Article Figures & Data
Figures
- Figure 1 Expression of PGRN in Patients With aBD and aVKH
(A-B) PGRN levels were detected by ELISA in serum collected from patients and normal controls (A: 34 aBD patients VS 31 normal controls, B: 37 aVKH patients VS 34 normal controls). (C-D) PGRN mRNA expression was measured by RT- PCR in PBMCs from patients and normal controls. (C: 22 aBD patients VS 20 normal controls, D: 17 aVKH patients VS 18 normal controls). Data are expressed as mean ± SEM, and dots represent individual participants. Mann-Whitney U tests or independent t tests were used for statistical analyses. BD = Behcet disease; mRNA = messenger RNA; PGRN = progranulin; RT-PCR = real-time PCR; VKH = Vogt-Koyanagi-Harada.
- Figure 2 PGRN Attenuates EAU and Inhibits Th1 and Th17 Immune Response
(A) Quantitative RT-PCR analysis of PGRN expression in the retinal tissues from EAU mice at different time points after immunization. (B) B10RIII mice were immunized for EAU and injected intraperitoneally with rPGRN or PBS every other day from days 7–13 postimmunization. Quantification of clinical score of 2 EAU groups from day 7 to day 13 after immunization was shown. (C) Quantification of intracellular expression of IL-17, IFN-γ, and CD25+Foxp3+ by CD4+ T cells (Th1, Th17, and Treg, respectively) in the spleen from the 2 groups. (D) Quantitative RT-PCR analysis of IFN-γ, IL-17, IL-10, and foxp3 in the splenocytes from PGRN- or PBS-treated mice after immunization on day 13. Data are shown as mean ± SEM from 2 to 3 independent experiments with total of 10 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant. EAU = experimental autoimmune uveitis; IL = interleukin; IFN = interferon; PBS = phosphate-buffered saline; PGRN = progranulin; RT-PCR = real-time PCR; Th = T helper; Treg = regulatory T.
- Figure 3 PGRN−/− Mice Develop Exacerbated EAU
WT and PGRN−/− mice of C57BL/6 background were induced for EAU. (A) Quantification of clinical score of the 2 groups from day 7 to day 13. (B) Pathologic scores of the 2 EAU groups on day 13. (C) Detection of intracellular expression of IL-17, IFN-γ, and Foxp3 by CD4+ T cells in spleen by flow cytometry. (D) Quantitative RT-PCR analysis of IFN-γ, IL-17, IL-10, and foxp3 in the splenocytes. Data are shown as mean ± SEM from 2 to 3 independent experiments with a total of 10 mice per group. *p < 0.05, **p < 0.01, and ***p < 0.001. EAU = experimental autoimmune uveitis; IL = interleukin; IFN = interferon; PGRN = progranulin; RT-PCR = real-time PCR; Th = T helper; WT = wild type.
- Figure 4 Recombinant PGRN Inhibits IRBP-Reactive Th1 and Th17 Cell Expansion and Promotes IRBP-Reactive Treg Cell Expansion
(A and B) Spleen cells from EAU mice were stimulated with IRBP651–670 in the presence or absence of rPGRN for 72 hours and then assessed for intracellular expression of IFN-γ, IL-17, and Foxp3 by CD4+ T cells by flow cytometry (n = 8). (A) Representative flow cytometry dot plots. (B) Histograms of IFN-γ, IL-17, and Foxp3 by CD4+ T cells (Th1, Th17, and Treg, respectively). (C and D) Naive CD4+ T cells from spleen cells of normal C57BL/6 mice were stimulated with Th1, Th17, and Treg cell polarization conditions in the presence or absence PGRN for 72 hours and then assessed for intracellular expression of IFN-γ, IL-17, and Foxp3 by CD4+ T cells by flow cytometry (n = 8). (C) Representative flow cytometry dot plots of the 2 groups. (D) Histograms of Th1, Th17, and Treg of the 2 groups. Data are shown as mean ± SEM from 2 independent experiments. *p < 0.05, **p < 0.01, and NS, not significant. EAU = experimental autoimmune uveitis; IL = interleukin; IFN = interferon; PGRN = progranulin; RT-PCR = real-time PCR; Th = T helper; Treg = regulatory T.
- Figure 5 DEG Screening and GSEA Analysis
(A) Volcano plots showed DEGs in the spleen and retina between the PGRN−/− EAU group (knockout (KO) n = 4) and the WT EAU group (WT n = 4). The red dots represent upregulated DEGs, whereas the blue dots represent the downregulated DEGs. Nonchanged genes are shown in gray color. (B) Venn diagram showed the shared 13 DEGs both in the spleen and in the retina. (C and D) The bar graphs showed normalized enrichment scores of GSEA on KEGG gene sets for RNA-seq analysis in the (C) spleen and (D) retina between WT and PGRN−/− EAU group. The figure presented the top 5 of significant gene sets (FDR <0.05). Upregulated and downregulated gene sets are highlighted in red and blue, respectively. In the spleen, there were only 3 downregulated gene sets. Upper left of the figure shows the enrichment plot of MAPK signaling pathway and cytokine-cytokine receptor interaction gene sets, respectively. DEG = differentially expressed genes; EAU = experimental autoimmune uveitis; FDR = false discovery rate; GSEA = gene set enrichment analysis; PGRN = progranulin; WT = wild type.
- Figure 6 PGRN−/− Mice Develop More Severe EAE
WT and PGRN−/− mice of C57BL/6 background were induced for EAE. (A and B) Quantification of clinical score and body weight of the 2 groups from day 7 to day 22. (C) Quantification of inflammation and demyelination of the spinal cord from the 2 EAE mice groups at day 22 postimmunization and hematoxylin and eosin staining of the spinal cord sections for analyzing the degree of inflammation. Luxol fast blue staining of spinal cord sections for analyzing the degree of demyelination. (D) Quantitative analysis the frequency of IFN-γ+CD4+ T cells (Th1), IL-17+CD4+ T cells (Th17), and Foxp3+CD4+ T (Treg) cells in the CNS (brain and spinal cord) and spleen by flow cytometry. Data are shown as mean ± SEM from 3 independent experiments with a total of at least 10 mice per group. *p < 0.05, **p < 0.01. EAA = experimental autoimmune encephalomyelitis; IL = interleukin; IFN = interferon; PGRN = progranulin; Th = T helper; Treg = regulatory T; WT = wild type.
- Figure 7 PGRN Skews the Balance of Macrophage Polarization From M1 to M2
(A and B) Mononuclear cells were harvested from the CNS or spleen from PGRN−/− EAE mice and control EAE mice on day 22; then, the cells were stained for CD11b, F4/80, MHC-II, CD86, CD40, and CD206 (6 mice per group). (A) Flow cytometric analysis of the frequency of CD11b+F4/80+ macrophages in the CNS and spleen, respectively. (B and C) Flow cytometric analysis of the expression of MHC-II, CD86, CD40, and CD206 on the CD11b+F4/80+ cells in the CNS and spleen, respectively. (D) Bone marrow cells were cultured with the M-CSF for 7 days and then stimulated with or without PGRN on M1 or M2 polarizing conditions for another 2 days; then, the cells were analyzed for the expression of iNOS and Arg-1 by real-time PCR (n = 6). *p < 0.05, **p < 0.01; NS = not significant. Arg-1 = arginase 1; EAE = experimental autoimmune encephalomyelitis; iNOS = inducible nitric oxide synthase; PGRN = progranulin.
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