Mechanism Underlying Defective Interferon Gamma-Induced IDO Expression in Non-obese Diabetic Mouse Fibroblasts

Indoleamine 2,3-dioxygenase (IDO) can locally suppress T cell-mediated immune responses. It has been shown that defective self-tolerance in early prediabetic female non-obese diabetic (NOD) mice can be attributed to the impaired interferon-gamma (IFN-γ)- induced IDO expression in dendritic cells of these animals. As IFN-γ can induce IDO in both dendritic cells and fibroblasts, we asked the question of whether there exists a similar defect in IFN-γ-induced IDO expression in NOD mice dermal fibroblasts. To this end, we examined the effect of IFN-γ on expression of IDO and its enzymatic activity in NOD dermal fibroblasts. The results showed that fibroblasts from either prediabetic (8 wks of age) female or male, and diabetic female or male (12 and 24 wks of age respectively) NOD mice failed to express IDO in response to IFN-γ treatment. To find underlying mechanisms, we scrutinized the IFN- γ signaling pathway and investigated expression of other IFN-γ-modulated factors including major histocompatibility complex class I (MHC-I) and type I collagen (COL-I). The findings revealed a defect of signal transducer and activator of transcription 1 (STAT1) phosphorylation in NOD cells relative to that of controls. Furthermore, we found an increase in MHC-I and suppression of COL-I expression in fibroblasts from both NOD and control mice following IFN-γ treatment; indicating that the impaired response to IFN-γ in NOD fibroblasts is specific to IDO gene. Finally, we showed that an IFN-γ-independent IDO expression pathway i.e. lipopolysaccharide (LPS)-mediated-c-Jun kinase is operative in NOD mice fibroblast. In conclusion, the findings of this study for the first time indicate that IFN-γ fails to induce IDO expression in NOD dermal fibroblasts; this may partially be due to defective STAT1 phosphorylation in IFN-γ-induced-IDO signaling pathway

Highly Efficient Stable Expression of Indoleamine 2,3 Dioxygenase Gene in Primary Fibroblasts

Indoleamine 2,3 dioxygenase (IDO) is a potent immunomodulatory enzyme that has recently attracted significant attention for its potential application as an inducer of immunotolerance in transplantation. We have previously demonstrated that a collagen matrix populated with IDO-expressing fibroblasts can be applied successfully in suppressing islet allogeneic immune response. Meanwhile, a critical aspect of such immunological intervention relies largely on effective long-term expression of the IDO gene. Moreover, gene manipulation of primary cells is known to be challenging due to unsatisfactory expression of the exogenous gene. In this study, a lentiviral gene delivery system has been employed to transduce primary fibroblasts. We used polybrene to efficiently deliver the IDO gene into primary fibroblasts and showed a significant increase (about tenfold) in the rate of gene transfection. In addition, by the use of fluorescence-activated cell sorting, a 95% pure population of IDO-expressing fibroblasts was successfully obtained. The efficiency of the IDO expression and the activity of the enzyme have been confirmed by Western blotting, fluorescence-activated cell sorting analysis, and Kynurenine assay, respectively. The findings of this study revealed simple and effective strategies through which an efficient and stable expression of IDO can be achieved for primary cells which, in turn, significantly improves its potential as a tool for achieving immunotolerance in different types of transplantation.Medicine, Faculty ofSurgery, Department ofNon UBCReviewedFacult

Different effect of IFN-γ on IDO expression in dermal fibroblasts of C57BL/6 prediabetic NOD mice.

<p>Dermal fibroblasts from prediabetic (8 weeks of age) male and female NOD mice failed to respond to IFN-γ induced IDO. Dermal fibroblasts isolated from C57BL/6 male mice of 8 weeks of age as control (solid bars), and aged matched male (hatched bars) or female (open bars) prediabetic NOD mice were treated with 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: Kyn levels in CM of treated cells, <b>B</b>: IDO expression at the protein level, <b>C</b>: the Mean±SEM ratio of densities of IDO to β-actin at protein control group treated with IFN-γ (n = 3, p<0.01). β-actin expression showed equal loading of proteins. ND: not detected.</p

COL-I expression in dermal fibroblasts from control and NOD mice.

<p>COL-I expression in dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice was evaluated by western blot and RT-PCR analyses. Cells were exposed to 0 or 1000 U/ml of IFN-γ for 48 hours before analysis. <b>A</b>: COL-1 expression at the protein level. <b>C</b>: COL-1 expression at mRNA level. <b>B</b> and <b>D</b> represent the Mean±SEM ratio of COL-1 to β-actin at protein and mRNA levels respectively. β-actin was used as loading control in both western blotting and RT-PCR assays. *demonstrates significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of COL-1 expression. **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.05).</p

IDO protein and mRNA expression in Ad-IDO transfected cells.

<p>Dermal fibroblasts from C57BL/6 (solid bars) and NOD (open bars) mice were transduced with Ad-IDO or mock vector. <b>A</b>: IDO expression was analyzed by western blotting, <b>C</b>: IDO expression was analyzed by RT-PCR. <b>B</b> and <b>D</b>: the Mean±SEM ratio of IDO to β-actin at the protein and GAPDH at mRNA level (n = 3). β-actin and GAPDH were used as a loading control for protein and mRNA expression respectively. ND: not detected.</p

MHC-I mRNA expression in fibroblasts isolated from control and NOD mice.

<p>Cells were treated with 0 or 1000 U/ml of IFN-γ for 48 hours. <b>A</b>: RT-PCR analysis of MHC-I mRNA expression. <b>B</b>: the Mean±SEM ratio of densities of MHC-I to GAPDH. Solid and open bars represent C57BL/6 and NOD fibroblasts respectively. GAPDH was used as loading control. *denotes significant difference between C57BL/6 and NOD fibroblasts treated with IFN-γ in terms of MHC-I expression (n = 3, p<0.05). **corresponds to significant difference between cells from the same strain treated with 0 or 1000 U/ml of IFN-γ (n = 3, p<0.01).</p

IFN-γ-induced-STAT1 phosphorylation in C57BL/6 and NOD dermal fibroblasts.

<p>Following starvation for 18 hours, dermal fibroblasts from NOD (open bars) and C57BL/6 (solid bars) mice were remained untreated or treated with 1000 U IFN-γ per ml of DMEM plus 2% FBS for 15, 30 or 60 minutes. Cell lysates were collected for western blot analysis. <b>A</b>: STAT 1 phosphorylation shown by western blotting. <b>B</b>: the Mean±SEM ratio of phospho-STAT1 (P-STAT1), to the ratio of β-actin to total STAT1. Total STAT1 and β-actin expressions were used as loading controls. *denotes significant difference between related bars (p<0.05, n = 3). UT: untreated, ND: not detected.</p