TLR2/MyD88 pathway-dependent regulation of dendritic cells by dengue virus promotes antibody-dependent enhancement via Th2-biased immunity.

Possible risk mediators in primary dengue virus (DenV) infection that favor secondary DenV infection to life-threatening dengue hemorrhagic fever (DHF) and shock syndrome (DSS) via antibody-dependent enhancement (ADE) have not yet been described. Here, DenV infection enhanced the expression of inflammatory mediators and activation molecules in dendritic cells (DCs) through TLR2/MyD88 pathway. TLR2 appeared to facilitate DenV infection in DCs that were less permissive than macrophages for viral replication. In experiments using separate evaluations of DenV-infected and uninfected bystander DCs, infected DCs showed impaired maturation accompanied with TLR2-dependent production of inflammatory cytokines, by which uninfected bystander DCs showed increased expression of co-stimulatory molecules. Differential phosphorylation of MAPK and STAT3 was also detected between DenV-infected and uninfected DCs. Furthermore, DenV infection stimulated Th2-polarized humoral and cellular immunity against foreign and DenV Ag via TLR2/MyD88 pathway, and DenV-infected DCs were revealed to facilitate Th2-biased immune responses in TLR2-dependent manner. TLR2/MyD88-mediated Th2-biased Ab responses to primary DenV infection increased the infectivity of secondary homotypic or heterotypic DenV via ADE. Collectively, these results indicate that TLR2/MyD88 pathway in DC-priming receptors can drive Th2-biased immune responses during primary DenV infection, which could favor secondary DenV infection to DHF/DSS via ADE.

Exacerbation of Japanese Encephalitis by CD11chi Dendritic Cell Ablation Is Associated with an Imbalance in Regulatory Foxp3+ and IL-17+CD4+ Th17 Cells and in Ly-6Chi and Ly-6Clo Monocytes.

Japanese encephalitis (JE) is neuroinflammation characterized by uncontrolled infiltration of peripheral leukocytes into the central nervous system (CNS). We previously demonstrated exacerbation of JE following CD11chi dendritic cell (DC) ablation in CD11c-DTR transgenic mice. Moreover, CD11chi DC ablation led to abnormal differentiation of CD11b+Ly-6Chi monocytes and enhanced permeability of the blood-brain barrier (BBB), resulting in promoting the progression of JE. Here, we examined changes in lymphoid and myeloid-derived leukocyte subpopulations associated with pro- and anti-inflammation during JE progression. The analyses of this study focused on regulatory CD4+Foxp3+ regulatory T cells (Tregs), IL-17+CD4+ Th17 cells, and CD11b+Ly-6Chi and Ly-6Clo monocytes. CD11chi DC ablation resulted in the accumulation of IL-17+CD4+ Th17 cells in the CNS, thereby leading to lower ratio of Tregs to Th17 cells. This result was corroborated by the higher expression levels of IL-17 and RORγT in CD4+ T cells from the brains of CD11chi DC-ablated mice. In addition, CD11chi DC-ablated mice showed higher frequency and total number of inflammatory CD11b+Ly-6Chi monocytes, whereas CD11b+Ly-6Clo monocytes were detected with lower frequency and total number in CD11chi DC-ablated mice. Furthermore, CD11chi DC ablation altered the phenotype and function of CD11b+Ly-6Clo monocytes, resulting in lower levels of activation marker and anti-inflammatory cytokine (IL-10 and TGF-ß) expression. Collectively, these results indicate that CD11chi DC ablation caused an imbalance in CD4+ Th17/Treg cells and CD11b+Ly-6Chi/Ly-6Clo monocytes in the lymphoid tissue and CNS during JE progression. This imbalanced orchestration of pro- and anti-inflammatory leukocytes following CD11chi DC ablation may contribute to the exacerbation of JE.

Ablation of CD11c(hi) dendritic cells exacerbates Japanese encephalitis by regulating blood-brain barrier permeability and altering tight junction/adhesion molecules.

