Fig 1.
Yet40 mice are susceptible to experimental autoimmune encephalitis (EAE).
(A) To identify APC populations that express IL12/23 p40 in vivo during EAE, and that are potentially capable of initiating and perpetuating CNS inflammation, B6.129-Il12b strain of p40eYFP reporter mice (yet40 mice) were actively immunized with CFA/MOGp35-55. The onset of disease and the clinical disease course were similar in both experimental groups (please see the Material and Methods section for definitions of disease stages). Mean clinical scores of yet40 mice were less severe than those observed in C57BL/6 mice, although those differences were not significant. (B & C) There was no difference in the number of inflammatory cellular infiltrates between yet40 mice and C57BL/6 wild-type mice in the CNS. Representative sections of brain tissue are shown.
Table 1.
Experimental autoimmune encephalitis induced by active immunization.
Fig 2.
P40 is highly expressed in secondary lymphoid organs during experimental autoimmune encephalitis (EAE).
To investigate which APCs could express IL12/23p40 during the course of EAE, we examined APC subsets from B6.129-Il12b strain of p40eYFP reporter mice (yet40 mice) for expression of eYFP by multi-parameter flow cytometry. Splenocytes were assessed during the pre-clinical, acute, and chronic stages of EAE (please see the Material and Methods section for definitions of disease stages). In lymph nodes cells, p40 expression was only assessed during the induction and acute disease stages, as p40 was undetectable during the chronic disease stage in this EAE model (data not shown). (A & B) p40 eYFP expression was readily detectable by flow cytometry in lymph node cells, and (C-E) splenocytes from yet40, but not in control C57BL/6 mice during the pre-clinical, acute, and chronic EAE stages. (A-E) During most disease stages in both compartments, CD11c+Cd11b+ dendritic cells (DC) were the main expressers of p40-eYFP, followed by PDCA1+ plasmacytoid dendritic cells (pDCs), and GR1+CD11b+ immature myeloid cells.
Fig 3.
p40 is expressed during all stages of experimental autoimmune encephalitis (EAE) in the brain and spinal cord.
(A & B) p40 was detectable in the brain and spinal cord by confocal microscopy during the pre-clinical stage of EAE, (C & D) the acute stage of EAE, and the (E & F) chronic stage of EAE by confocal microscopy (please see in the Material and Methods section for definitions of disease stages). Expression of p40 was highest during the acute EAE stage in the spinal cord. (G) When pooled cells from brain and spinal cord were immunophenotyped by multi-parameter flow cytometry, the highest cellular expressers of p40-eYFP during the pre-clinical stage of EAE, (H) the acute stage of EAE, and the (I) chronic stage of EAE were CD11c+Cd11b+ DC, followed by GR1+CD11b+ immature myeloid cells during the pre-clinical stage, and the acute stage. (G-I) Interestingly, the percentage of PDCA1+ plasmacytoid DC increased as the disease progressed and decreased in severity.
Fig 4.
p40 expression in the brain is not required for experimental autoimmune encephalitis (EAE) induction after adoptive transfer of encephalitogenic T cells.
(A) Adoptive transfer of CD4+ T cells from C57BL/6 donor mice into C57BL/6 recipient mice, or into p40-deficient (p40ko) mice resulted in EAE, indicating that p40 expression in the CNS is not an absolute requirement for CNS autoimmunity to occur once antigen-specific peripheral CD4+ T cells are activated and differentiated. (B & C) When CD4+ T cells from 2DD donor mice were polarized into Th1 cells or Th17 cells, transfer of either phenotype into p40ko mice also resulted in severe EAE. (B) Peripheral antagonism of IL-2, a trophic factor required for proliferation and differentiation of Th precursor cells into effector Th1 cells with a monoclonal antibody resulted in slightly milder disease during the acute stage of Th1-mediated adoptively transferred EAE. IL-6 is a cytokine critical for the differentiation and maintenance of Th17 cells, and (C) peripheral antagonism of IL-6R with a monoclonal antibody also decreased the severity of EAE substantially during the acute stage of Th17-mediated adoptively transferred EAE.
Table 2.
Experimental autoimmune encephalitis induced by adoptive transfer.