The KD ameliorates EAE-mediated CNS-inflammation and oxidative stress.

<p>Comparison of the frequencies of (A) CD4⁺/CD8⁺ T cells, (B) CD11b⁺/CD45⁺ cells on gated lymphocyte populations in EAE with and without KD treatment. (C) Frequencies of CD4⁺CD25⁺ Foxp3⁺ regulatory T cells. The KD suppressed CNS cellular infiltration and myelin-reactive T cell responses in EAE. Groups of mice (receiving standard diet [SD] and KD diet) were immunized with MOG/CFA/PT, and sacrificed between day 16–19 p.i. Lymph node, spleen and CNS cells were isolated. (D) Lymphoid (LN) or CNS cells were re-stimulated with MOG_35–55 peptide overnight and IFN-γ- and IL-17-expressing CD4⁺ T cells were measured by intracellular staining. The number seen in the box indicated the average percentage of individual markers on the cells. (E) Summary data of the percentage of individual marker in the cells (LN <i>vs.</i> CNS) in SD-treated EAE mice or KD-treated EAE mice. Data are representative of two independent experiments (n = 4–8/group). P values, Student’s <i>t</i>-test; *, <i>p</i><0.05. (E) Visualization and quantification of brain inflammation by <i>in vivo</i> bioluminescence imaging. This is achieved by imaging reactive oxygen species (ROS) levels in the brain. Bioluminescent images in live mice were captured during a 1 min acquisition time using the Xenogen IVIS system at several time points after injection of 27 mg/kg dihydroethidium. Representative images of ROS seen in the left panel were captured in naïve mice and EAE mice with and without KD treatment. All experiments were conducted on groups of mice (n = 4–8) between days 14–19 p.i. Data are representative of two independent experiments (mean and SEM). P values, one way ANOVA; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p

Impairment of NK cell-dependent suppression of B16 tumor cell metastasis by nicotine is mediated by nAChR β2.

<p>Mice of different genotypes received nicotine (Nic) or PBS for 21 days and engrafted with the B16 melanoma cell line (1×10⁶ cells/mouse). <b>A</b>. Seven or 14 days later, a portion of mice were euthanized and the lung dissected. Total numbers of melanoma nodules counted in these organs are shown (n = 12 mice/group). <b>B, C</b>. Quantification of tumor growth in these animals during the period are achieved via bioluminescence imaging (B) and high field MRI (C for T1 images; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057495#pone.0057495.s002" target="_blank">Figure S2</a> denotes Th2 images. N = 15–18/group. P values, ANOVA, *p<0.05. <b>D</b>. Survival for the remaining animals are monitored for 60 days (n = 15–18 mice/group). <b>E</b>. Mice were engrafted with melanoma cells lines (1×10⁶/mouse) in the presence or absence of nicotine, and NK cells (5×10⁵ NK cells/mouse) from wild type or nAChR β2^−/− mice. Mice were then monitored for survival up to 60 days. Results are pooled from three experiments with similar results. N = 6–8 mice/group.</p

Nicotine alters NK cell phenotype via nAChR β2.

<p>Wild type (β2^+/+) or nAChR β2 knock-out (β2^−/−) mice received nicotine (Nic) or PBS for 21 days before they were scarified. Single cell suspensions were prepared from spleens. Frequency and phenotypes of NK cells within the mononuclear cells were analyzed by FACS. <b>A.</b> Expression of NKG2D, NKG2A and Ly49I on gated NK1.1⁺CD3⁻ cells. <b>B</b>. Expression of perforin and granzyme B secreted by gated NK1.1⁺CD3⁻ cells. The plot data represents results from two separate experiments (n = 3–4 mice/group). P values, Student’s <i>t</i>-test, *p<0.05.</p

NK1.1⁺ CD3⁻ NK cell receptor expression in lung and spleen of different groups.

