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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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Apoptosis induction in Raw264.7 cells in response to tryptophan deficiency.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) in the presence or absence of 50 µg/ml kynurenine (K) for 24 hours. Presence of PARP, cleaved PARP (cl. PARP), Pro-caspase-3 and cleaved caspase-3 (cl. caspase-3) were 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 four independent experiments. (B) The ratio of cleaved-caspase-3/β-actin expression in Raw264.7 cells. (C) The ratio of cleaved PARP/β-actin expression in Raw264.7 cells. Data is mean±SEM of four independent experiments (*P-value<0.05 and **P-value<0.01, n = 4).
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pone-0071044-g003: Apoptosis induction in Raw264.7 cells in response to tryptophan deficiency.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) in the presence or absence of 50 µg/ml kynurenine (K) for 24 hours. Presence of PARP, cleaved PARP (cl. PARP), Pro-caspase-3 and cleaved caspase-3 (cl. caspase-3) were 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 four independent experiments. (B) The ratio of cleaved-caspase-3/β-actin expression in Raw264.7 cells. (C) The ratio of cleaved PARP/β-actin expression in Raw264.7 cells. Data is mean±SEM of four independent experiments (*P-value<0.05 and **P-value<0.01, n = 4).

Mentions: To determine the mechanisms involved in Raw264.7 cell death, the presence of cleaved caspase-3 and cleaved PARP, as two indicators for apoptosis were examined. To achieve this, caspase-3 activation and PARP cleavage were analyzed on cell lysates from cells cultured in RPMI (R), tryptophan-deficient medium (T), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) in the presence or absence of 50 µg/ml kynurenine (K). In the western blots probed with anti PARP and anti caspase-3 antibodies, the upper bands are the full length PARP protein (116 KD) and pro-caspase-3 (35 KD), respectively. The lower bands are the cleaved PARP (cl. PARP, 89 KD), signature fragment produced by caspase cleavage during apoptosis, and cleaved-caspase-3 (cl. caspase-3, 17 KD). As shown in figure 3A, while no PARP or caspase-3 cleavage is detectable in Raw264.7 cell cultured in RPMI (lane R), a marked increase in the levels of cleaved-PARP (cl. PARP) and cleaved-caspase-3 (cl. caspase-3) was observed in Raw264.7 cell cultured in tryptophan-deficient medium (lane T) or tryptophan-deficient medium supplemented with kynurenine (lane T+K) relative to the cells cultured in RPMI (lane R). It was also shown that cleavage of pro-caspase-3 and PARP was inhibited by addition of excessive amounts of tryptophan to the tryptophan-deficient medium (lane T+T). ‘Addition of kynurenine to RPMI or Trp-D+Trp medium did not have any noticeable effect on the detectable levels of cleaved caspase-3 and cleaved PARP (lane R+K and lane T+T+K, respectively) compared to cells cultured in RPMI (lane R). Jurkat cells and dermal fibroblasts were cultured in the same conditions as positive and negative controls for apoptosis induction in response to tryptophan deficiency and kynurenine accumulation, respectively. As it was shown previously by our group [22], [23], tryptophan deficiency induced a noticeable increase in PARP and pro-caspase-3 cleavage in Jurkat cells. This apoptotic response was inhibited by addition of excessive amounts of tryptophan to the Trp-D medium (Fig. 3A, middle panel). Dermal fibroblasts did not demonstrate detectable levels of apoptotic indicators, activated caspase-3 and cleaved PARP, in response to tryptophan starvation or high levels of kynurenine (Fig. 3A, right panel). For control of protein loading and quantitative analysis, all corresponding blots for Raw264.7 cells were re-probed for beta-actin (β-actin). The signals were then quantified by densitometry and the ratio of cleaved-caspase-3/β-actin or cleaved-PARP/β-actin was determined as shown in figure 3B and figure 3C, respectively. The finding clearly indicates a significant increase in the cleaved caspase-3 (Fig. 3B, ** P-value<0.01, n = 4) and cleaved PARP (Fig. 3C, * P-value<0.05, ** P-value<0.01, n = 4) level in Raw264.7 cells cultured in tryptophan-deficient medium, in the presence or absence of kynurenine, relative to cells cultured in RPMI. It was also shown that addition of excessive amounts of tryptophan (50 µg/ml) to the Trp-D medium reduced the detected levels of cleaved caspase-3 (Fig. 3B) and cleaved PARP (Fig. 3C) to the levels comparable to the cells cultured in RPMI. The addition of high levels of kynurenine (50 µg/ml) to RPMI or Trp-D+Trp medium did not increase the cleaved caspase-3 (Fig. 3B) or cleaved PARP (Fig. 3C) level in Raw264.7 cells.


