Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

doi: 10.4049/jimmunol.1203363

Figure Lengend Snippet: FIGURE 1. PTPN22 is induced in M2 but not M1 macrophages. (A) Macrophages were purified from spleens of WT and PTPN22 KO mice and stained with Abs against CD11b and F4/80. Representative F4/80 and CD11b plots from at least three independent experiments are shown. (B) Cell lysate was prepared from WT and PTPN22 KO Th cells and splenic macrophages, which were stimulated with LPS for 24 h. The level of PTPN22 and Hsp90 was measured with Western blotting. The experiment was done twice and the data from one of the two experiments are shown. (C and D) Splenic macrophages obtained from WT and STAT6-deficient mice were left unstimulated (R) or stimulated with LPS/IFN-g (M1) or IL-4/IL-13 (M2) for 24 h. The level of PTPN22 and lamin B was examined with Western blotting (C) or real-time PCR (D). The levels of PTPN22 protein shown in (C) were quantified with densitometer and normalized against the density of lamin B. The normalized value of resting WT macrophages was arbitrarily set as 1. The means and SDs from three independent experiments are shown in the right panel. The data shown in (D) are the means and SDs of three independent experiments. Statistical analysis of (C) and (D) was performed with one-way ANOVA followed by a Tukey test. (E) Human macrophages were enriched from the peripheral blood of two healthy donors, who are homozygous for the C allele of the C1858T SNP. The cells were left unstimulated (R) or stimulated with LPS/IFN-g (M1) or IL-4/IL-13 (M2) for 24 h. The level of PTPN22 and lamin B was examined with Western blotting. ***p , 0.001.

Article Snippet: The following Abs were used: human PTPN22 Ab AF3428 (R&D Systems, Minneapolis, MN); Hsp90 a/b (sc-7947) and lamin B (sc-6216) Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and murine PTPN22 Ab (Genentech).

Techniques: Staining, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

doi: 10.4049/jimmunol.1203363

Figure Lengend Snippet: FIGURE 2. PTPN22 inhibits M1 polarization but promotes the expression of M2-associated genes. (A and B) Splenic macrophages of WT and PTPN22 KO mice were left unstimulated (R) or polarized into M1 or M2 cells for 24 h. The expression of indicated genes was examined with ELISA or real-time PCR in duplicate. The transcript level of each gene was normalized against the level of unstimulated WT cells, which was arbitrarily set as 1. The data shown are the means and SDs of at least three independent experiments. (C and D) Human primary macrophages were enriched from the peripheral blood of healthy donors, who are homozygous for the C allele of the C1858T SNP, and subjected to mock transfection or transfected with PTPN22 siRNA or scrambled siRNA. The level of PTPN22 and Hsp90 protein was examined with Western blotting (C). Cell extract from Jurkat cells and HT-29 cells was used as positive and negative controls, respectively, of PTPN22. The transfected human macrophages were then left unstimulated (R) or polarized into M1 or M2 cells. The concentration of the indicated cytokines was quantified with ELISA in duplicate (D). The data are cumulative results of four independent experiments. Statistical analysis was performed with one-way ANOVA followed by a Tukey test. (E) Resting WT and PTPN22 KO mouse macrophages were transfected with indicated luciferase reporters and then left unstimulated or stimulated with LPS/IFN-g (M1) or IL-4/IL-13 (M2). Luciferase activity in cell extract was measured 24 h later and was normalized against that of unstimulated WT cells. The data shown are the means and SDs of three in- dependent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used: human PTPN22 Ab AF3428 (R&D Systems, Minneapolis, MN); Hsp90 a/b (sc-7947) and lamin B (sc-6216) Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and murine PTPN22 Ab (Genentech).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Transfection, Western Blot, Concentration Assay, Luciferase, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

doi: 10.4049/jimmunol.1203363

Figure Lengend Snippet: FIGURE 3. PTPN22 is present in the cytoplasm and nucleus of mac- rophages. (A) Comparison of the amino acid sequence of the putative N- terminal bipartite nuclear localization signal (boxed) of PTPN22 from difference species. (B) Splenic macrophages were purified from WT or PTPN22 KO mice and stimulated with LPS for 24 h or left unstimulated. The levels of PTPN22, Hsp90 (a cytoplasmic protein), and Lamin B (a nuclear protein) in whole cells (W) and cytoplasmic (C) and nuclear (N) compartments were examined with Western blotting. (C) Human primary macrophages were enriched from the peripheral blood of healthy donors and stimulated with LPS for the indicated length of time. The level of PTPN22, Hsp90, and Lamin B was analyzed with Western blotting. Jurkat and HT-29 cells were used as positive and negative controls of PTPN22, respectively. The levels of PTPN22 protein shown in (B) and (C) were quantified with a densitometer. The level of cytoplasmic PTPN22 was normalized against that of Hsp90, whereas the level of nuclear PTPN22 was normalized against that of Lamin B. The normalized level of cyto- plasmic PTPN22 of umstimulated cells was arbitrarily set as 1. The means and SDs of relative PTPN22 level from three independent experiments are shown in the right panels. (D) 293T cells were transfected with a plasmid vector expressing WT EYFP-PTPN22, EYFP-D17–33, or EYFP-L11A mutant of PTPN22. The transfected cells were stained with DAPI and subjected to confocal microscope analyses. Scale bars, 20 mm.

