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Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

Kim SB, Choi JY, Uyangaa E, Patil AM, Hossain FM, Hur J, Park SY, Lee JH, Kim K, Eo SK - J Neuroinflammation (2016)

Bottom Line: Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function.Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells.Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.

ABSTRACT

Background: Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis.

Methods: Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed.

Results: Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c(+) dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c(+) DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment.

Conclusions: Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

No MeSH data available.


Related in: MedlinePlus

IDO expression is correlated with clinical signs of JE. a IDO expression at the early stage of JE. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues, including the spleen (Spl), mesenteric LN (MLN), bone marrow (BM), brain (Br), spinal cord (SC), and liver (Liv), 1, 2, and 3 days following JEV infection. b Comparison of IDO expression between aparalytic and paralytic hosts. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) 5 dpi. c Detection of IDO protein during JE progression. IDO protein levels in several tissues of JEV-infected mice were evaluated by Western blot using a monoclonal antibody specific for IDO. Differences in IDO protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (AP) or paralytic symptoms (P) 5 dpi. d IDO expression in primary myeloid cells, microglia, and cortical neurons after JEV infection. Bone marrow-derived DCs (BMDCs), plasmacytoid DCs (pDCs), macrophages (BMDMs), microglia, and primary cortical neurons were infected with JEV (10 MOI for BMDCs, pDCs, BMDMs, and microglia; 0.01 MOI for neurons) and IDO expression was determined by real-time qRT-PCR at the indicated time points. The levels of IDO expression were expressed as fold relative to mock-infected cells (0 h). *p < 0.05; **p < 0.01; ***p < 0.001 compared to levels in the indicated groups
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Fig1: IDO expression is correlated with clinical signs of JE. a IDO expression at the early stage of JE. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues, including the spleen (Spl), mesenteric LN (MLN), bone marrow (BM), brain (Br), spinal cord (SC), and liver (Liv), 1, 2, and 3 days following JEV infection. b Comparison of IDO expression between aparalytic and paralytic hosts. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) 5 dpi. c Detection of IDO protein during JE progression. IDO protein levels in several tissues of JEV-infected mice were evaluated by Western blot using a monoclonal antibody specific for IDO. Differences in IDO protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (AP) or paralytic symptoms (P) 5 dpi. d IDO expression in primary myeloid cells, microglia, and cortical neurons after JEV infection. Bone marrow-derived DCs (BMDCs), plasmacytoid DCs (pDCs), macrophages (BMDMs), microglia, and primary cortical neurons were infected with JEV (10 MOI for BMDCs, pDCs, BMDMs, and microglia; 0.01 MOI for neurons) and IDO expression was determined by real-time qRT-PCR at the indicated time points. The levels of IDO expression were expressed as fold relative to mock-infected cells (0 h). *p < 0.05; **p < 0.01; ***p < 0.001 compared to levels in the indicated groups

