1. Volume 144, Issue 5, Pages 978-988.e10 (May 2013)
Loss of Protein Tyrosine Phosphatase Nonreceptor Type 22 Regulates Interferon-γ– Induced Signaling in Human Monocytes 
Marianne R. Spalinger, Silvia Lang, Achim Weber, Pascal Frei, Michael Fried, Gerhard Rogler, Michael Scharl 
Gastroenterology 
Volume 144, Issue 5, Pages e10 (May 2013)
DOI: /j.gastro
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2. Figure 1
PTPN22 is increased upon IFN-γ treatment and decreased in Crohn's disease. (A–C) THP-1 cells were treated with IFN-γ (100 U/mL) for the indicated time points. (A) The graph shows relative PTPN22 mRNA levels compared with untreated control, normalized to β-actin (n = 3). (B) Representative Western blots show levels of PTPN22 expression and the loading control, β-actin; the graph depicts densitometric analysis (n = 3). (C) PTPN22 phosphatase activity relative to untreated control. Representative Western blot shows the PTPN22 amount in the used precipitates (n = 3). (D) Peripheral blood mononuclear cells from healthy donors were treated with 1000 U/mL IFN-γ for the indicated time. The graph depicts PTPN22 mRNA levels normalized to β-actin and relative to nontreated control cells (n = 4). (E and F) Graphs show (E) PTPN22 mRNA expression in UC (severe, n = 5; inactive, n = 5), CD (severe, n = 6; moderate, n = 9; quiescent, n = 14), and control patients (n = 21) normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (F) PTPN22 protein levels in control (n = 6) and CD (n = 7) patients normalized to β-actin. Pictures show representative PTPN22 and β-actin Western blots. Asterisks denote significant differences from the respective control (*P < .05, **P < .01, ***P < .001).
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3. Figure 2
PTPN22 protein is reduced in IBD patients. Intestinal tissue specimen from (A) non-IBD control patients (n = 11), patients with (B) UC in remission (n = 7), (C) active UC (n = 13), (D) CD in remission (n = 10), and (E and F) active CD (n = 12) were immunohistochemically stained for PTPN22 and representative pictures are shown. Overview images, 10-fold magnification. Higher resolution image scale bars: 20 μm.
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4. Figure 3
Cell type–specific reduction of PTPN22 in IBD patients. Representative pictures (magnification, 40×) of intestinal specimen from non-IBD control, active CD, or active UC patients (n = 5 each), co-stained for PTPN22 and (A) CD68, (B) CD3, (C) CD20, or (D) CD56. Red arrows indicate PTPN22 and CD-antigen double-stained cells, and black arrows indicate CD-antigen single-positive cells.
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5. Figure 4
PTPN22 knockdown promotes IFN-γ–induced p38 phosphorylation and IL-6 secretion. (A) THP-1 cells were treated with 100 ng/mL TNF-α, 50 ng/mL IL-1β, 25 ng/mL IL-6, or 100 ng/mL IL-23 for the indicated time. The graph shows PTPN22 mRNA levels normalized to β-actin. (B–F) THP-1 cells were transfected with either nonspecific control or PTPN22-specific siRNA and treated with IFN-γ (1000 U/mL) for the indicated time. Representative Western blots show levels of (B) PTPN22 and β-actin (n = 3); (C) phospho-Src (Tyr416) and Src (n = 3); and (D) phospho-p38 (Thr180/Tyr182) and p38 (n = 3). Numbers above the blots show results from densitometric analysis ± standard error of the mean. (E) IL-6 mRNA expression levels normalized to β-actin (n = 3) and (F) IL-6 secretion relative to untreated control-transfected cells (n = 3). Asterisks denote significant differences from the respective control (*P < .05, **P < .01, ***P < .001). ##P < .01, ###P < .001 vs IFN-γ treatment of cells transfected with control siRNA.
