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Immuno-regulatory function of indoleamine 2,3 dioxygenase through modulation of innate immune responses.

Poormasjedi-Meibod MS, Jalili RB, Hosseini-Tabatabaei A, Hartwell R, Ghahary A - PLoS ONE (2013)

Bottom Line: Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction.Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression.Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Plastic Surgery, Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO-expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11 ± 1.47 vs. 70.5 ± 7.57 cells/HPF), T-cells (8.75 ± 1.03 vs. 75.75 ± 5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.

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Specific activation of GCN2 kinase pathway mediates selective suppressive effects of tryptophan deficiency on Raw264.7cells.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T) or tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) for 24 hours. CHOP expression was analyzed by Western blotting. β-actin was used as the protein loading control. Jurkat cells and dermal fibroblasts were cultured in the same conditions. Results are representative of five independent experiments. (B) CHOP/β-actin expression ratio was calculated in Raw264.7 cells. Data is mean±SEM of five independent experiments (*P-value<0.05 and **P-value<0.01, n = 5).
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pone-0071044-g004: Specific activation of GCN2 kinase pathway mediates selective suppressive effects of tryptophan deficiency on Raw264.7cells.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T) or tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) for 24 hours. CHOP expression was analyzed by Western blotting. β-actin was used as the protein loading control. Jurkat cells and dermal fibroblasts were cultured in the same conditions. Results are representative of five independent experiments. (B) CHOP/β-actin expression ratio was calculated in Raw264.7 cells. Data is mean±SEM of five independent experiments (*P-value<0.05 and **P-value<0.01, n = 5).

Mentions: It was previously shown [23] that the selective sensitivity of immune cells (T cells) to the IDO-induced low-tryptophan, high-kynurenine environment compared to fibroblasts was governed by their difference in GCN2 kinase pathway activation. GCN2 kinase pathway activation in response to tryptophan starvation results in proliferative arrest, anergy induction and apoptosis in T cells [22], [31]. In order to determine the effect of tryptophan deficiency on GCN2 kinase pathway activation in Raw264.7 cells, the expression of CHOP protein, a well-accepted marker for GCN2 activation, was evaluated in cells cultured in RPMI, tryptophan-deficient medium or tryptophan deficient medium supplemented with excessive amounts of tryptophan. As shown in figure 4A, left panel, CHOP expression was increased in response to tryptophan deficiency (lane T) in Raw264.7 cells relative to cells cultured in RPMI (lane R). It was also shown that CHOP expression was inhibited by addition of tryptophan to the Trp-D medium (lane T+T). As it was shown previously by our group [22], [23], tryptophan starvation (lane T) induced a marked increase in CHOP expression in Jurkat cells compared with cells cultured in RPMI (Fig. 4A, middle panel). This increase in CHOP expression by Jurkat cells was suppressed by the addition of excessive amounts of tryptophan to the Trp-D medium (Fig. 4A, middle panel, lane T+T). Dermal fibroblast did not express detectable levels of CHOP in response to tryptophan starvation (lane T), indicating the selective activation of GCN2 kinase pathway in immune cells (Fig. 4A, right panel). For quantitative analysis, the intensity of signals was determined by densitometry, and the mean±SEM of the CHOP expression/β-actin was plotted for Raw264.7 cells cultured in different media. A significant increase in CHOP expression was observed in response to tryptophan starvation (Trp-D), relative to those cells cultured in either RPMI (Fig. 4B, **P-value<0.01, n = 5) or tryptophan-deficient medium supplemented with tryptophan (Trp-D+Trp, Fig. 4B, *P-value<0.05, n = 5).


Immuno-regulatory function of indoleamine 2,3 dioxygenase through modulation of innate immune responses.

Poormasjedi-Meibod MS, Jalili RB, Hosseini-Tabatabaei A, Hartwell R, Ghahary A - PLoS ONE (2013)

Specific activation of GCN2 kinase pathway mediates selective suppressive effects of tryptophan deficiency on Raw264.7cells.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T) or tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) for 24 hours. CHOP expression was analyzed by Western blotting. β-actin was used as the protein loading control. Jurkat cells and dermal fibroblasts were cultured in the same conditions. Results are representative of five independent experiments. (B) CHOP/β-actin expression ratio was calculated in Raw264.7 cells. Data is mean±SEM of five independent experiments (*P-value<0.05 and **P-value<0.01, n = 5).
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pone-0071044-g004: Specific activation of GCN2 kinase pathway mediates selective suppressive effects of tryptophan deficiency on Raw264.7cells.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T) or tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) for 24 hours. CHOP expression was analyzed by Western blotting. β-actin was used as the protein loading control. Jurkat cells and dermal fibroblasts were cultured in the same conditions. Results are representative of five independent experiments. (B) CHOP/β-actin expression ratio was calculated in Raw264.7 cells. Data is mean±SEM of five independent experiments (*P-value<0.05 and **P-value<0.01, n = 5).
Mentions: It was previously shown [23] that the selective sensitivity of immune cells (T cells) to the IDO-induced low-tryptophan, high-kynurenine environment compared to fibroblasts was governed by their difference in GCN2 kinase pathway activation. GCN2 kinase pathway activation in response to tryptophan starvation results in proliferative arrest, anergy induction and apoptosis in T cells [22], [31]. In order to determine the effect of tryptophan deficiency on GCN2 kinase pathway activation in Raw264.7 cells, the expression of CHOP protein, a well-accepted marker for GCN2 activation, was evaluated in cells cultured in RPMI, tryptophan-deficient medium or tryptophan deficient medium supplemented with excessive amounts of tryptophan. As shown in figure 4A, left panel, CHOP expression was increased in response to tryptophan deficiency (lane T) in Raw264.7 cells relative to cells cultured in RPMI (lane R). It was also shown that CHOP expression was inhibited by addition of tryptophan to the Trp-D medium (lane T+T). As it was shown previously by our group [22], [23], tryptophan starvation (lane T) induced a marked increase in CHOP expression in Jurkat cells compared with cells cultured in RPMI (Fig. 4A, middle panel). This increase in CHOP expression by Jurkat cells was suppressed by the addition of excessive amounts of tryptophan to the Trp-D medium (Fig. 4A, middle panel, lane T+T). Dermal fibroblast did not express detectable levels of CHOP in response to tryptophan starvation (lane T), indicating the selective activation of GCN2 kinase pathway in immune cells (Fig. 4A, right panel). For quantitative analysis, the intensity of signals was determined by densitometry, and the mean±SEM of the CHOP expression/β-actin was plotted for Raw264.7 cells cultured in different media. A significant increase in CHOP expression was observed in response to tryptophan starvation (Trp-D), relative to those cells cultured in either RPMI (Fig. 4B, **P-value<0.01, n = 5) or tryptophan-deficient medium supplemented with tryptophan (Trp-D+Trp, Fig. 4B, *P-value<0.05, n = 5).

Bottom Line: Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction.Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression.Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity.

View Article: PubMed Central - PubMed

Affiliation: Division of Plastic Surgery, Department of Surgery, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO-expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11 ± 1.47 vs. 70.5 ± 7.57 cells/HPF), T-cells (8.75 ± 1.03 vs. 75.75 ± 5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.

Show MeSH
Related in: MedlinePlus