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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 ablation results in early and increased CNS infiltration by myeloid and lymphoid leukocytes. a, b The frequency and number of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS. The frequency (a) and total number (b) of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS were determined by flow cytometric analyses 3 and 5 dpi using vigorous heart perfusion. Values presented in the representative dot plots denote the average percentage of the indicated population after gating on CD11b+ cells (n = 4–5). c, d Accumulated number of NK cells, CD4+, and CD8+ T-cells in the CNS. Total accumulated number of NK cells (CD3−NK1.1+DX5+), CD4+ (CD3+CD4+), and CD8+ (CD3+CD8+) T-cells in the CNS were enumerated by flow cytometric analysis 3 and 5 dpi. e The expression of TNF-α and CC chemokines in the CNS. The expression levels of TNF-α and CC chemokines were determined by real-time qRT-PCR using total RNA extracted from brain tissue 2 dpi. Data show the average ± SD of the indicated cell populations derived from at least five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups
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Fig3: IDO ablation results in early and increased CNS infiltration by myeloid and lymphoid leukocytes. a, b The frequency and number of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS. The frequency (a) and total number (b) of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS were determined by flow cytometric analyses 3 and 5 dpi using vigorous heart perfusion. Values presented in the representative dot plots denote the average percentage of the indicated population after gating on CD11b+ cells (n = 4–5). c, d Accumulated number of NK cells, CD4+, and CD8+ T-cells in the CNS. Total accumulated number of NK cells (CD3−NK1.1+DX5+), CD4+ (CD3+CD4+), and CD8+ (CD3+CD8+) T-cells in the CNS were enumerated by flow cytometric analysis 3 and 5 dpi. e The expression of TNF-α and CC chemokines in the CNS. The expression levels of TNF-α and CC chemokines were determined by real-time qRT-PCR using total RNA extracted from brain tissue 2 dpi. Data show the average ± SD of the indicated cell populations derived from at least five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups

Mentions: CNS infiltration by CD11b+Ly-6Chi monocytes is a hallmark of CNS inflammation caused by neurotropic viral infection [29]. These cells migrate into the infected brain, where they differentiate into DC, macrophage, and microglia populations [30–33]. Although the potential contribution of CD11b+Ly-6Chi monocytes to neuroinflammation remains controversial, CNS infiltration by CD11b+Ly-6Chi monocytes is believed to support their protective role during lethal neuroinflammation [34–37]. Therefore, in order to better understand the amelioration of JE in IDO KO mice, we examined CNS infiltration by leukocytes, including CD11b+Ly-6Chi monocytes, during JE progression. Wild-type BL/6 and IDO KO mice contained comparable levels of CD11b+Ly-6Chi monocytes and CD11b+Ly-6Ghi granulocytes in the brain before JEV infection. However, the frequency of CD11b+Ly-6Chi monocytes gradually increased in the CNS of IDO KO mice during JE progression (Fig. 3a). Also, CNS infiltration by CD11b+Ly-6Ghi granulocytes was transiently increased in IDO KO mice 3 dpi, after which the Ly-6Ghi granulocyte frequency was comparable in both BL/6 and IDO KO mice 5 dpi. Similarly, the accumulated total number of CNS-infiltrated Ly-6Chi monocytes and Ly-6Ghi granulocytes was increased in IDO KO mice, compared to those of BL/6 mice (Fig. 3b). Furthermore, because CD4+ Th1, CD8+, and NK cells may play beneficial roles in controlling JE progression [38–41], we examined CNS infiltration of NK, CD4+, and CD8+ T-cells. As with CNS infiltration by Ly-6Chi monocytes, CNS infiltration by NK, CD4+, and CD8+ T-cells was observed to gradually increase in IDO KO mice compared to levels observed in BL/6 mice (Fig. 3c, d). Notably, CD8+ T-cells showed marked infiltration with threefold increased levels in the CNS of IDO KO mice. With respect to CNS inflammation, the expression of cytokines and chemokines within the CNS may be required to fully explain encephalitis, because encephalitis caused by neurotropic viruses is indirectly derived from CNS degeneration caused by robust immunological responses, such as the uncontrolled secretion of cytokines, including TNF-α, and the resultant activation of microglia and astrocytes [22, 23]. Therefore, we examined the expression of TNF-α and CC chemokines in the CNS. Our results revealed that the expression of CC chemokines was comparable in both BL/6 and IDO KO mice, except that the expression levels of TNF-α and CCL2 were modestly decreased in IDO KO mice (Fig. 3e). This result implies that CC chemokine expression may not play a role in increased CNS infiltration by Ly-6Chi monocytes, NK, CD4+, and CD8+ T-cells. Collectively, these results suggest that IDO ablation allows for early and increased CNS infiltration by myeloid and lymphoid leukocytes, which may mediate the early control of viral replication in the CNS.Fig. 3


