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

Fibroblast cell-based therapy prevents induction of alopecia areata in an experimental model

YesAlopecia areata (AA) is an autoimmune hair loss disease with infiltration of proinflammatory cells into hair follicles. Current therapeutic regimens are unsatisfactory mainly because of the potential for side effects and/or limited efficacy. Here we report that cultured, transduced fibroblasts, which express the immunomodulatory molecule indoleamine 2,3-dioxygenase (IDO), can be applied to prevent hair loss in an experimental AA model. A single intraperitoneal (IP) injection of IDO-expressing primary dermal fibroblasts was given to C3H/HeJ mice at the time of AA induction. While 60–70% of mice that received either control fibroblasts or vehicle injections developed extensive AA, none of the IDO-expressing fibroblast-treated mice showed new hair loss up to 20 weeks post injection. IDO cell therapy significantly reduced infiltration of CD4+ and CD8+ T cells into hair follicles and resulted in decreased expression of TNF-α, IFN-γ and IL-17 in the skin. Skin draining lymph nodes of IDO fibroblast-treated mice were significantly smaller, with more CD4+ CD25+ FoxP3+ regulatory T cells and fewer Th17 cells than those of control fibroblast and vehicle-injected mice. These findings indicate that IP injected IDO-expressing dermal fibroblasts can control inflammation and thereby prevent AA hair loss.Canadian Institutes of Health Researches (Funding Reference Number: 134214 and 136945)

Fibroblast Cell-Based Therapy for Experimental Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of insulin producing β cells of the pancreatic islets. Curbing autoimmunity at the initiation of T1D can result in recovery of residual β cells and consequently remission of diabetes. Here we report a cell-based therapy for autoimmune diabetes in non-obese diabetic (NOD) mice using dermal fibroblasts. This was achieved by a single injection of fibroblasts, expressing the immunoregulatory molecule indoleamine 2,3 dioxygenase (IDO), into peritoneal cavity of NOD mice shortly after the onset of overt hyperglycemia. Mice were then monitored for reversal of hyperglycemia and changes in inflammatory/regulatory T cell profiles. Blood glucose levels dropped into the normal range in 82% of NOD mice after receiving IDO-expressing fibroblasts while all control mice remained diabetic. We found significantly reduced islet inflammation, increased regulatory T cells, and decreased T helper 17 cells and β cell specific autoreactive CD8+ T cells following IDO cell therapy. We further showed that some of intraperitoneal injected fibroblasts migrated to local lymph nodes and expressed co-inhibitory molecules. These findings suggest that IDO fibroblasts therapy can reinstate self-tolerance and alleviate β cell autoreactivity in NOD mice, resulting in remission of autoimmune diabetes

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

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

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

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

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