Mentions: To determine whether IDO expression changes during JE progression, several tissues obtained from JEV-infected BL/6 mice were used to evaluate IDO mRNA expression at the early stage of infection (from 0 to 3 dpi) prior to the presentation of neurological disorders. As shown in Fig. 1a, JEV infection induced no apparent changes in the expression of IDO in the examined tissues, including lymphoid (spleen, mesenteric LN, bone marrow), extraneural (liver), and CNS tissues (brain, spinal cord) prior to the onset of neurological disorders. In general, infected mice showed clinical signs starting with generalized piloerection, paresis, and rigidity, which then progressed to severe neurological signs, such as postural imbalance, ataxia, and generalized tonic-clonic seizure, by 4 to 5 dpi. Thus, we were interested in testing whether IDO expression varies between mice showing clinical signs, such as paralysis, and mice displaying no clinical signs. To this end, mice were divided into two populations showing either paralysis or no paralysis 5 dpi, the point at which approximately 30–40 % of infected mice showed neurological disorders, and the expression of IDO mRNA was evaluated in several tissues. Interestingly, enhanced induction of IDO expression was observed only in lymphoid tissues (spleen and bone marrow) and the CNS (brain and spinal cord) of mice showing paralysis compared to mice showing no paralysis (Fig. 1b). However, other extraneural tissues, including mesenteric LN and liver, showed no apparent increases in expression of IDO in paralyzed mice. IDO expression at the protein level was also confirmed by Western blotting using total lysates derived from several tissues. In agreement with the enhanced induction of IDO mRNA expression in paralyzed mice, mice showing paralysis displayed an apparent increase in the expression of IDO protein in lymphoid tissues (spleen, bone marrow) and neural tissues (brain and spinal cord) (Fig. 1c). DCs and macrophages in peripheral tissues are the primary target cells of JEV, and neuron cells support JEV replication after the virus gains access into the CNS. Also, microglia, tissue-resident macrophages in the CNS are believed to regulate neuroinflammation caused by various insults. Therefore, we evaluated IDO expression in primary myeloid-derived DCs, pDCs, macrophages, microglia, and primary cortical neuron cells after JEV infection. Conventional DCs (BMDCs), pDCs, macrophages (BMDMs), microglia, and neuron cells displayed different dynamic patterns of IDO expression, depending on the time after JEV infection (Fig. 1d). pDCs showed the most rapid expression of IDO, with levels peaking at 24 h pi, whereas BMDCs displayed a delayed expression pattern with the levels peaking at 48 h pi. IDO was also expressed in macrophages with gradual and moderate increases in levels up to 72 h pi, and microglia showed basal levels of IDO expression up to 24 h pi and increased levels thereafter. Neuron cells showed a gradual increase in IDO expression with approximately fivefold higher levels at 72 h pi compared to levels in the mock-infected group. This result indicates that the primary target cells in the periphery and CNS tissues can actively express IDO with different intrinsic patterns in response to JEV infection. Collectively, these results indicate that IDO expression in myeloid cells and neurons of the lymphoid and CNS tissues is closely associated with the clinical signs of JE. Notably, IDO expression in the neural tissues (brain and spinal cord) was likely to be coupled with neurological disorders such as paralysis.Fig. 1


Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

Kim SB, Choi JY, Uyangaa E, Patil AM, Hossain FM, Hur J, Park SY, Lee JH, Kim K, Eo SK - J Neuroinflammation (2016)

IDO expression is correlated with clinical signs of JE. a IDO expression at the early stage of JE. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues, including the spleen (Spl), mesenteric LN (MLN), bone marrow (BM), brain (Br), spinal cord (SC), and liver (Liv), 1, 2, and 3 days following JEV infection. b Comparison of IDO expression between aparalytic and paralytic hosts. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) 5 dpi. c Detection of IDO protein during JE progression. IDO protein levels in several tissues of JEV-infected mice were evaluated by Western blot using a monoclonal antibody specific for IDO. Differences in IDO protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (AP) or paralytic symptoms (P) 5 dpi. d IDO expression in primary myeloid cells, microglia, and cortical neurons after JEV infection. Bone marrow-derived DCs (BMDCs), plasmacytoid DCs (pDCs), macrophages (BMDMs), microglia, and primary cortical neurons were infected with JEV (10 MOI for BMDCs, pDCs, BMDMs, and microglia; 0.01 MOI for neurons) and IDO expression was determined by real-time qRT-PCR at the indicated time points. The levels of IDO expression were expressed as fold relative to mock-infected cells (0 h). *p < 0.05; **p < 0.01; ***p < 0.001 compared to levels in the indicated groups
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Related In: Results  -  Collection