Japanese encephalitis (JE), characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV), is becoming a leading cause of viral encephalitis due to rapid changes in climate and demography. The blood-brain barrier (BBB) plays an important role in restricting neuroinvasion of peripheral leukocytes and virus, thereby regulating the progression of viral encephalitis. In this study, we explored the role of CD11c(hi) dendritic cells (DCs) in regulating BBB integrity and JE progression using a conditional depletion model of CD11c(hi) DCs. Transient ablation of CD11c(hi) DCs resulted in markedly increased susceptibility to JE progression along with highly increased neuro-invasion of JEV. In addition, exacerbated JE progression in CD11c(hi) DC-ablated hosts was closely associated with increased expression of proinflammatory cytokines (IFN-ß, IL-6, and TNF-α) and CC chemokines (CCL2, CCL3, CXCL2) in the brain. Moreover, our results revealed that the exacerbation of JE progression in CD11c(hi) DC-ablated hosts was correlated with enhanced BBB permeability and reduced expression of tight junction and adhesion molecules (claudin-5, ZO-1, occluding, JAMs). Ultimately, our data conclude that the ablation of CD11c(hi) DCs provided a subsidiary impact on BBB integrity and the expression of tight junction/adhesion molecules, thereby leading to exacerbated JE progression. These findings provide insight into the secondary role of CD11c(hi) DCs in JE progression through regulation of BBB integrity and the expression of tight junction/adhesion molecules.

CCR5 ameliorates Japanese encephalitis via dictating the equilibrium of regulatory CD4(+)Foxp3(+) T and IL-17(+)CD4(+) Th17 cells.

BACKGROUND: CCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4(+)Foxp3(+) regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4(+)Foxp3(+) Treg homing has not been investigated. METHODS: Infected wild-type (Ccr5(+/+)) and CCR5-deficient (Ccr5(-/-)) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5(+)CD4(+)Foxp3(+) Tregs was used to evaluate the role of Tregs in JE progression. RESULTS: CCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. Compared to Ccr5(+/+) mice, Ccr5(-/-) mice unexpectedly showed increased responses of IFN-γ(+)NK and CD8(+) T cells in the spleen, but not CD4(+) T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17(+)CD4(+) Th17 cells and correspondingly reduced CD4(+)Foxp3(+) Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5(+)CD4(+)Foxp3(+) Tregs into Ccr5(-/-) mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17(+)CD4(+) Th17 cells, myeloid-derived Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. Instead, adoptive transfer of CCR5(+)CD4(+)Foxp3(+) Tregs into Ccr5(-/-) mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-ß) in the spleen and brain, and transferred CCR5(+) Tregs were found to produce IL-10. CONCLUSIONS: CCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4(+)Foxp3(+) Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.

CCL2, but not its receptor, is essential to restrict immune privileged central nervous system-invasion of Japanese encephalitis virus via regulating accumulation of CD11b(+) Ly-6C(hi) monocytes.

Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2-CCL2 axis plays an important role in trafficking CD11b(+) Ly-6C(hi) monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2- and CCL2-ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2-ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2- and CCL2-ablated mice was unlikely to be mediated by innate natural killer and adaptive T-cell responses. Furthermore, CCL2 produced by haematopoietic stem cell-derived leucocytes played a dominant role in CNS accumulation of Ly-6C(hi) monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.

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

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.

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade.

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6Chi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6Clo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6Chi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I-dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6Chi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6Ghi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11bhiF4/80hi macrophages and CD11chiEpCAM+ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6Chi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I-dependent pathway that establishes orchestrated mobilization of Ly-6Chi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident-to-hematopoietic-to-resident cells that drives cytokine-to-chemokine-to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.

CD11c(hi) Dendritic Cells Regulate Ly-6C(hi) Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation.

