Revised: December 9, 2011
Accepted: December 20, 2011
Published online: February 27, 2012
AIM: To investigate the temporal onset and dynamic interplay of CD4+ T helper cell subsets in experimental autoimmune encephalomyelitis (EAE).
METHODS: EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide p35-55. The clinical signs were scored and the tissue samples and immune cells isolated for analysis at different phases of EAE. The expression levels of inflammatory cytokines and related transcription factors were detected by quantitative reverse transcription polymerase chain reaction (PCR) and enzyme linked immunosorbant assay (ELISA). The percentages of Th1, Th17, Th2, Treg and memory T cell subsets in EAE were analyzed by immunostaining and flow cytometry. The data were analyzed by statistical techniques.
RESULTS: Quantitative real-time PCR analysis showed that EAE mice express elevated levels of Th1 [interferon gamma (IFNγ), interleukin (IL)-12p40], Th17 [IL-17, related orphan receptor gamma (RORγ), IL-12p40] and Treg [Foxp3, Epstein-Barr virus induced gene 3 (EBI3), IL-10] genes in the central nervous system at the peak of the disease. Whereas, the expression of Th1 (IFNγ, T-bet, IL-12p35, IL-12p40), Th17 (RORγ, IL-12p40), Th2 (IL-4) and Treg (Foxp3, EBI3) response genes was reduced in the spleen during pre-disease but gradually recovered at the later phases of EAE. ELISA and flow cytometry analyses showed an increase in Th17 response in the periphery, while Th1 response remained unchanged at the peak of disease. The mRNA levels of IFNγ, IL-17 and IL-12p40 in the brain were increased by 23 (P < 0.001), 9 (P < 0.05) and 14 (P < 0.01) fold, respectively, on day 21 of EAE. Conversely, the mRNA expression of IL-10 was increased by 2 fold (P < 0.05) in the spleen on day 21. CD4+CD25+Foxp3+Treg response was reduced at pre-disease but recovered to naïve levels by disease onset. The percentage of CD25+Foxp3+ regulatory T cells decreased from 7.7% in the naïve to 3.2% (P < 0.05) on day 7 of EAE, which then increased to 8.4% by day 28. Moreover, the CD4+CD127+CD44high memory T cell response was increased during the onset and recovery phases of EAE. The memory and effector cells showed an inverse relationship in EAE, where the memory T cells increased from 12.3% in naïve to 20% by day 21, and the effector cells decreased from 32% in naïve to 21% (P < 0.01) by day 21. The wild type C57BL/6 mice with EAE showed elevated levels of effector-memory T cells (TEM) with concomitant reduction in central-memory T cells (TCM), but the EAE-resistant IL-7R deficient mice showed elevated TCM with no effect on TEM cells in EAE.
CONCLUSION: Our findings highlight the temporal onset and dynamic interplay of effector, memory and regulatory CD4+ T cell subsets and its significance to clinical outcome in EAE and other autoimmune diseases.