Article Figures & Data
Figures
- FIGURE 1.
Chemokine production by NOD.SCID pancreatic islets. a, Gel electrophoresis of CCL2, CCL3, CCL17, CCL21, CCL22, CXCL10, and HPRT-amplified products. Total RNA was isolated from handpicked NOD.SCID pancreatic islets. b, Quantification of CXCL10, CCL2, CCL22, CCL3, CCL17, and CCL21 expression by SYBRgreen chemistry on the same preparation of NOD.SCID islets. The levels of mRNA are shown as arbitrary units normalized to HPRT expression. Data are from a representative experiment of three performed. c, Constitutive secretion of CXCL10, CCL2, CCL5, CCL3, CCL17, and CCL21 by unstimulated NOD.SCID pancreatic islets. Islets were cultured for 72 h, and the chemokine concentration in the supernatants was measured by ELISA. The data represent the mean ± SE of three to five independent islet culture wells per group from three to eight individual experiments. d, Constitutive secretion of CXCL10, CCL2, CCL5, and CCL3 by NOR (▦) and BALB/c (▨) compared with NOD.SCID (▪) pancreatic islets. Islets were cultured for 72 h, and the chemokine concentration in the supernatants was measured by ELISA. Values are represented as a percentage of the NOD.SCID pancreatic islet secretion. The data represent the mean ± SE of three to five independent islet culture wells per group from two to three individual experiments.
- FIGURE 2.
TLR expression in pancreatic islets. a, Quantification of TLR mRNA expression by real-time RT-PCR on freshly isolated NOD.SCID (▪), NOR (▦), or BALB/c (▨) pancreatic islets. The levels of mRNA are shown as arbitrary units normalized to GAPDH expression. Data are from one representative experiment of three performed. b, Individual and merged stainings for TLR3, TLR4, and insulin in NOD.SCID islet cells. No staining was revealed in slides incubated with isotype-matched primary Ab controls (data not shown). c, Quantification of TLR mRNA expression by real-time RT-PCR in handpicked human islets. The levels of mRNA are shown as arbitrary units normalized to GAPDH expression. d, Individual and merged stainings for TLR3, TLR4, and insulin in human islet cells. No staining was revealed in slides incubated with isotype-matched primary Ab controls (data not shown). Bars correspond to 20 μm.
- FIGURE 3.
Enhancement of CXCL10, CCL2, CCL5, and CCL3 production by NOD.SCID islets stimulated with TLR ligands. NOD.SCID islets were cultured with peptidoglycan, poly(I:C), LPS, flagellin, R848, CpG 1668, or with CpG 1826 for 72 h and chemokine concentration in the supernatants was measured by ELISA. The data represent the mean ± SE of three to five independent islet culture wells per group from three to eight individual experiments. ∗∗∗, p < 0.0005; ∗∗, p < 0.005; and ∗, p < 0.05 by Mann-Whitney U test.
- FIGURE 4.
BXL-219 down-regulates chemokine production in vitro by mouse and human islets. CXCL10 mRNA expression detected by real-time RT-PCR using TaqMan chemistry, shown as arbitrary units (A.U.) normalized to GAPDH expression, was measured in NOD.SCID and NOD islets (a and c, respectively, left panels) cultured for 2 h with 100 nM BXL-219 (▦) or without (▪). NOD.SCID (right panel, b), NOD (c), or human (d) islets were cultured for 72 h with 100 nM BXL-219 (▦) or without (▪). The concentration of the indicated chemokines was measured in culture supernatants by two-site ELISA. Bars represent mean ±SE from three independent experiments. ∗, p < 0.05 and ∗∗∗, p < 0.001 by one-tailed Student’s t test. e shows, as arbitrary units normalized to GAPDH expression, TLR, and CYP24 mRNA levels detected by real-time RT-PCR using TaqMan chemistry in NOD islets cultured for 2 h with 100 nM BXL-219 (▦) or without (▪).
- FIGURE 5.
BXL-219 treatment down-regulates islet chemokine production in vivo, reduces T cell recruitment to the pancreas, and delays T1D development. Eight-week-old NOD.SCID mice were treated with vehicle (▪) or with BXL-219 (▦). Bars represent mean ±SE from individual mice. ∗, p < 0.05 and ∗∗∗, p < 0.001 by one-tailed Student’s t test. a, Constitutive CXCL10 mRNA expression (expressed as arbitrary units, A.U.) and protein production by islets purified from vehicle (n = 8) or BXL-219-treated (n = 7) NOD.SCID mice. b, Constitutive CCL2 and CCL5 secretion by islets purified from vehicle (n = 7) or BXL-219-treated (n = 8) NOD.SCID mice. c, CXCL10 secretion by islets purified from vehicle (n = 7) or BXL-219-treated (n = 8) NOD.SCID mice, unstimulated or stimulated in vitro with a mixture of proinflammatory cytokines (IFN-γ, TNF-α, IL-1β) or with LPS. d, At the end of the treatment, NOD.SCID mice were injected i.v. with 107 positively selected CD4+ and CD8+ T splenocytes from early-diabetic NOD mice. No further treatment was given to spleen cell recipients. Fourteen days after cell transfer, pancreatic cells isolated from vehicle (n = 5) and BXL-219-treated (n = 5) NOD.SCID mice were stained with anti-CD45 mAb and analyzed by cytofluorimetry. e, Recovery of CD45+ cells from the pancreas of a representative NOD.SCID mouse not receiving cell transfer (dotted line) or transferred with 107 positively selected CD4+ and CD8+ T splenocytes from early-diabetic NOD mice following treatment of the recipient with vehicle (broken line) or BXL-219 (solid line). f, Cumulative T1D incidence in vehicle (n = 10) and BXL-219-treated (n = 9) NOD.SCID mice transferred with 107 positively selected CD4+ and CD8+ T splenocytes from early-diabetic NOD mice.
- FIGURE 6.
BXL-219 enhances IκBα transcript expression in islet cells and traps NF-κBp65 into the cytoplasm. a, Islets from NOD.SCID mice were cultured for 18 h with (▦) or without (▪) 100 nM BXL-219. mRNA levels were detected by real-time RT-PCR using TaqMan chemistry. b, After an 18-h culture, islets were incubated with 1 μg/ml LPS for 45 min, fixed, and stained with anti-NF-κBp65 (p65) Ab and propidium iodide (PI). Individual and merged stainings are shown. Original magnifications, ×63; insets, ×1000.
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