title: 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.
abstract: 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.
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.
authors.forename: Seong Bum
authors.initials: SB
authors.lastname: Kim
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Choi
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.
authors.forename: Jin Hyoung
authors.initials: JH
authors.lastname: Kim
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.
authors.forename: Erdenebelig
authors.initials: E
authors.lastname: Uyangaa
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.
authors.forename: Ajit Mahadev
authors.initials: AM
authors.lastname: Patil
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.|Department of Bioactive Material Sciences, Graduate School, Chonbuk National University, Jeonju, 54896, Republic of Korea.
authors.forename: Sang-Youel
authors.initials: SY
authors.lastname: Park
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.|Department of Bioactive Material Sciences, Graduate School, Chonbuk National University, Jeonju, 54896, Republic of Korea.
authors.forename: John Hwa
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Pharmacology, School of Medicine, Pusan National University, Yangsan, 50612, Republic of Korea.
authors.forename: Koanhoi
authors.initials: K
authors.lastname: Kim
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea.
authors.forename: Young Woo
authors.initials: YW
authors.lastname: Han
authors.affiliation: College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Iksan, 54596, Republic of Korea. vetvirus@chonbuk.ac.kr.|Department of Bioactive Material Sciences, Graduate School, Chonbuk National University, Jeonju, 54896, Republic of Korea. vetvirus@chonbuk.ac.kr.
authors.forename: Seong Kug
authors.initials: SK
authors.lastname: Eo
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000096:biosynthesis*; D002454:Cell Differentiation / Q000502:physiology; D004672:Encephalitis, Japanese / Q000276:immunology / Q000378:metabolism* / Q000517:prevention & control; D016898:Interferon-alpha / Q000096:biosynthesis*; D016899:Interferon-beta / Q000096:biosynthesis*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009000:Monocytes / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9 / Q000037:antagonists & inhibitors / Q000172:deficiency*
chemical_list: D000950:Antigens, Ly; D016898:Interferon-alpha; C494192:Ly-6C antigen, mouse; D053261:Tumor Necrosis Factor Receptor Superfamily, Member 9; D016899:Interferon-beta