Figure 1.
TIMP1 is expressed in T cells activated under Th17 conditions.
(A) Naïve CD4+ T cells were stimulated for three days in media alone, iTreg conditions or Th17(β) conditions. Subsequently, mRNA was isolated and Il17a and Timp1 mRNA analysed by quantitative-PCR. (B) Naïve CD4+ T cells were stimulated in media alone, IL-6 alone, iTreg conditions, Th17(β) conditions or Th17(23) conditions. After three days, secreted TIMP1 was measured by ELISA. (C) Naïve CD4+ T cells were polyclonally stimulated in media alone (Th0) or in either Th17(β) conditions or Th17(23) conditions in the presence or absence of IL-1β. After three days, secreted TIMP1 was measured by ELISA. (D) Naïve CD4+ T cells were stimulated in media alone or under Th1 conditions, Th2 conditions or Th17(β) conditions. After three days, secreted TIMP1 was measured by ELISA. Histograms represent mean values (n = 3 per group), error bars represent s.d. Data are representative of two independent experiments.
Figure 2.
Activated T cells secrete active TIMP proteins by Zymography.
Naïve CD4+ wild type B6 T cells (A) or Timp1−/− T cells (B) were stimulated either under Th0 (media alone), IL-6 alone, Th1 conditions, Th2 conditions, iTreg conditions, Th17(β) or Th17(23) conditions. After three days, samples of supernatant were run on a protein gel together with control samples of TIMP1 and TIMP2. TIMP activity was determined by the ability of the protein gel to resist digestion by metaloprotease. Naïve CD4+ wild type B6 T cells (C) or Timp1−/− T cells (D) were stimulated either under Th0 (media alone), IL-6 alone, Th1 conditions, Th2 conditions, iTreg conditions, Th17(β) or Th17(23) conditions. After three days, samples of supernatant were run on a protein gel together with control samples of MMP9. MMP9 activity was determined by its ability to digest protein within the gel. Results are representative of two independent experiments.
Figure 3.
TIMP1 is induced in Th1 and Th17 cells by different mechanisms.
(A) Naïve CD4+ T cells from Stat3fl/fl (black bars) or Stat3fl/fl;CD4-Cre (white bars) mice were stimulated in media alone (Th0), Th1 conditions or Th17(β) conditions. After three days, secreted TIMP1 was measured by ELISA. (B) Naïve CD4+ T cells from wild type (black bars) or Stat4−/− (grey bars) were stimulated in media alone (Th0), Th1 conditions or Th17(β) conditions. After three days secreted TIMP1 was measured by ELISA. Histograms represent mean values (n = 3 per group), error bars represent s.d. Data are representative of two independent experiments.
Figure 4.
STAT3 and STAT4 are able to bind to the Timp1 promoter site.
Chromatin signatures as defined by the presence of H3K4Me3 or STAT transcription factor binding at the Timp1 gene locus, illustrated at the top of the figure. Th17(β) polarized cells were re-stimulated with IL-6 and immunoprecipitated with anti-H3K4Me3 or STAT3 (upper two panels). The STAT3 dataset is taken from [47], the H3K4Me3 dataset is representative of two independent experiments. Th1 polarised cells were re-stimulated with CD3/CD28 and IL-12, immunoprecipitated with anti-STAT4 or with anti-STAT1 (lower two panels). The STAT4 dataset is taken from [18] and the STAT1 dataset is taken from [48].
Figure 5.
TIMP1 does no alter T cell cytokine expression in vitro.
Naïve CD4+ T cells from wild type (A) or Timp1−/− (B) mice were stimulated under Th0, Th1 or Th17(β) conditions in the presence or absence of TIMP1. After three days, cells were fixed and assessed for IFN-γ and IL-17 expression. Naïve CD4+ T cells from wild type or Timp1−/− mice were stimulated under Th1 (C) or Th17(β) (D) conditions for three days. The percentage IFN-γ+ (C) or IL-17+ (D) cells were determined by intracellular staining. The histograms indicate mean ±s.e.m and the data were obtained from four independent experiments.
Figure 6.
Mice receiving Timp1 deficient auto-reactive Th1 or Th17 cells are partially resistant to the development of EAE.
Naïve CD4+ T cells from 2D2 or Timp1−/−2D2 mice were stimulated under Th1 conditions (A), Th17(β) conditions (B) or Th17(23) conditions (C). After three days cells were fixed and assessed for IFN-γ and IL-17 expression (top panels). Next 106 polarized cells from each group were adoptively transferred into Rag2−/− recipients, which were followed for signs of neurological disease. Data show mean ±s.e.m of the EAE clinical score of 5 mice per group. After 20 days (Mice receiving Th1 cells) or 35 days (Mice receiving Th17 cells), lymphocytes were isolated from the spinal chords from animals with a clinical score of 3.5 and IL-17 and IFN-γ expression were determined by intracellular staining (lower panels). Significance was determined by a Mann-Whitney U test, *P<0.05. Data are representative of two independent experiments.