Immune Netw.  2017 Jun;17(3):192-200. 10.4110/in.2017.17.3.192.

Exacerbation of Japanese Encephalitis by CD11c(hi) Dendritic Cell Ablation Is Associated with an Imbalance in Regulatory Foxp3⁺ and IL-17⁺CD4⁺ Th17 Cells and in Ly-6C(hi) and Ly-6C(lo) Monocytes

• Choi JY
• Kim JH
• Patil AM
• Kim SB
• Uyangaa E
• Hossain FM
• Eo SK

• KMID: 2383358
• DOI: http://doi.org/10.4110/in.2017.17.3.192

Abstract

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 CD11c(hi) dendritic cell (DC) ablation in CD11c-DTR transgenic mice. Moreover, CD11c(hi) DC ablation led to abnormal differentiation of CD11b⁺Ly-6C(hi) 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-6C(hi) and Ly-6C(lo) monocytes. CD11c(hi) 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 CD11c(hi) DC-ablated mice. In addition, CD11c(hi) DC-ablated mice showed higher frequency and total number of inflammatory CD11b⁺Ly-6C(hi) monocytes, whereas CD11b⁺Ly-6C(lo) monocytes were detected with lower frequency and total number in CD11c(hi) DC-ablated mice. Furthermore, CD11c(hi) DC ablation altered the phenotype and function of CD11b⁺Ly-6C(lo) monocytes, resulting in lower levels of activation marker and anti-inflammatory cytokine (IL-10 and TGF-β) expression. Collectively, these results indicate that CD11c(hi) DC ablation caused an imbalance in CD4⁺ Th17/Treg cells and CD11b⁺Ly-6C(hi)/Ly-6C(lo) monocytes in the lymphoid tissue and CNS during JE progression. This imbalanced orchestration of pro- and anti-inflammatory leukocytes following CD11c(hi) DC ablation may contribute to the exacerbation of JE.

Keyword

Japanese encephalitis; CD4⁺Foxp3⁺ Treg; IL-17⁺CD4⁺ Th17; CD11b⁺Ly-6C(hi) monocytes; CD11b⁺Ly-6C(lo) monocytes; Dendritic cells

MeSH Terms

Animals
Asian Continental Ancestry Group*
Blood-Brain Barrier
Brain
Central Nervous System
Dendritic Cells*
Encephalitis, Japanese*
Humans
Interleukin-17
Leukocytes
Lymphoid Tissue
Mice
Mice, Transgenic
Monocytes*
Permeability
Phenotype
T-Lymphocytes
T-Lymphocytes, Regulatory
Th17 Cells*
Interleukin-17

Figure

• 

  Figure 1 CD11chi DC ablation leads to an imbalance in Foxp3+ Tregs and IL-17+ Th17 cells during JE progression. (A and B) Frequency and absolute number of IL-17+ and IFN-γ+CD4+ T cells in the spleen. (C and D) Frequency and absolute number of Foxp3+CD4+ Tregs in the spleen. (E-H) Frequency and absolute number of CNS-infiltrated IL-17+, IFN-γ+, and Foxp3+CD4+ Th cell subsets. The frequency and absolute number of IL-17+CD4+ Th17 and IFN-γ+CD4+ Th1 cells were determined by intracellular cytokine staining in response to PMA plus ionomycin stimulation of splenocytes or brain leukocytes prepared from JEV-infected CD11c-DTR mice at 5 dpi. CD4+Foxp3+ Tregs were detected by intracellular Foxp3 and surface CD4 staining. (I) The ratio of Foxp3+ Tregs to IL-17+ Th17 cells in the CNS. The ratio of Tregs to Th17 CD4+ Th cells was calculated using absolute numbers of CNS-infiltrated Tregs and Th17 cells in the CNS at 5 dpi. Data are presented as the mean±SE of values derived from at least three independent experiments (n=3~5). *p<0.05 and **p<0.01 compared to levels in the indicated groups.

• 

  Figure 2 Expression of transcription factors specific for CD4+ Th cell subsets in the CNS. After vigorous heart perfusion, CD4+ T cells from CNS-infiltrated leukocytes were briefly stimulated with PMA plus ionomycin for 3 h. The expression of transcription factors specific for each CD4+ Th cell subset was determined by real-time qRT-PCR using total RNA extracted from stimulated CD4+ T cells. Data are presented as the mean±SE of values derived from at least three independent experiments (n=3~5). *p<0.05 and **p<0.01 compared to levels in the indicated groups.

• 

  Figure 3 CD11chi DC ablation results in less accumulation of CD11b+Ly-6Clo monocytes during JE progression. (A and B) The frequency and absolute number of splenic CD11b+Ly-6Chi and Ly-6Clo monocytes during JE progression. (C and D) The frequency and absolute number of CD11b+Ly-6Chi and Ly-6Clo monocytes in the brain during JE. The frequency and absolute number of CD11b+Ly-6Chi and Ly-6Clo monocytes were determined by flow cytometric analysis using splenocytes or brain leukocytes prepared from JEV-infected CD11c-DTR mice at 3 dpi. (E) The ratio of Ly-6Chi to Ly-6Clo monocytes in the spleen and brain. The ratio of CD11b+Ly-6Chi to CD11b+Ly-6Clo monocytes was calculated using absolute numbers of Ly-6Chi and Ly-6Clo monocytes in the spleen and brain at 3 dpi. Data are presented as the mean±SE of values derived from at least three independent experiments (n=3~5). *p<0.05 and **p<0.01 compared to levels in the indicated groups.

• 

  Figure 4 Altered characteristics of CD11b+Ly-6Clo monocytes by CD11chi DC ablation. (A) Cytokine and growth factor expression profiles in sorted CD11b+Ly-6Clo monocytes. Total RNA extracted from splenic CD11b+Ly-6Clo monocytes sorted by FACSAria at 3 dpi were used for real-time qRT-PCR. (B) Phenotypes of splenic CD11b+Ly-6Clo monocytes during JE progression. The levels of activation markers in splenic CD11b+Ly-6Clo monocytes were determined by flow cytometric analysis at 3 dpi. Data are presented as the mean±SE of values derived from at least three independent experiments (n=3~5). *p<0.05 and **p<0.01 compared to levels in the indicated groups.

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