Fig. 4.

TLR9-deficiency promotes beta cell development. (a) Photographs were taken of pancreatic sections after staining with haematoxylin alone to visualise the islets better. Islet area was evaluated by ImageJ software. More islets were present in the pancreases of Tlr9^−/− NOD mice (black diamonds) compared with Tlr9^+/+ NOD mice (black circles) (p < 0.001). (b) Representative pancreas sections after staining with H&E are shown. Scale bar, 100 μm. (c) Beta cells from pancreatic islets of 4-week-old female Tlr9^+/+ NOD and Tlr9^−/− NOD mice (n = 4 mice for both groups) were harvested after treatment with Cell Dissociation Solution (Sigma). After staining with anti-CD45 and FluoZin-AM, beta cells (CD45⁻FluoZin⁺ cells) were enumerated by flow cytometry. Beta cell number was increased in the pancreas of Tlr9^−/− NOD mice when compared with Tlr9^+/+ NOD mice. (d, e) Total cellular RNA was extracted from islets of ~4-week-old female Tlr9^+/+ NOD and Tlr9^−/− NOD mice and qPCR was conducted with specific primers for transcription factors for beta cell development, Pdx-1 (d) and Ngn3 (e). The relative expression level of mRNA was determined by normalisation with the housekeeping gene, Gapdh. The expression of both Pdx-1 and Ngn3 was increased in pancreatic islets of Tlr9^−/− NOD mice when compared with Tlr9^+/+ NOD mice (p = 0.01). The experiments shown in (c–e) were performed twice, with results from one of the experiments being shown. Data in (c–e) are expressed as means (SD). Data were analysed in (a) by two-way ANOVA and in (c–e) by two-tailed unpaired Student’s t test. *p < 0.05, ***p < 0.001