Quantitative RT-PCR analysis of <i>P</i>16 mRNA expression in thymic lymphoma tissue samples radiation induced irradiation and normal control non-irradiated thymus tissue samples.

<p>N: normal control non-irradiated thymus tissue samples. T: radiation induced thymic lymphoma tissue samples. (T vs N, <i>P</i><0.05).</p

Data of mRNA expression with significant difference in G₁/S cell cycle by the Illumina Sentrix Mouse-6 Expression Bead Chip.

<p>N: normal control non-irradiated thymus tissue samples. T: radiation induced thymic lymphoma tissue samples.</p

The P16 promoter region and Methylation at the 23 CpG sites.

<p>(A) Schematic of the P16 gene promoter region (from 400 nucleotides upstream to 200 nucleotides downstream of the transcription start site) is presented in the upper diagram. The two CpG islands are boxed. The CpG dinucleotides are indicated as |. The major transcription start site is located at −355 upstream to 78 downstream of the ATG translation start site. The 16 CpG sites analyzed for cytosine methylation (bold) with their position relative to the A (chr4: NC_000070, 12509) of the ATG translation start site (underlined) are indicated. (B) Analysis of cloned amplified bisulfite-treated DNA from 6 pairs of radiation induced carcinogenesis tissue samples and normal control non-irradiated thymus tissues. Solid circles are methylated CpG sites. The location of these sites is shown relative to their location in the amplified P16 region. (C) Percent methylation of 16 CpG dinucleotides in the p16 promoter region. CpG sites in Sp1, USF-1, NF-Y, HSF2 and E2F-1 binding sites are indicated. *<i>p</i><0.05. The numbers 1 to 23 represented 23 methylation sites sequentially (−355 to 78). N: normal control non-irradiated thymus tissue samples. T: radiation induced thymic lymphoma tissue samples.</p

Analysis of mRNA expression profile by the Illumina Sentrix Mouse-6 Expression BeadChip in G1/S cell cycle.

<p>Blue: expression down-regulated; Pink: expression up-regulated.</p

Increased <i>P16 - Figure 1 </i> DNA Methylation in Mouse Thymic Lymphoma Induced by Irradiation

<p>A. Gross image of a thymic lymphoma in a dissected mouse. B. Histological section of a thymic lymphoma stained with Hematoxylin and Eosin (H&E). N: normal control non-irradiated thymus tissue samples. T: radiation induced thymic lymphoma tissue samples.</p

Microglia inhibit maDC-initiated proliferation of CD4 T cells.

<p>CD4 T cells from DO11.10×C57BL/6 F1 hybrid mice were cocultured with maDCs or/and microglia for 5 days. (a)The photos were taken in bright field (objective ×20). (b) The proliferation of CFSE-labeled CD4 T cells was detected by FACS after 3 days coculture. (c) The live CD4 T cells in the coculture system were counted by FACS. Data are presented as mean±s.d. of triplicate wells and representative of three independent experiments.^***<i>P</i><0.01.</p

NO is involved in the immune inhibition by microglia and MLCs.

<p>(a) The inhibitory function of supernatants(SN) of microglia or MLCs cultured for 48 hours and paraformaldehyde-fixed microglia or MLCs were tested in the mDC/CD4 coculture system. (b) NO donor NOC-18 at a dosage of 40 µg/ml, NO synthetase inhibitor PBIT at a dosage of 20 µg/ml and neutralization antibodies against IL-10 were added into the maDCs/T cells coculture system and then the live CD4 T cells were detected by FACS. Data are presented as mean±s.d. of triplicate wells and representative of three independent experiments.^*, <i>P</i>>0.05;^**, <i>P</i><0.05; ^***, <i>P</i><0.01.</p

The characteristics of the endothelial cells from the CNS.

<p>(a) The CNS endothelial stroma cells before and after CD11b⁻ CD31⁺ selection were observed under the microscopy. The purity of CD11b⁻ CD31⁺ cells was tested by expression of CD31 and CD106. (b, c) The secretion of cytokines including IL-7, TGF-β, GM-CSF, M-CSF and VEGF(b), and chemokines including MCP-1, MCP-3, SDF-1α, TECK and MDC(c) was detected. (d)The chemotactic ability to monocytes or DCs of the supernatant of LPS-treated EC was assayed.</p

A model of dynamic transformation of antigen presenting cells in CNS.

<p>Some special infection in the CNS can destroy neural tissues and endothelial cells of capillary, in this status, BBB was impaired followed by the entrance of monocytes and autoreactive T cells from peripheral blood. Monocytes will become mature DCs in the stimulation of inflammation factors secreted by the infected cells, after crossing the endothelia. New mature DCs derived from monocytes will capture the antigens of neural tissue, prime autoreactive T cells and lead to immunopathological injury. On the contrary, microglia activated by the inflammation factors can secrete high level of NO to inhibit T cell proliferation, even induce T cell apoptosis. The role of IL-10 secreted by microglia in the inhibitory function remains to be investigated. The infiltrated dendritic cells after priming might become inhibitory microglia like cells or microglia under the sustained influence of microenvironment. The cell-to-cell interaction played a key role in the differentiation of DC. The effects of TGF-β, M-CSF, VEGF secreted by activated endothelial cells remain to be investigated. Whether MLC will differentiate into microglia is unknown. Microglia and the transformation from priming dendritic cells to inhibitory microglia like cells and even microglia contribute to the remission of the autoimmune diseases of CNS.</p

Table_1_Acute stress induces an inflammation dominated by innate immunity represented by neutrophils in mice.xls

It is well known that psychological stress could affect the immune system and then regulate the disease process. Previous studies mostly focused on the effects of chronic stress on diseases and immune cells. How acute stress affects the immune system remains poorly understood. In this study, after 6 hours of restraint stress or no stress, RNA was extracted from mouse peripheral blood followed by sequencing. Through bioinformatics analysis, we found that when compared with the control group, differentially expressed genes in the stress group mainly displayed up-regulated expression. Gene set enrichment analysis results showed that the enriched gene terms were mainly related to inflammatory response, defense response, wounding response, wound healing, complement activation and pro-inflammatory cytokine production. In terms of cell activation, differentiation and chemotaxis, the enriched gene terms were related to a variety of immune cells, among which neutrophils seemed more active in stress response. The results of gene set variation analysis showed that under acute stress, the inflammatory reaction dominated by innate immunity was forming. Additionally, the concentration of serum IL-1β and IL-6 increased significantly after acute stress, indicating that the body was in an inflammatory state. Importantly, we found that acute stress led to a significant increase in the number of neutrophils in peripheral blood, while the number of T cells and B cells decreased significantly through flow cytometric analysis. Through protein-protein interaction network analysis, we screened 10 hub genes, which mainly related to inflammation and neutrophils. We also found acute stress led to an up-regulation of Ccr1, Ccr2, Xcr1 and Cxcr2 genes, which were involved in cell migration and chemotaxis. Our data suggested that immune cells were ready to infiltrate into tissues in emergency through blood vessels under acute stress. This hypothesis was supported in LPS-induced acute inflammatory models. After 48 hours of LPS treatment, flow cytometric analysis showed that the lungs of mice with acute stress were characterized by increased neutrophil infiltration, decreased T cell and B cell infiltration. Immunohistochemical analysis also showed that acute stress led to more severe lung inflammation. If mice received repeat acute stress and LPS stimulation, the survival rate was significantly lower than that of mice only stimulated by LPS. Altogether, acute stress led to rapid mobilization of the immune system, and the body presented an inflammatory state dominated by innate immune response represented by neutrophils.</p