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 ablation results in early and increased CNS infiltration by myeloid and lymphoid leukocytes. a, b The frequency and number of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS. The frequency (a) and total number (b) of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS were determined by flow cytometric analyses 3 and 5 dpi using vigorous heart perfusion. Values presented in the representative dot plots denote the average percentage of the indicated population after gating on CD11b+ cells (n = 4–5). c, d Accumulated number of NK cells, CD4+, and CD8+ T-cells in the CNS. Total accumulated number of NK cells (CD3−NK1.1+DX5+), CD4+ (CD3+CD4+), and CD8+ (CD3+CD8+) T-cells in the CNS were enumerated by flow cytometric analysis 3 and 5 dpi. e The expression of TNF-α and CC chemokines in the CNS. The expression levels of TNF-α and CC chemokines were determined by real-time qRT-PCR using total RNA extracted from brain tissue 2 dpi. Data show the average ± SD of the indicated cell populations derived from at least five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups
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Fig3: IDO ablation results in early and increased CNS infiltration by myeloid and lymphoid leukocytes. a, b The frequency and number of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS. The frequency (a) and total number (b) of Ly-6Chi monocytes and Ly-6Ghi granulocytes in the CNS were determined by flow cytometric analyses 3 and 5 dpi using vigorous heart perfusion. Values presented in the representative dot plots denote the average percentage of the indicated population after gating on CD11b+ cells (n = 4–5). c, d Accumulated number of NK cells, CD4+, and CD8+ T-cells in the CNS. Total accumulated number of NK cells (CD3−NK1.1+DX5+), CD4+ (CD3+CD4+), and CD8+ (CD3+CD8+) T-cells in the CNS were enumerated by flow cytometric analysis 3 and 5 dpi. e The expression of TNF-α and CC chemokines in the CNS. The expression levels of TNF-α and CC chemokines were determined by real-time qRT-PCR using total RNA extracted from brain tissue 2 dpi. Data show the average ± SD of the indicated cell populations derived from at least five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 compared with levels in the indicated groups
Mentions: CNS infiltration by CD11b+Ly-6Chi monocytes is a hallmark of CNS inflammation caused by neurotropic viral infection [29]. These cells migrate into the infected brain, where they differentiate into DC, macrophage, and microglia populations [30–33]. Although the potential contribution of CD11b+Ly-6Chi monocytes to neuroinflammation remains controversial, CNS infiltration by CD11b+Ly-6Chi monocytes is believed to support their protective role during lethal neuroinflammation [34–37]. Therefore, in order to better understand the amelioration of JE in IDO KO mice, we examined CNS infiltration by leukocytes, including CD11b+Ly-6Chi monocytes, during JE progression. Wild-type BL/6 and IDO KO mice contained comparable levels of CD11b+Ly-6Chi monocytes and CD11b+Ly-6Ghi granulocytes in the brain before JEV infection. However, the frequency of CD11b+Ly-6Chi monocytes gradually increased in the CNS of IDO KO mice during JE progression (Fig. 3a). Also, CNS infiltration by CD11b+Ly-6Ghi granulocytes was transiently increased in IDO KO mice 3 dpi, after which the Ly-6Ghi granulocyte frequency was comparable in both BL/6 and IDO KO mice 5 dpi. Similarly, the accumulated total number of CNS-infiltrated Ly-6Chi monocytes and Ly-6Ghi granulocytes was increased in IDO KO mice, compared to those of BL/6 mice (Fig. 3b). Furthermore, because CD4+ Th1, CD8+, and NK cells may play beneficial roles in controlling JE progression [38–41], we examined CNS infiltration of NK, CD4+, and CD8+ T-cells. As with CNS infiltration by Ly-6Chi monocytes, CNS infiltration by NK, CD4+, and CD8+ T-cells was observed to gradually increase in IDO KO mice compared to levels observed in BL/6 mice (Fig. 3c, d). Notably, CD8+ T-cells showed marked infiltration with threefold increased levels in the CNS of IDO KO mice. With respect to CNS inflammation, the expression of cytokines and chemokines within the CNS may be required to fully explain encephalitis, because encephalitis caused by neurotropic viruses is indirectly derived from CNS degeneration caused by robust immunological responses, such as the uncontrolled secretion of cytokines, including TNF-α, and the resultant activation of microglia and astrocytes [22, 23]. Therefore, we examined the expression of TNF-α and CC chemokines in the CNS. Our results revealed that the expression of CC chemokines was comparable in both BL/6 and IDO KO mice, except that the expression levels of TNF-α and CCL2 were modestly decreased in IDO KO mice (Fig. 3e). This result implies that CC chemokine expression may not play a role in increased CNS infiltration by Ly-6Chi monocytes, NK, CD4+, and CD8+ T-cells. Collectively, these results suggest that IDO ablation allows for early and increased CNS infiltration by myeloid and lymphoid leukocytes, which may mediate the early control of viral replication in the CNS.Fig. 3

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