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b(+)Ly-6C(hi) monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11c(hi)PDCA-1(int/lo) DCs without alteration in CD11c(int)PDCA-1(hi) plasmacytoid DC number, we found that CD11c(hi) DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b(+)Ly-6C(hi) monocytes and higher expression of CC chemokines. More interestingly, selective CD11c(hi) DC ablation provided altered differentiation and function of infiltrated CD11b(+)Ly-6C(hi) monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b(+)Ly-6C(hi) monocytes generated in CD11c(hi) DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11c(hi) DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b(+)Ly-6C(hi) monocytes.

Amelioration of Japanese encephalitis by blockage of 4-1BB signaling is coupled to divergent enhancement of type I/II IFN responses and Ly-6C(hi) monocyte differentiation.

BACKGROUND: Japanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model. METHODS: Infected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed. RESULTS: Blocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4(+) Th1 cells as well as increased infiltration of mature Ly-6C(hi) monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host. CONCLUSIONS: Blocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4(+) Th1 cells and mature Ly-6C(hi) monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.

Functional restoration of exhausted CD4(+) and CD8(+) T cells in chronic viral infection by vinegar-processed flos of Daphne genkwa.

T-cell exhaustion has become an important issue in chronic infection because exhausted antigen-specific T cells show impaired abilities to eradicate persistently infected pathogens and produce effector cytokines, such as IFN-γ and TNF-α. Thus, strategies to either restore endogenous exhausted T cell responses or provide functional T cells are needed for therapeutics of chronic infection. Despite promising developments using antibodies and cell immunotherapy, there have been no reported attempts to restore exhausted T cells using treatment with materials derived from natural resources. Here, using a mouse model of chronic infection with lymphocytic choriomeningitis virus (LCMV), we found that vinegar-processed flowers (flos) of Daphne genkwa (vp-genkwa), which was composed mainly of four index components, restored exhausted CD4(+) and CD8(+) T cells significantly, as corroborated by evidence that vp-genkwa treatment enhanced functional LCMV-specific CD4(+) and CD8(+) T cells, both quantitatively and qualitatively. Furthermore, pretreatment with vp-genkwa prevented the generation of exhausted LCMV-specific CD8(+) T cells. Such restorations of exhausted LCMV-specific CD4(+) and CD8(+) T cells by vp-genkwa were closely associated with reduced viral burden in sera and tissues. More interestingly, vp-genkwa treatment induced down-regulation of negative molecules, such as PD-1 and Tim-3, in exhausted CD4(+) and CD8(+) T cells with more apparent down-regulation of Tim-3, suggesting that Tim-3 molecule may be a major target in restoring exhausted T cell responses. Collectively, these results provide valuable new insights into the use of vp-genkwa to develop a therapeutic strategy for chronic human diseases, such as hepatitis B and C virus, human immunodeficiency virus, and cancers.

Distinct dictation of Japanese encephalitis virus-induced neuroinflammation and lethality via triggering TLR3 and TLR4 signal pathways.

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3(-/-) and TLR4(-/-) mice, i.e. TLR3(-/-) mice were highly susceptible to JE, whereas TLR4(-/-) mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b(+)Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4(-/-) mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3(-/-) myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4(+) and CD8(+) T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b(+)Ly-6C(high) monocytes) and CD4(+)Foxp3(+) Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components during JE progression could be responsible for determining disease outcome through regulating negative and positive factors.

Enhancement of Th1-biased protective immunity against avian influenza H9N2 virus via oral co-administration of attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 along with an inactivated vaccine.

BACKGROUND: Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) as natural immunomodulators. RESULTS: Oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens. CONCLUSIONS: Our results indicate the value of combined administration of chIFN-α and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2.

Impaired cross-presentation of CD8α+ CD11c+ dendritic cells by Japanese encephalitis virus in a TLR2/MyD88 signal pathway-dependent manner.

