcriptase Kit (Life Technologies) according to the manufacturer’s instructions, and qRT-PCR was performed on a LightCycler 480 with SYBR Green Master Mix according to the manufacturer’s instructions. Gapdh was used as a reference gene to calculate the relative abundance of the indicated genes of interest. RNA was quantified with the NanoDrop 100 Spectrophotometer (Thermo Scientific). Statistical analysis of gene expression obtained by qRT-PCR was performed by one-way analysis of variance (ANOVA) with a Tukey posttest.

Flow cytometry Fc receptors on purified macrophages were blocked before antibody labeling with anti-CD16 antibodies (BD Biosciences). Surface antigens were detected with the following antibodies diluted in Dulbecco’s PBS supplemented with 2.5% fetal calf serum: PE (phycoerythrin)–Cy5–conjugated anti-F4/80, PE-conjugated anti-CD11b, APC (allophycocyanin)–conjugated anti-MHCII, and FITC (fluorescein isothiocyanate)–conjugated anti-CD80 (BioLegend). When necessary to detect cytokine production by flow cytometry, GolgiStop (BD Biosciences) was added to cultures 4 hours before cells were harvested. Accumulated intracellular cytokines were detected by initially fixing and permeabilizing cells with BD Cytofix/Cytoperm (BD Biosciences). TNF-a and IL-6 were then detected with APC- and PE-conjugated antibodies specific for TNF-a and IL-6, respectively (BD Biosciences). All samples were collected on a FACSCalibur instrument (BD Biosciences) and analyzed with FlowJo software (Tree Star). One-way ANOVA with a Tukey posttest was performed on independent replicate experiments detecting TNF-a+ cells or IL-4RaHIGH cells.

Immunoprecipitation and Western blotting Total cell lysates were generated with modified radioimmunoprecipitation assay (RIPA) buffer [50 mM tris-HCl (pH 7.5), 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40]. Lysates were resolved by SDS– polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to membranes for Western blotting analysis. The following antibodies used for Western blotting were purchased from Cell Signaling Technology and were used at a 1:1000 dilution: rabbit anti–phospho-STAT3 (Y705), rabbit antiSTAT3 (79D7), rabbit anti–phospho-STAT1 (Y701) (58D6), rabbit antiSTAT1, rabbit anti–phospho-STAT5 (Y694), and rabbit anti-STAT5 (3H7). The mouse anti-calnexin antibody (used at a 1:5000 dilution) was provided by J. J. M. Bergeron (McGill University), whereas the rabbit anti-PTP1B


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it is possible that this response will be converted into a proinflammatory response if the inhibition is too efficient or prolonged. The potential for the loss of PTP1B to cause an alteration in IL-10R– dependent transcription became evident from our analysis of the RNA-seq libraries generated from unstimulated and IL-10–stimulated PTP1B+/+ and PTP1B–/– macrophages. In the absence of PTP1B, two primary gene sets were identified as having changed. The first gene set reflects a normal antiinflammatory response in which genes were induced in both PTP1B+/+ and PTP1B–/– macrophages, but to a greater extent in the latter. The second gene set, which was unique to PTP1B–/– macrophages, was identified and functionally characterized as a proinflammatory gene set. The expression of this second gene set correlated with the activation of STAT1. Thus, PTP1B is an important inhibitor of the IL-10R that sets the upper threshold of its activity. In the absence of PTP1B, this threshold is exceeded, resulting in enhanced phosphorylation of STAT1 and STAT3, thereby altering the IL-10 transcriptional program (fig. S5). Whether STAT1 phosphorylation, and the transcription of proinflammatory genes, occurs as a mechanism to compensate for the enhanced STAT3-dependent anti-inflammatory response is unclear. An advantage of the experimental approach that we took is that it enabled us to isolate and compare the actions of pro- and anti-inflammatory cytokines on JAK-STAT3 signaling in the absence of PTP1B, and to identify any changes in the transcriptional program of the cell. We demonstrated that PTP1B was not required for inhibition of the proinflammatory receptor IL-6R. Whereas IL-10R activates the JAK1/TYK2-STAT3 cascade, the IL-6R activates the JAK1/JAK2-STAT3 cascade. Both JAK2 and TYK2 are targets of PTP1B. Hence, why is IL-6R not regulated by PTP1B? It is possible that because the IL-6R predominantly uses JAK1 rather than JAK2, it is less dependent on PTP1B. In addition, alternative phosphatases may compensate for the loss of PTP1B downstream of the IL-6R. Alternatively, because SOCS3 is the major inhibitor of the IL-6R–STAT3 activation pathway, PTP1B may act more to fine-tune IL-6–dependent, STAT3mediated transcription. In comparison, PTP1B may be particularly important in regulating IL-10R given that this receptor does not contain a SOCS3-binding site and, as such, is not susceptible to inhibition by SOCS3 (6). Given the limited number of JAK and STAT family members, differential use of inhibitors may be necessary to ensure the specificity of transcriptional responses in response to a given cytokine. The dependence of the IL-10R on PTP1B, rather than SOCS, may be required to enable a sufficiently high and prolonged activation of STAT3 to elicit an anti-inflammatory gene transcription profile. However, an unregulated anti-inflammatory response is detrimental to the host and thus must be maintained below a given threshold. Although PTPs are important regulators of JAK-STAT signaling, the fundamental question whether these phosphatases are necessary to ensure that the correct STAT-dependent genes are expressed and the required transcriptional profiles are generated has not been addressed. The requirement for PTP1B to safeguard the IL-10–dependent anti-inflammatory response suggests that this is indeed the case. We suggest that PTP1B is capable of modulating the relative strengths of the anti-inflammatory and inflammatory responses.