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6. Figure 5
PTPN22 knockdown prevents IFN-γ–induced phosphorylation of STAT molecules and expression of STAT target genes. THP-1 cells were transfected with nonspecific control or PTPN2-specific siRNA and treated with IFN-γ (1000 U/mL) for the indicated time. Representative Western blots show (A) phospho-Jak1 (Tyr1022/Tyr1023) and Jak1 (n = 3), (B) phospho-STAT1 (Tyr701) and STAT1 (n = 3), and (C) phospho-STAT3 (Tyr705) and STAT3 (n = 3). Numbers above the blots show results from densitometric analysis ± standard error of the mean. (D) ICAM-1 (n = 3), (E) NOD2 (n = 3), and (F) T-bet (n = 5) mRNA levels normalized to β-actin. Significant differences from the controls are denoted by asterisks (*P < .05, **P < .01, ***P < .001). #P < .05, ##P < .01, ###P < .001 vs IFN-γ–treated control-transfected cells.
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7. Figure 6
PTPN22 knockdown reduces NF-κB–p65, but enhances SOCS1 phosphorylation. THP-1 cells were transfected with either nonspecific control or PTPN22-specific siRNA and treated with IFN-γ (1000 U/mL) for the indicated time. Representative Western blots show (A) phospho-NF-κB–p65 (Ser536) and NF-κB–p65 in whole-cell lysates (n = 3), (B) phospho-tyrosine and SOCS1 in SOCS1 immunoprecipitates (n = 3), and (C) SOCS1 expression and β-actin in whole-cell lysates (n = 3). (D) Representative Western blots show SOCS1 and PTPN22 levels in SOCS1 (top) and PTPN22 (bottom) immunoprecipitates, (E) levels of phospho-tyrosine and SOCS3 in SOCS3 immunoprecipitates (n = 3), and (F) SOCS3 and β-actin (n = 3). Numbers above the blots show results from densitometric analysis ± standard error of the mean. Asterisks denote significant differences from the respective control (*P < .05, **P < .01, ***P < .001). ###P < .001 vs IFN-γ–treated control-transfected cells.
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8. Figure 7
PTPN22 knockdown alters secretion of inflammatory mediators. THP-1 cells were transfected with either nonspecific control or PTPN22-specific siRNA and treated with IFN-γ (1000 U/mL) for 48 hours. Graphs depict secretion (pg/mL) of (A) MCP-1, (B) IL-8, (C) IL-12p40, (D) IL-17, (E) IL-2, and (F) IL-4 into the cell culture medium. Values were normalized to RNA content in harvested cells. Significant differences from the controls are denoted by asterisks (*P < .05, **P < .01); #P < .05, ###P < .001 vs IFN-γ–treated control-transfected cells.
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9. Supplementary Figure 1
Comparable PTPN22 mRNA levels in different intestinal segments. (A and B) PTPN22 mRNA levels in biopsy specimens from UC (severe, n = 5; inactive, n = 5), CD (severe, n = 6; moderate, n = 9; quiescent, n = 14), and control patients (n = 21) normalized to (A) β-actin or (B) HuPo. The graphs depict PTPN22 mRNA levels normalized to (C) glyceraldehyde-3-phosphate dehydrogenase (GAPDH), (D) HuPo, or (E) β-actin in biopsy specimens from healthy control patients collected from the indicated intestinal segments. (F–H) PTPN22 expression in sections from non-IBD control (n = 11), UC (active, n = 13; quiescent, n = 7), or CD (active, n = 12; quiescent, n = 10) patients were analyzed by a pathologist in a blinded manner. Slides with (F) strong PTPN22 staining were counted as positive, (G) weakly stained slides or slides without PTPN22 staining were counted as negative. (H) The graph depicts the percentage of positive slides for each subgroup. Asterisks denote statistic significant differences (*P < .5, **P < .01) as assessed by the Mann–Whitney U test. NS, not specified.
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10. Supplementary Figure 2
Differential effect of proinflammatory cytokines on PTPN22 expression in THP-1 monocytes. THP-1 cells were treated with (A) 100 ng/mL TNF-α, (B) 50 ng/mL IL-1β, (C) 25 ng/mL IL-6, or (D) 100 ng/mL IL-23 for the indicated amounts of time. Representative Western blots show protein levels of PTPN22 and β-actin loading control.
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11. Supplementary Figure 3
IFN-γ induces MAPK and STAT signaling in THP-1 cells. THP-1 cells were treated with IFN-γ for the indicated time points (n = 3 each). Representative Western blots and densitometric analysis show levels of (A) phospho-p38 (Thr180/Tyr182) and p38, (B) phospho-JNK (Thr183/Tyr185) and JNK (n = 3), (C) phospho-ERK (Tyr42/Tyr44) and ERK (n = 3), (D) phospho-STAT1 (Tyr701) and STAT1, and (E) phospho-STAT3 (Tyr705) and STAT3.