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)

Apoptosis induction in Raw264.7 cells in response to tryptophan deficiency.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) in the presence or absence of 50 µg/ml kynurenine (K) for 24 hours. Presence of PARP, cleaved PARP (cl. PARP), Pro-caspase-3 and cleaved caspase-3 (cl. caspase-3) were 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 four independent experiments. (B) The ratio of cleaved-caspase-3/β-actin expression in Raw264.7 cells. (C) The ratio of cleaved PARP/β-actin expression in Raw264.7 cells. Data is mean±SEM of four independent experiments (*P-value<0.05 and **P-value<0.01, n = 4).
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Related In: Results  -  Collection

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pone-0071044-g003: Apoptosis induction in Raw264.7 cells in response to tryptophan deficiency.(A) Raw264.7 cells were cultured in RPMI (R), tryptophan-deficient medium (T), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) in the presence or absence of 50 µg/ml kynurenine (K) for 24 hours. Presence of PARP, cleaved PARP (cl. PARP), Pro-caspase-3 and cleaved caspase-3 (cl. caspase-3) were 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 four independent experiments. (B) The ratio of cleaved-caspase-3/β-actin expression in Raw264.7 cells. (C) The ratio of cleaved PARP/β-actin expression in Raw264.7 cells. Data is mean±SEM of four independent experiments (*P-value<0.05 and **P-value<0.01, n = 4).
Mentions: To determine the mechanisms involved in Raw264.7 cell death, the presence of cleaved caspase-3 and cleaved PARP, as two indicators for apoptosis were examined. To achieve this, caspase-3 activation and PARP cleavage were analyzed on cell lysates from cells cultured in RPMI (R), tryptophan-deficient medium (T), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan (T+T) in the presence or absence of 50 µg/ml kynurenine (K). In the western blots probed with anti PARP and anti caspase-3 antibodies, the upper bands are the full length PARP protein (116 KD) and pro-caspase-3 (35 KD), respectively. The lower bands are the cleaved PARP (cl. PARP, 89 KD), signature fragment produced by caspase cleavage during apoptosis, and cleaved-caspase-3 (cl. caspase-3, 17 KD). As shown in figure 3A, while no PARP or caspase-3 cleavage is detectable in Raw264.7 cell cultured in RPMI (lane R), a marked increase in the levels of cleaved-PARP (cl. PARP) and cleaved-caspase-3 (cl. caspase-3) was observed in Raw264.7 cell cultured in tryptophan-deficient medium (lane T) or tryptophan-deficient medium supplemented with kynurenine (lane T+K) relative to the cells cultured in RPMI (lane R). It was also shown that cleavage of pro-caspase-3 and PARP was inhibited by addition of excessive amounts of tryptophan to the tryptophan-deficient medium (lane T+T). ‘Addition of kynurenine to RPMI or Trp-D+Trp medium did not have any noticeable effect on the detectable levels of cleaved caspase-3 and cleaved PARP (lane R+K and lane T+T+K, respectively) compared to cells cultured in RPMI (lane R). Jurkat cells and dermal fibroblasts were cultured in the same conditions as positive and negative controls for apoptosis induction in response to tryptophan deficiency and kynurenine accumulation, respectively. As it was shown previously by our group [22], [23], tryptophan deficiency induced a noticeable increase in PARP and pro-caspase-3 cleavage in Jurkat cells. This apoptotic response was inhibited by addition of excessive amounts of tryptophan to the Trp-D medium (Fig. 3A, middle panel). Dermal fibroblasts did not demonstrate detectable levels of apoptotic indicators, activated caspase-3 and cleaved PARP, in response to tryptophan starvation or high levels of kynurenine (Fig. 3A, right panel). For control of protein loading and quantitative analysis, all corresponding blots for Raw264.7 cells were re-probed for beta-actin (β-actin). The signals were then quantified by densitometry and the ratio of cleaved-caspase-3/β-actin or cleaved-PARP/β-actin was determined as shown in figure 3B and figure 3C, respectively. The finding clearly indicates a significant increase in the cleaved caspase-3 (Fig. 3B, ** P-value<0.01, n = 4) and cleaved PARP (Fig. 3C, * P-value<0.05, ** P-value<0.01, n = 4) level in Raw264.7 cells cultured in tryptophan-deficient medium, in the presence or absence of kynurenine, relative to cells cultured in RPMI. It was also shown that addition of excessive amounts of tryptophan (50 µg/ml) to the Trp-D medium reduced the detected levels of cleaved caspase-3 (Fig. 3B) and cleaved PARP (Fig. 3C) to the levels comparable to the cells cultured in RPMI. The addition of high levels of kynurenine (50 µg/ml) to RPMI or Trp-D+Trp medium did not increase the cleaved caspase-3 (Fig. 3B) or cleaved PARP (Fig. 3C) level in Raw264.7 cells.

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