Article Snippet: The following Abs were used: human PTPN22 Ab AF3428 (R&D Systems, Minneapolis, MN); Hsp90 a/b (sc-7947) and lamin B (sc-6216) Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and murine PTPN22 Ab (Genentech).

Techniques: Comparison, Sequencing, Western Blot, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Staining, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

doi: 10.4049/jimmunol.1203363

Figure Lengend Snippet: FIGURE 4. Cytoplasmic PTPN22 is functionally distinct from nuclear PTPN22. (A and B) PTPN22 KO macrophages were transfected with vectors expressing indicated mouse PTPN22 proteins. Whole-cell extract was prepared from a fraction of the transfected PTPN22 KO and WT cells and subjected to Western blotting with indicated Abs (top panels). The remaining cells were stimulated with LPS/IFN-g (M1) or IL-4/IL-13 (M2) for 24 h. The expression of indicated genes was examined with ELISA or real-time PCR in duplicate. The transcript level of each gene was first normalized against that of actin in the same sample and then against the normalized value of resting WT cells, which was arbitrarily set as 1. The data shown are the means and SDs of at least three independent experi- ments. The PTPN22 KO samples in each plot were analyzed with one-way ANOVA followed by a Dunnett test using vector-transfected PTPN22 KO cells as controls. *p , 0.05, **p , 0.01.

Article Snippet: The following Abs were used: human PTPN22 Ab AF3428 (R&D Systems, Minneapolis, MN); Hsp90 a/b (sc-7947) and lamin B (sc-6216) Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and murine PTPN22 Ab (Genentech).

Techniques: Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

doi: 10.4049/jimmunol.1203363

Figure Lengend Snippet: FIGURE 5. PTPN22 KO mice are more susceptible to DSS-induced colitis. WT and PTPN22 KO mice were fed plain water (four mice per group) or 1.8% DSS-containing water (eight mice per group) according to the protocol described in Materials and Methods. Their weight loss (A) and clinical scores (B) over the period of the experiment are shown. Sta- tistical analysis was performed with serial Student t tests. All mice were sacrificed on day 10. Sections of large intestines were subjected to H&E staining. The histological scores (C) and representative sections (D) are shown. Original magnification 340. Macrophages were purified from the lamina propria of the experimental mice. A fraction of the cells were stained with CD11b and F4/80. Representative FACS plots are shown in (E). The lamina propria macrophages were cultivated in vitro in the ab- sence of any stimulation for 24 h. The expression of indicated cytokines and genes was quantified with ELISA or real-time PCR in duplicate (F). The transcript level of each gene of WT cells from untreated mice was arbitrarily set as 1. The data shown are from one of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used: human PTPN22 Ab AF3428 (R&D Systems, Minneapolis, MN); Hsp90 a/b (sc-7947) and lamin B (sc-6216) Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and murine PTPN22 Ab (Genentech).

Techniques: Staining, In Vitro, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: PTPN22 modulates macrophage polarization and susceptibility to dextran sulfate sodium-induced colitis.

doi: 10.4049/jimmunol.1203363

Figure Lengend Snippet: FIGURE 6. Impacts of the C1858T SNP on macrophages. (A and B) Cell extract of PTPN22 KO macrophages expressing indicated mouse PTPN22 proteins was examined with Western blotting (A). The cells were polarized to M1 or M2 cells. The expression of IL-12 and IL-10 was quantified with ELISA in duplicate and is shown in (B). (C–H) Peripheral blood macrophages of healthy donors described in Table I were left unstimulated (R) or polarized to M1 or M2 cells. PTPN22 protein was visualized with Western blotting. A representative gel including three donors is shown in (C). The level of PTPN22 protein was normalized against that of Lamin B and further normalized against the level of resting macrophages of no. 3 CC donor, which was set as 1. The relative level of PTPN22 protein and transcript of all samples are shown in (D) and (E), respectively. C and T represent CC and CT plus TT groups, respectively. The level of PTPN22 protein of resting macrophages was plotted against the level of PTPN22 transcript of corresponding M1 cells and subjected to linear regression analysis (F). (G) Resting macrophages of a CC donor and a TT donor were transfected with indicated expression vectors. The level of total PTPN22 transcript is shown in the left panel. The transfected cells were polarized to M1 or M2 cells, and the level of endogenous PTPN22 transcript is shown in the right panel. (H) The expression of indicated genes expressed by macrophages described in (C)–(E) was measured in duplicate and is shown. The two open squares in (D), (E), and (H) indicate the two TT donors. The data shown in (B) and (G) are from three independent experiments. Statistical analysis was performed with one-way ANOVA followed by a Tukey test in (B) and (G) and with Student t test in (D), (E), and (H). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The following Abs were used: human PTPN22 Ab AF3428 (R&D Systems, Minneapolis, MN); Hsp90 a/b (sc-7947) and lamin B (sc-6216) Ab (Santa Cruz Biotechnology, Santa Cruz, CA); and murine PTPN22 Ab (Genentech).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection