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Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling.

Holmes DA, Suto E, Lee WP, Ou Q, Gong Q, Smith HR, Caplazi P, Chan AC - J. Exp. Med. (2015)

Bottom Line: Pep(-/-) hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep(+/+) progenitors in response to IFN-α.In addition, Pep(-/-) mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α-treated Pep(+/+) mice.As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Department of Translational Immunology, and Department of Pathology, Genentech, Inc., South San Francisco, CA 94080.

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PEP is a negative regulator of IFNAR signaling. (A) Freshly isolated Pep+/+ and Pep−/− lineage-depleted BM cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes) and whole-cell extracts were isolated and analyzed for STAT1 activation (phosphorylated STAT1, top; total STAT1, middle) or ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from A is shown. (C) Pep+/+ and Pep−/− lineage-depleted BM cells were expanded for 3-4 d in vitro with IL-3 (25 ng/ml), IL-6 (25 ng/ml), and SCF (50 ng/ml), treated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes), and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 (top) and ACTIN (bottom). (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of ACTIN from C is shown. (E) Pep+/+ and Pep−/− mice were injected with poly(I:C) for 16 h, and BM progenitors isolated and analyzed for expression of IFN-responsive genes by PCR array. Data are represented as fold up-regulation of Pep−/− relative to Pep+/+ progenitor cells. Relative gene expression is expressed by 2DCt method. P < 0.01 for Irf4, Mx1, and Osmr; P = 0.02 for Isg15. (F) BMDMs from Pep+/+ and Pep−/− mice were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts were isolated and analyzed for phosphorylated STAT1 (top), total STAT1 (middle), or ACTIN (bottom). (G) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from E is shown. (H) Pep+/+ and Pep−/− BMDMs were stimulated with rIFN-α4 (2,000 U/ml) for 4 h and analyzed for expression of IFN-responsive genes by PCR array as described in E. Data shown (A, C, E, F, and H) are representative of three independent experiments. Values in graphs represent means ± SEM. Statistical analysis was done by two-tailed paired Student’s t test. P < 0.05 for Prlr, Irf8, and Il7.
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fig5: PEP is a negative regulator of IFNAR signaling. (A) Freshly isolated Pep+/+ and Pep−/− lineage-depleted BM cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes) and whole-cell extracts were isolated and analyzed for STAT1 activation (phosphorylated STAT1, top; total STAT1, middle) or ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from A is shown. (C) Pep+/+ and Pep−/− lineage-depleted BM cells were expanded for 3-4 d in vitro with IL-3 (25 ng/ml), IL-6 (25 ng/ml), and SCF (50 ng/ml), treated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes), and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 (top) and ACTIN (bottom). (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of ACTIN from C is shown. (E) Pep+/+ and Pep−/− mice were injected with poly(I:C) for 16 h, and BM progenitors isolated and analyzed for expression of IFN-responsive genes by PCR array. Data are represented as fold up-regulation of Pep−/− relative to Pep+/+ progenitor cells. Relative gene expression is expressed by 2DCt method. P < 0.01 for Irf4, Mx1, and Osmr; P = 0.02 for Isg15. (F) BMDMs from Pep+/+ and Pep−/− mice were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts were isolated and analyzed for phosphorylated STAT1 (top), total STAT1 (middle), or ACTIN (bottom). (G) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from E is shown. (H) Pep+/+ and Pep−/− BMDMs were stimulated with rIFN-α4 (2,000 U/ml) for 4 h and analyzed for expression of IFN-responsive genes by PCR array as described in E. Data shown (A, C, E, F, and H) are representative of three independent experiments. Values in graphs represent means ± SEM. Statistical analysis was done by two-tailed paired Student’s t test. P < 0.05 for Prlr, Irf8, and Il7.

Mentions: Our data and that of others (Essers et al., 2009) suggest an intrinsic requirement for IFNAR signaling on progenitor cells during IFN-α–mediated disease and stress hematopoiesis. To determine the role of PEP in IFNAR signaling, lineage-depleted BM cells from Pep+/+ and Pep−/− mice were treated with IFN-α and STAT1 phosphorylation was assessed by Western blot using an anti–phospho-specific STAT1 antibody. Addition of recombinant mouse IFN-α4 (rIFN-α4) to Pep−/− progenitors demonstrated a twofold increase in STAT1 phosphorylation on tyrosine 701 compared to Pep+/+ progenitors (Fig. 5, A and B). Increased STAT1 phosphorylation was also observed using in vitro–generated Pep−/− myeloid progenitors (Fig. 5, C and D). To determine the downstream consequences of enhanced STAT phosphorylation in Pep−/− progenitor cells, we treated Pep+/+ and Pep−/− mice with poly(I:C) for 16 h and sorted lineage-negative progenitor cells for transcriptome analysis. Consistent with increased phosphorylation and activation of STAT1, IFN-responsive genes Isg15, Irf4, Mx1, and Osmr were significantly up-regulated in Pep−/− compared to Pep+/+ progenitor cells (Fig. 5 E and Table S1).


Autoimmunity-associated protein tyrosine phosphatase PEP negatively regulates IFN-α receptor signaling.