<p>NK1.1⁺CD3⁻ NK cells in lungs of high-metastatic group were significantly higher than that in spleen (<i>p</i> = 0.003). NK1.1⁺CD3⁻ NK cells in spleen of high-metastasis group (0.10±.06) were significantly lower than that in low-metastasis group (<i>p</i> = 0.017) and control group (<i>p</i> = 0.025).</p

Structural brain changes in EAE mice with and without KD treatment.

<p>(A) T2-weighted images were obtained with a 7T MR scanner during the peak stage of motor disability. EAE mice showed abnormal signal intensities adjacent to the lateral ventricles. Arrows indicate focal lesions on T2-weighted imaging. In contrast, lesion volumes were significantly reduced in KD-fed EAE mice (N = 4–8). (B) In contrast to the observed hippocampal volume loss in EAE mice at 30 days post immunization, neither naïve mice nor KD-fed EAE mice showed changes in hippocampal volume.</p

Effects of the ketogenic diet on motor disability and memory deficit in EAE mice.

<p>(A) Starting from day 17 post immunization (p.i.), KD-fed EAE mice (n = 11) showed significantly less motor disability than SD-fed EAE mice (**, <i>p<</i>0.01, Mann-Whitney <i>U</i> test). In KD- fed and SD- fed EAE mice, the mean clinical scores (1.9±0.20 vs 2.7±0.35 and 0.43±0.12 vs 1.0±0.15) were measured at 17 days and 25 days p.i., respectively. (B, C) Restoration of spatial learning and memory by the KD in EAE mice. MWM testing was conducted in groups of EAE mice either prior to the occurrence of motor deficits (before 10 days p.i., left panel, B) or from 30 days to 35 days p.i. (right panel, C). The graph illustrates average latencies to finding the submerged escape platform. Each group consisted of 5–8 mice. Values represent mean±SEM. One way ANOVA followed by Tukey test. (D) Change in the synaptic plasticity at 13–16 days p.i. with or without the KD. EAE mice exerted impairment in LTP after high-frequency stimulation (10 slices form 5 mice). Both the naïve and KD-fed EAE mice demonstrated intact LTP induction and maintenance (9 slices from 4 mice). (E) Change in synaptic plasticity at 25–35 days p.i. with or without the KD. Neither the naïve nor KD-fed EAE mice showed changes in LTP maintenance (10 slices from 5 mice). The dotted line indicates the baseline field potential amplitude. Synaptic plasticity seen during peak and mild stages of motor disability are depicted in the left and right panels, respectively.</p

Transplanted tumors-lung metastasis models of high (L9981)/low (NL9980) metastatic human large cell lung cancer cell lines.

<p>A: Living fluorescence imaging detect mouse model (the left is the right inguen subcutaneously tumor for 1 week and the middle is for 6 weeks after injection, the right is the lung metastases tumors in six weeks after inoculation.), B: Growth curve of subcutaneously transplant tumor in SCID mouse, C: lung metastases luminescence imaging of tumor-bearing mouse in vitro, D: The comparison of luminescence value between transplanted tumor and lung metastases of high (L9981)/low (NL9980) metastatic human large cell lung cancer cell lines.</p

NK cells phenotype mRNA expression levels in different histologic type of lung cancer.

<p>NK cells receptors CD56 and CD16 phenotype mRNA in different histologic types of lung cancer have statistical significance.</p

CD56⁺CD16⁺NK cells infiltration extent in different pathological type of lung cancer.

<p>*<i>p</i><0.05, Difference have statistical significance.</p><p>CD56⁺CD16⁺NK cells infiltration extent in different pathological type of lung cancer.</p

Nicotine inhibits NF-κB activation in NK cells through nAChR β2.

<p>NK cells were sorted from splenocyte single cell suspensions of RAG2^−/− and RAG2^−/−β2^−/− mice. NF-κB and IKKα mRNA of sorted NK cells in β2^+/+ or β2^−/− mice received nicotine (Nic) was measured by qRT-PCR. The data of one experiment out of two performed is shown, n = 3–4 mice/group. P values, Student’s <i>t</i>-test, *p<0.05.</p