Cross-presentation is the pathway by which exogenous antigens are routed for presentation by MHC class I molecules leading to activation of antiviral CD8(+) T-cell responses. However, there is little information describing the modulation of cross-presentation and the impact of pathogen-derived signals associated with Japanese encephalitis virus (JEV), which is one of the most common causes of encephalitis in humans. In this study, we demonstrate that JEV infection could suppress in vivo cross-presentation of soluble and cell-associated antigens, thereby generating weak CD8(+) T-cell responses to exogenous antigens, as evaluated by CFSE dilution of adoptively transferred CD8(+) T cells and in vivo CTL killing activity. Furthermore, CD8α(+) CD11c(+) dendritic cells (DCs), which are known to be far more efficient at cross-presenting soluble antigens, played a specific role in contributing to JEV-mediated inhibition of the cross-presentation of exogenous antigens through interference with effective antigen uptake. Finally, this study provides evidence that TLR2-MyD88 and p38 MAPK signal pathway might be involved in JEV-mediated inhibition of cross-presentation of soluble and cell-associated antigens. These observations suggest that the modulation of cross-presentation of exogenous antigens through TLR signaling has important implications for antiviral immune responses against JEV infection and the development of effective vaccination strategies.

Co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interleukin-18 and interferon-α provides enhanced Th1-biased protective immunity against inactivated vaccine of pseudorabies virus.

The co-administration of two or more cytokines may generate additive or synergistic effects for controlling infectious diseases. However, the practical use of cytokine combinations for the modulation of immune responses against inactivated vaccine has not been demonstrated in livestock yet, primarily due to protein stability, production, and costs associated with mass administration. In light of the current situation, we evaluated the immunomodulatory functions of the combined administration of swine interleukin-18 (swIL-18) and interferon-α (swIFN-α) against an inactivated PrV vaccine using attenuated Salmonella enterica serovar Typhimurium as a cytokine delivery system. Co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α produced enhanced Th1-biased humoral and cellular immune responses against the inactivated PrV vaccine, when compared to single administration of S. enterica serovar Typhimurium expressing either swIL-18 or swIFN-α. Also, enhanced immune responses in co-administered piglets occurred rapidly after virulent PrV challenge, and piglets that received co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α displayed a greater alleviation of clinical severity following the virulent PrV challenge, as determined by clinical scores and cumulative daily weight gain. Furthermore, this enhancement was confirmed by reduced nasal shedding of PrV following viral challenge. Therefore, these results suggest that oral co-administration of S. enterica serovar Typhimurium expressing swIL-18 and swIFN-α provide enhanced Th1-biased immunity against inactivated PrV vaccine to alleviate clinical signs caused by PrV challenge.

Oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 enhances the alleviation of clinical signs caused by respiratory infection with avian influenza virus H9N2.

The combined use of cytokines has shown synergistic and/or additive effects in controlling several viral infections of livestock animals. However, little is known concerning the practical use of chicken cytokine combinations to control avian diseases. Here, we investigated the antiviral efficacy of oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) using attenuated Salmonella enterica serovar Typhimurium in chickens infected with avian influenza virus (AIV) H9N2. Our results demonstrate that oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 produced a greater alleviation of clinical signs caused by respiratory infection with AIV H9N2 in chickens, when compared to administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18 alone. Mortality, clinical symptom severity, and feed and water intake were used to access treatment effectiveness. This enhancement of antiviral immunity was further confirmed by evidence of reduced rectal shedding and decreased replication of AIV H9N2 in several different tissues of challenged chickens including trachea, lung, cecal tonsil, and brain. Furthermore, oral co-administration of chIFN-α and chIL-18 more efficiently modulated the immune responses of chickens against AIV H9N2 by enhancing both humoral and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results suggest that the combined administration of two chicken cytokines, chIFN-α and chIL-18, using attenuated S. enterica serovar Typhimurium as an oral carrier, provides an effective means for controlling respiratory disease caused by AIV H9N2 infection.

