Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge

Abstract

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1B-deficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.

Figures

Figure 1

Effect of PTP1B deficiency on the polarization of macrophages. Peritoneal macrophages from wild-type (WT) or PTP1B-deficient (KO) mice were isolated after thioglycollate eliciting. Cells were stimulated with 200 ng/ml of LPS or 25 μg/ml polyI:C (M1 stimuli) and the levels of the indicated proteins were determined at 24 h (a). The response to M2 stimuli was analyzed after challenge for 24 h with a mixture of 20 ng/ml each IL-4+IL-13 (b). The levels of PTP1B in M1 and M2 polarized macrophages were determined (c). The accumulation of NO, PGE2 and TNF-α in the incubation medium was determined after 24 h of activation with 200 ng/ml LPS, 25 μg/ml polyI:C, 5 μg/ml LTA, 20 ng/ml IL-10 or 20 ng/ml of IL-4+IL-13 (d). The time course of mRNA levels of IL-6 and NOS-2 after activation with 200 ng/ml LPS (e) and of cells undergoing apoptosis by annexin V binding criteria (f) were determined. Results show the mean±S.D. of three independent experiments. *P<0.05; **P<0.01 versus the same condition in the WT cells or non-treated cells (c). (a–c) The band intensities of NOS-2, COX-2 (a), HO-1, Arg-1 (b) and PTP1B (c) after normalization using p85 and β-actin, respectively, are shown

Figure 2

PTP1B deficiency enhances IκBα degradation and impairs IκBα recovery. Macrophages from WT and PTP1B KO mice were stimulated with 200 ng/ml of LPS for the indicated times and the NF-κB and IRF3 pathways were analyzed (a). The upregulation of IκBα mRNA expression as a sensor of resetting of NF-κB was determined by qPCR (b). The nuclear translocation of p65 was determined by western blotting using p85 and lamin B as markers of the corresponding cytosolic and nuclear fractions, respectively (c). Results show the mean±S.D. of four independent experiments (b) or a representative blot out of four (a, c). *P<0.05; **P<0.01 versus the same condition in the WT cells

Figure 3

PTP1B deficiency enhances AKT and MAPK activation. Macrophages from WT and PTP1B KO mice were stimulated with 200 ng/ml of LPS and the phosphorylation of AKT, ERK, p38 and JNK was determined at the indicated times (a). To evaluate the effect of the PI3K pathway in the modulation of AKT and MAPK activities, macrophages from PTP1B KO mice were treated for 10 min before LPS activation with 10 μM LY29004. At the indicated times, the phosphorylation state of these proteins was determined (b). In parallel to the precedent assays, the levels of IκBα were determined using a lower LPS concentration (50 ng/ml) as stimulus (c). The effect of recombinant PTP1B on the dephosphorylation of extracts from WT and PTP1B macrophages activated for 30 min with 200 ng/ml of LPS was assayed in vitro after 30 min of incubation at 30 °C with recombinant PTP1B (d). Results show the mean±S.D. of three independent experiments, or a representative dephosphorylation experiment (d). *P<0.05; **P<0.01 versus the same condition in the WT cells

Figure 4

Gene expression profiling of WT and PTP1B-deficient macrophages treated with LPS. Macrophages were activated with 200 ng/ml of LPS for 24 h and the RNA was extracted, analyzed for quality in a Bioanalyzer and submitted to microarray hybridization (Agilent). Heatmap showing the microarray probes with differential response (FDRa). Gene set enrichment analysis (GSEA) of KEGG and REACTOME pathways in macrophages from PTP1B KO mice after pro-inflammatory stimulation (b). The genes in significantly enriched pathways (FDR<0.25) are shown and the color code corresponds to the standardized gene expression per row (not LPS fold change as in a). Red indicates higher expression and blue lower expression

Figure 5

Effect of PTP1B silencing on the response of human macrophages. Human monocytes from buffy coats were differentiated into macrophages and treated with a mixture of sc- or siRNA oligonucleotides to silence PTP1B. After incubation for 24 h with 200 ng/ml of LPS plus 20 ng/ml of human IFN-γ, 25 μg/ml of polyI:C or 20 ng/ml of each human IL-4+IL-13, the levels of PTP1B and COX2 were determined (a). The time course of the phosphorylation and total levels of proteins related to NF-κB, AKT and MAPKs were determined (b). The release of TNF-α and PGE2 to the culture medium was determined with specific kits (c). The induction of apoptosis, measured as the annexin V-positive population, was determined in both resting and LPS+IFN-γ activated cells (d). In addition to this, to determine the sensitivity of cells silenced for PTP1B to apoptosis in response to pro-inflammatory dependent stimuli the time course and the sensitivity at 36 h to different concentrations of staurosporine (as an stressor to promote mitochondrial-dependent apoptosis) were determined (d). Results show a representative experiment or the mean±S.D. of three independent experiments. *P<0.05; **P<0.01 versus the same condition in cells treated with scRNA

Figure 6

In vivo effect of PTP1B deficiency on the response of mice to different pro-inflammatory challenges. WT or PTP1B KO male mice matched in age were used in these experiments. The serum levels of TNF-α of untreated animals (n=12) were determined (a). The ear MPO activity and edema (n=10 animals per condition) after topic administration of TPA was calculated as ratio versus the ear treated with vehicle (b). Fixed ears were stained with eosin/hematoxylin to show the structure and with (sagittal sections; c). The serum levels of TNF-α of untreated animals or i.p. injected LPS (1 mg/kg body weight) or zymosan (30 mg/kg body weight) were determined at 1 h (n=7 animals per condition) (d). The sensitivity to irradiation after a unique dose of 10 Gy or two doses of 5 Gy administered in an interval of 4 h (n=15 per group) was evaluated as survival after 7 days post transplantation (Tx) of the corresponding bone marrow; P=0.0005 versus the WT at 10 Gy (e). The liver injury, determined by the measurement of the ALT activity in the serum (f) and structure (eosin/hematoxylin staining at 4 h; white bar=100 μm; g), and the survival of animals after i.p. administration of a single dose of LPS/D-GalN (n=12 animals per condition) were determined; P=0.074 versus the WT (h, left). Alternatively, a series of WT animals underwent irradiation (10 Gy) as depicted in (e), and were subsequently submitted to bone marrow transplantation from WT or PTP1B KO mice. After restitution of the immune system (12 weeks), these animals were submitted to LPS/D-GalN challenge and survival was determined (h, right). The TNF-α levels after 1 h of treatment with LPS/D-GalN were determined in serum from animals of (i). Results show the mean±S.D. of the indicated number of animals and/or the Kaplan–Meier representation of survival (e and h). *P<0.05; **P<0.01 versus the same condition in the WT animals or samples