RESEARCH ARTICLE

RNA-seq and data analysis RNAwas purified from thioglycollate-elicited peritoneal macrophages, purified by TRIzol, and subjected to RNA-seq on an Illumina HiSeq 2000. Paired-end reads were aligned to the mouse transcriptome (Ensembl v67) with RSEM software v1.1.21 (51) and bowtie software v0.12.7 (52). Only genes with at least 20 mapping sequence tags in at least two biological samples were considered for downstream analysis. RNA-seq data were then normalized for GC (guanine-cytosine) content with EDASeq software v1.4.0 (53), and differential expression was determined with edgeR software v3.0.8 (54) with a q value of 0.05. Genes were then further filtered on the basis of twofold increase in expression and were assigned to specific categories depending on their fold change in the different genotypes. GO enrichment analysis was performed with PANTHER. STAT1 ChIP-seq data were extracted from GSE38377 (28). Other RNA-seq data were taken from GSE29278 (55), GSE38371 (28), GSE34550 (27), and GSE34550 (56). RNA-seq data were reanalyzed with bowtie and RSEM software, as described earlier. Raw data were deposited in Gene Expression Omnibus with accession number GSE49449.

SUPPLEMENTARY MATERIALS www.sciencesignaling.org/cgi/content/full/7/324/ra43/DC1 Fig. S1. IL-10 stimulates the production of PTP1B protein. Fig. S2. Phenotypic and transcriptional comparison of peritoneal macrophages from PTP1B+/+, PTP1B+/–, and PTP1B–/– mice. Fig. S3. IL-6 does not induce Ptpn1 expression. Fig. S4. Loss of PTP1B does not induce IFN-g production. Fig. S5. PTP1B is required to regulate the IL-10–induced transcriptional program. Table S1. Selected genes from the RNA-seq gene set.

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(ABS40, 1:1000) antibody was obtained from Millipore. Secondary antibodies, horseradish peroxidase (HRP)–conjugated goat anti-mouse immunoglobulin and goat anti–rabbit-HRP (Jackson Laboratories), were used at a final dilution of 1:5000. The pixel intensity of bands of interest was calculated with Quantity One Software (Bio-Rad). Quantification was determined by normalizing the signal intensity of the band corresponding to pSTAT3 to that of the band corresponding to total STAT3. One-way ANOVA with a Tukey posttest was performed on normalized pSTAT3 pixel intensities from independent replicate experiments. Positive control total cell lysates for pSTAT1 and pSTAT5 were generated by stimulating macrophages with IFN-g or in vitro differentiated T cell progenitors with IL-7, respectively. For immunoprecipitations, cell lysates were generated with standard RIPA buffer. TYK2 was immunoprecipitated with 4 mg of antiTYK2 antibody (Santa Cruz, C-20) for 2 hours. Immunoprecipitates were then captured with protein G–agarose (Bio-Rad) overnight. After washing, beads were boiled in SDS-PAGE loading dye, and supernatants were resolved by SDS-PAGE. Western blotting was performed with anti-phosphotyrosine (4G10, 1:1000) and anti-TYK2 (Abnova, 1:1000) antibodies.

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Protein Tyrosine Phosphatase 1B Is a Regulator of the Interleukin-10−Induced Transcriptional Program in Macrophages Kelly A. Pike, Andrew P. Hutchins, Valerie Vinette, Jean-François Théberge, Laurent Sabbagh, Michel L. Tremblay and Diego Miranda-Saavedra (May 6, 2014) Science Signaling 7 (324), ra43. [doi: 10.1126/scisignal.2005020]

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