Oral administration of Salmonella enterica serovar Typhimurium expressing swine interleukin-18 induces Th1-biased protective immunity against inactivated vaccine of pseudorabies virus.

Enhancing and/or modulating innate and adaptive immunity by cytokines appears to be greatly useful to provide effective protective immunity against infectious diseases. However, an effective delivery system for mass administration in livestock industry is needed because of limitations such as cost, labor, time, and protein stability. Here the immunomodulatory functions of swine interleukine-18 (swIL-18), known as IFN-γ-inducing factor (IGIF), were evaluated in a vaccination model of pseudorabies virus (PrV) using attenuated Salmonella enterica serovar Typhimurium as the oral delivery system. The oral administration of S. enterica serovar Typhimurium expressing swIL-18 prior to vaccination with inactivated PrV vaccine induced enhanced levels of serum PrV-specific IgG and its IgG2 isotype, compared to administration of S. enterica serovar Typhimurium harboring the empty vector. Furthermore, S. enterica serovar Typhimurium expressing swIL-18 mounted Th1-biased cellular immune responses against PrV antigen, as evaluated by the production of IFN-γ and IL-4 from peripheral blood mononuclear cells of piglets. Subsequently, Th1-biased immunity induced by S. enterica serovar Typhimurium expressing swIL-18 showed rapid response and rendered piglets displayed more alleviated clinical signs following the virulent PrV challenge. Also, this alleviation of clinical signs was further confirmed by the reduction of nasal excretion of PrV after challenge. The present study demonstrates the extended use of immunomodulatory functions of swIL-18 orally delivered by attenuated S. enterica serovar Typhimurium.

Enhanced protection against infection with transmissible gastroenteritis virus in piglets by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-α and interleukin-18.

The enhanced effect of cytokine combinations has been assessed empirically, based on their immunobiological mechanisms. However, far less is known of the enhanced protection of practical cytokine combinations against viral infection in the livestock industry, due to cost and production issues associated with mass administration. This study demonstrates the enhanced protection of oral co-administration of swine interferon-α (swIFN-α) and interleukin-18 (swIL-18) against infection with transmissible gastroenteritis virus (TGEV) in piglets using attenuated Salmonella enterica serovar Typhimurium as carrier of cytokine proteins. A single oral co-administration of S. enterica serovar Typhimurium expressing swIFN-α and swIL-18 induced enhanced alleviation of the severity of diarrhea caused by TGEV infection, compared to piglets administered S. enterica serovar Typhimurium expressing swIFN-α or swIL-18 alone. This enhancement was further observed by the reduction of TGEV shedding and replication, and the expression of IFN-stimulated gene products in the intestinal tract. The results suggest that the combined administration of the swIFN-α and swIL-18 cytokines using attenuated S. enterica serovar Typhimurium as an oral carrier provides enhanced protection against intestinal tract infection with TGEV.

Oral administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α alleviates clinical signs caused by respiratory infection with avian influenza virus H9N2.

Low pathogenic avian influenza (LPAI) H9N2 has attracted considerable attention due to severe commercial losses in the poultry industry. Furthermore, avian influenza virus (AIV) H9N2-infected chickens can be a reservoir for viral transmission to mammals including pigs and humans, complicating control of viral mutants. Chicken interferon-alpha (chIFN-α) may be useful as an exogenous antiviral agent to control AIV H9N2 infection. However, a superior vehicle for administration of chIFN-α is needed because of challenges of protein stability, production cost, and labor associated with mass administration. Presently, oral administration of single dose of attenuated Salmonella enterica serovar Typhimurium expressing chIFN-α alleviated clinical signs and histopathological changes caused by respiratory infection with AIV H9N2 and reduced the excretion of virus in cloacal swab samples. Similarly, chickens administered S. enterica serovar Typhimurium expressing chIFN-α showed inhibited replication of AIV H9N2 in several different tissues including trachea, lung, cecal tonsil, and brain. Furthermore, immune responses specific for challenged AIV H9N2 were enhanced in chickens administered S. enterica serovar Typhimurium expressing chIFN-α, as determined by hemagglutination inhibition assay of sera, proliferation and IFN-γ and interleukin-4 expression by AIV H9N2 antigen-stimulated peripheral blood mononuclear cells and splenocytes. Therefore, oral administration of S. enterica serovar Typhimurium expressing chIFN-α can successfully control clinical signs caused by respiratory infection with AIV H9N2, which provides valuable insight into the use of attenuated Salmonella vaccine as an oral delivery system of chIFN-α to prevent the replication of AIV H9N2 in respiratory tract.