Holmes DA, Suto E, Lee WP, Ou Q, Gong Q, Smith HR, Caplazi P, Chan AC - J. Exp. Med. (2015)

PEP is a negative regulator of IFNAR signaling. (A) Freshly isolated Pep+/+ and Pep−/− lineage-depleted BM cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes) and whole-cell extracts were isolated and analyzed for STAT1 activation (phosphorylated STAT1, top; total STAT1, middle) or ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from A is shown. (C) Pep+/+ and Pep−/− lineage-depleted BM cells were expanded for 3-4 d in vitro with IL-3 (25 ng/ml), IL-6 (25 ng/ml), and SCF (50 ng/ml), treated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes), and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 (top) and ACTIN (bottom). (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of ACTIN from C is shown. (E) Pep+/+ and Pep−/− mice were injected with poly(I:C) for 16 h, and BM progenitors isolated and analyzed for expression of IFN-responsive genes by PCR array. Data are represented as fold up-regulation of Pep−/− relative to Pep+/+ progenitor cells. Relative gene expression is expressed by 2DCt method. P < 0.01 for Irf4, Mx1, and Osmr; P = 0.02 for Isg15. (F) BMDMs from Pep+/+ and Pep−/− mice were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts were isolated and analyzed for phosphorylated STAT1 (top), total STAT1 (middle), or ACTIN (bottom). (G) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from E is shown. (H) Pep+/+ and Pep−/− BMDMs were stimulated with rIFN-α4 (2,000 U/ml) for 4 h and analyzed for expression of IFN-responsive genes by PCR array as described in E. Data shown (A, C, E, F, and H) are representative of three independent experiments. Values in graphs represent means ± SEM. Statistical analysis was done by two-tailed paired Student’s t test. P < 0.05 for Prlr, Irf8, and Il7.
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fig5: PEP is a negative regulator of IFNAR signaling. (A) Freshly isolated Pep+/+ and Pep−/− lineage-depleted BM cells were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes) and whole-cell extracts were isolated and analyzed for STAT1 activation (phosphorylated STAT1, top; total STAT1, middle) or ACTIN (bottom). (B) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from A is shown. (C) Pep+/+ and Pep−/− lineage-depleted BM cells were expanded for 3-4 d in vitro with IL-3 (25 ng/ml), IL-6 (25 ng/ml), and SCF (50 ng/ml), treated with rIFN-α4 (2,000 U/ml) for the indicated times (minutes), and whole-cell extracts were analyzed by immunoblotting for phosphorylated STAT1 (top) and ACTIN (bottom). (D) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of ACTIN from C is shown. (E) Pep+/+ and Pep−/− mice were injected with poly(I:C) for 16 h, and BM progenitors isolated and analyzed for expression of IFN-responsive genes by PCR array. Data are represented as fold up-regulation of Pep−/− relative to Pep+/+ progenitor cells. Relative gene expression is expressed by 2DCt method. P < 0.01 for Irf4, Mx1, and Osmr; P = 0.02 for Isg15. (F) BMDMs from Pep+/+ and Pep−/− mice were stimulated with rIFN-α4 (2,000 U/ml) for the indicated times and whole-cell extracts were isolated and analyzed for phosphorylated STAT1 (top), total STAT1 (middle), or ACTIN (bottom). (G) Relative band intensity by densitometry of pSTAT1 (α-isoform) normalized to relative band intensity of total STAT1 (α-isoform) from E is shown. (H) Pep+/+ and Pep−/− BMDMs were stimulated with rIFN-α4 (2,000 U/ml) for 4 h and analyzed for expression of IFN-responsive genes by PCR array as described in E. Data shown (A, C, E, F, and H) are representative of three independent experiments. Values in graphs represent means ± SEM. Statistical analysis was done by two-tailed paired Student’s t test. P < 0.05 for Prlr, Irf8, and Il7.
Mentions: Our data and that of others (Essers et al., 2009) suggest an intrinsic requirement for IFNAR signaling on progenitor cells during IFN-α–mediated disease and stress hematopoiesis. To determine the role of PEP in IFNAR signaling, lineage-depleted BM cells from Pep+/+ and Pep−/− mice were treated with IFN-α and STAT1 phosphorylation was assessed by Western blot using an anti–phospho-specific STAT1 antibody. Addition of recombinant mouse IFN-α4 (rIFN-α4) to Pep−/− progenitors demonstrated a twofold increase in STAT1 phosphorylation on tyrosine 701 compared to Pep+/+ progenitors (Fig. 5, A and B). Increased STAT1 phosphorylation was also observed using in vitro–generated Pep−/− myeloid progenitors (Fig. 5, C and D). To determine the downstream consequences of enhanced STAT phosphorylation in Pep−/− progenitor cells, we treated Pep+/+ and Pep−/− mice with poly(I:C) for 16 h and sorted lineage-negative progenitor cells for transcriptome analysis. Consistent with increased phosphorylation and activation of STAT1, IFN-responsive genes Isg15, Irf4, Mx1, and Osmr were significantly up-regulated in Pep−/− compared to Pep+/+ progenitor cells (Fig. 5 E and Table S1).

Bottom Line: Pep(-/-) hematopoietic progenitors demonstrate increased IFNAR signaling, increased IFN-inducible gene expression, and enhanced proliferation and activation compared to Pep(+/+) progenitors in response to IFN-α.In addition, Pep(-/-) mice treated with IFN-α display a profound defect in hematopoiesis, resulting in anemia, thrombocytopenia, and neutropenia when compared to IFN-α-treated Pep(+/+) mice.As SLE patients carrying the PTPN22(C1858T) risk variant have higher serum IFN-α activity, these data provide a molecular basis for how type I IFNs and PTPN22 may cooperate to contribute to lupus-associated cytopenias.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Department of Translational Immunology, and Department of Pathology, Genentech, Inc., South San Francisco, CA 94080.

Show MeSH
Related in: MedlinePlus