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Fig1: IDO expression is correlated with clinical signs of JE. a IDO expression at the early stage of JE. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues, including the spleen (Spl), mesenteric LN (MLN), bone marrow (BM), brain (Br), spinal cord (SC), and liver (Liv), 1, 2, and 3 days following JEV infection. b Comparison of IDO expression between aparalytic and paralytic hosts. IDO expression was determined by real-time qRT-PCR using total RNA extracted from several tissues in mice showing a neurological disorder such as paralysis (paralytic) and mice showing no paralytic symptoms (aparalytic) 5 dpi. c Detection of IDO protein during JE progression. IDO protein levels in several tissues of JEV-infected mice were evaluated by Western blot using a monoclonal antibody specific for IDO. Differences in IDO protein levels between aparalytic and paralytic hosts were evaluated after mice were divided into groups showing either aparalytic (AP) or paralytic symptoms (P) 5 dpi. d IDO expression in primary myeloid cells, microglia, and cortical neurons after JEV infection. Bone marrow-derived DCs (BMDCs), plasmacytoid DCs (pDCs), macrophages (BMDMs), microglia, and primary cortical neurons were infected with JEV (10 MOI for BMDCs, pDCs, BMDMs, and microglia; 0.01 MOI for neurons) and IDO expression was determined by real-time qRT-PCR at the indicated time points. The levels of IDO expression were expressed as fold relative to mock-infected cells (0 h). *p < 0.05; **p < 0.01; ***p < 0.001 compared to levels in the indicated groups
Mentions: To determine whether IDO expression changes during JE progression, several tissues obtained from JEV-infected BL/6 mice were used to evaluate IDO mRNA expression at the early stage of infection (from 0 to 3 dpi) prior to the presentation of neurological disorders. As shown in Fig. 1a, JEV infection induced no apparent changes in the expression of IDO in the examined tissues, including lymphoid (spleen, mesenteric LN, bone marrow), extraneural (liver), and CNS tissues (brain, spinal cord) prior to the onset of neurological disorders. In general, infected mice showed clinical signs starting with generalized piloerection, paresis, and rigidity, which then progressed to severe neurological signs, such as postural imbalance, ataxia, and generalized tonic-clonic seizure, by 4 to 5 dpi. Thus, we were interested in testing whether IDO expression varies between mice showing clinical signs, such as paralysis, and mice displaying no clinical signs. To this end, mice were divided into two populations showing either paralysis or no paralysis 5 dpi, the point at which approximately 30–40 % of infected mice showed neurological disorders, and the expression of IDO mRNA was evaluated in several tissues. Interestingly, enhanced induction of IDO expression was observed only in lymphoid tissues (spleen and bone marrow) and the CNS (brain and spinal cord) of mice showing paralysis compared to mice showing no paralysis (Fig. 1b). However, other extraneural tissues, including mesenteric LN and liver, showed no apparent increases in expression of IDO in paralyzed mice. IDO expression at the protein level was also confirmed by Western blotting using total lysates derived from several tissues. In agreement with the enhanced induction of IDO mRNA expression in paralyzed mice, mice showing paralysis displayed an apparent increase in the expression of IDO protein in lymphoid tissues (spleen, bone marrow) and neural tissues (brain and spinal cord) (Fig. 1c). DCs and macrophages in peripheral tissues are the primary target cells of JEV, and neuron cells support JEV replication after the virus gains access into the CNS. Also, microglia, tissue-resident macrophages in the CNS are believed to regulate neuroinflammation caused by various insults. Therefore, we evaluated IDO expression in primary myeloid-derived DCs, pDCs, macrophages, microglia, and primary cortical neuron cells after JEV infection. Conventional DCs (BMDCs), pDCs, macrophages (BMDMs), microglia, and neuron cells displayed different dynamic patterns of IDO expression, depending on the time after JEV infection (Fig. 1d). pDCs showed the most rapid expression of IDO, with levels peaking at 24 h pi, whereas BMDCs displayed a delayed expression pattern with the levels peaking at 48 h pi. IDO was also expressed in macrophages with gradual and moderate increases in levels up to 72 h pi, and microglia showed basal levels of IDO expression up to 24 h pi and increased levels thereafter. Neuron cells showed a gradual increase in IDO expression with approximately fivefold higher levels at 72 h pi compared to levels in the mock-infected group. This result indicates that the primary target cells in the periphery and CNS tissues can actively express IDO with different intrinsic patterns in response to JEV infection. Collectively, these results indicate that IDO expression in myeloid cells and neurons of the lymphoid and CNS tissues is closely associated with the clinical signs of JE. Notably, IDO expression in the neural tissues (brain and spinal cord) was likely to be coupled with neurological disorders such as paralysis.Fig. 1

Bottom Line: Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function.Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells.Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

View Article: PubMed Central - PubMed

Affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.

ABSTRACT

Background: Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis.

Methods: Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed.

Results: Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c(+) dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c(+) DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment.

Conclusions: Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

No MeSH data available.


Related in: MedlinePlus