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12. Supplementary Figure 4
IFN-γ does not induce NF-κB phosphorylation. (A–D) THP-1 cells were treated with IFN-γ (1000 U/mL) for the indicated time points (n = 3 each). Representative Western blots and densitometric analysis are shown for (A) phospho-Src (Tyr416) and total Src; (B) phospho-IκB (Ser32) and total IκB (C) phospho-p65 (Ser536) and p65; (D) phospho-p105 (Ser933) and p105/p50. (E) THP-1 cells were treated for 30 minutes with 100 ng/mL TNF-α (n = 3). Representative Western blots show levels of phopsho–NF-κB–p105 (Ser933), total NF-κB–p105, phospho-NF-κB–p65 (Ser536), and total NF-κB–p65. (F–H) THP-1 cells were treated with IFN-γ (1000 U/mL) for increasing times. Depicted are relative mRNA levels normalized to β-actin for (F) IL-6, (G) ICAM-1, and (H) NOD2. Asterisks denote significant differences from the respective control (*P < .05, **P < .01).
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13. Supplementary Figure 5
PTPN22 knockdown does not alter PTPN2 or PTP1B expression. (A and B) THP-1 cells were transfected with either nonspecific control siRNA or PTPN22-specific siRNA and treated with IFN-γ (1000 U/mL) for 48 hours (n = 3 each). (A) The graph depicts PTPN22 mRNA levels normalized to β-actin and nontreated control-transfected cells. (B) Representative Western blots show levels of PTPN22 and β-actin loading control, results of densitometric analysis are shown in the graphs below. (C and D) THP-1 cells were treated with IFN-γ (1000 U/mL) for the indicated time points. Representative Western blots show protein levels of β-actin loading control and (C) PTPN2 or (D) PTP1B, the graphs depict results of densitometric analysis (n = 3). (E and F) THP-1 cells were treated as in panel B. Western blots and respective densitometric analysis show levels of (E) PTPN2 or (F) PTP1B and β-actin loading control (n = 3). Asterisks denote significant differences from the respective control (*P < .05, **P < .01, ***P < .001).
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14. Supplementary Figure 6
PTPN22 knockdown does not interfere with JNK, ERK, or Jak2 phosphorylation. THP-1 cells were transfected with nontargeting control or with PTPN22-specific siRNA and treated with 1000 U/mL IFN-γ for (A–C) 30 minutes or (D) 24 hours. Western blots and densitometric analysis show levels of (A) phospho-JNK (Thr183/Tyr185) and JNK, (B) phospho-ERK (Tyr42/Tyr44) and ERK, or (C) phospho-Jak2 and Jak2 protein expression (n = 3 each). (D) mRNA levels normalized to β-actin are shown for TGFβ mRNA expression relative to nonstimulated control transfected cells (n = 7). Asterisks denote significant differences from the respective control (**P < .01, ***P < .001). ###P < .001 vs IFN-γ treatment of cells transfected with control siRNA.
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15. Supplementary Figure 7
Phosphorylation of NF-κB p105 is not influenced by PTPN22 knockdown; PTPN22 does not interact directly with SOCS3. THP-1 cells were transfected with nonspecific control siRNA or PTPN22-specific siRNA and treated for the indicated time with 1000 U/mL IFN-γ. Representative Western blots and respective densitometric analysis show (A) phospho-IκB (Ser32) and IκB (n = 3), and (B) phospho–NF-κB–p105 (Ser933) and NF-κB–p105/p50 (n = 3). (C) Representative Western blots from 3 independent experiments show SOCS3 and PTPN22 levels in SOCS3 (upper panels) and PTPN22 (lower panels) immunoprecipitates. (D) Densitometric analysis of SOCS1 levels in PTPN22 precipitates, normalized to precipitated PTPN22 and IFN-γ–treated control siRNA-transfected cells. (E) Densitometric analysis of PTPN22 levels in SOCS1 precipitates, normalized to precipitated SOCS1 and IFN-γ–treated control siRNA-transfected cells. (F) Representative Western blot and densitometric analysis of PTPN22 expression normalized to β-actin in 3-hour IFN-γ–treated cells.
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