Jin Hyoung Kim

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-6Chigh 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.

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.

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

BackgroundCCR5 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.MethodsInfected 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.ResultsCCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6Chi monocytes and Ly-6Ghi 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-6Chi monocytes and Ly-6Ghi granulocytes. Instead, adoptive transfer of CCR5+CD4+Foxp3+ Tregs into Ccr5−/− mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-β) in the spleen and brain, and transferred CCR5+ Tregs were found to produce IL-10.ConclusionsCCR5 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.

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

BackgroundControl 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.ResultsOral 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.ConclusionsOur 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.

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.

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.

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, Peyers 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-α 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.

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.