Systemic and mucosal immunity induced by attenuated Salmonella enterica serovar Typhimurium expressing ORF7 of porcine reproductive and respiratory syndrome virus.

Oral administration of attenuated Salmonella vaccine may provide valuable advantages such as low cost, easy preparation, and safety. Attenuated Salmonella vaccines also serve as carriers of foreign antigens and immunomodulatory cytokines. Presently, an attenuated Salmonella enterica serovar Typhimurium strain was used as a carrier for open reading frame 7 (ORF7) protein of porcine reproductive and respiratory syndrome virus (PRRSV), a swine pathogen of significant global economic importance. Initially, an attenuated S. enterica serovar Typhimurium expressing ORF7 gene derived from PRRSV Korean isolate was constructed. Following oral administration of a single dose of the attenuated Salmonella vaccine expressing PRRSV ORF7, humoral and cell-mediated immune responses specific for ORF7 were induced at both systemic and mucosal sites including spleen, mesenteric lymph node, Peyer's patch, and laminar propria, as evaluated by determining serum ORF7-specific IgG and mucosal IgA responses, as well as Th1- and Th2-type cytokine production from antigen-stimulated T cells. The induced humoral responses were sustained for at least 12weeks post-immunization. In particular, the immunized mice displayed immune responses to both the foreign ORF7 antigen and Salmonella itself. The results indicate the value of attenuated S. enterica serovar Typhimurium as an oral carrier of PRRSV antigenic proteins to induce effective systemic and mucosal immunity.

Live attenuated Salmonella enterica serovar Typhimurium expressing swine interferon-alpha has antiviral activity and alleviates clinical signs induced by infection with transmissible gastroenteritis virus in piglets.

Enhancing innate and acquired immunity by cytokines such as IFN-alpha appears to be useful as a first line of defense against viral infection. However, the practical use of cytokines in livestock is not evident due to cost and production issues associated with mass administration. In this study, we tested the efficacy of live attenuated Salmonella enterica serovar Typhimurium designed to secrete swine IFN-alpha (swIFN-alpha) protein for preventing the clinical signs caused by infection with transmissible gastroenteritis virus (TGEV), one of the diarrhea-causing viruses in the swine industry. Attenuated Salmonella vaccine (chi8501) containing swIFN-alpha-encoding pYA3560 vector (chi8501/swIFN-alpha) successfully induced the secretion of swIFN-alpha protein into the culture supernatants, as confirmed by SDS-PAGE and Western blot. The culture supernatants of chi8501/swIFN-alpha had antiviral activity against TGEV with 50% effective dose (ED(50)) of 320 per mg of supernatant protein. In addition, oral administration of chi8501/swIFN-alpha reduced the severity of clinical signs caused by TGEV infection with the effect more apparent at 6-8 days post-infection, and reduced excretion of TGEV in feces. Similarly, the amount of TGEV in intestinal tissues and mesenteric lymph node of chi8501/swIFN-alpha-administered piglets was lower than in piglets that were treated with control bacteria. These results indicate the value of attenuated Salmonella vaccines as delivery systems of cytokines that can be used for mass administration, thereby overcoming cost and production issues.