To detect possible changes of cytokines expression level in the brain tissue during prion infection, 10% brain homogenates from four C57 mice infected with scrapie strains 139A or ME7 at terminal stage of infection (around 180 dpi) were subjected to Luminex assay using a commercial kit that could measure 17 different cytokines. Four agematched healthy mice were used as control. Nine out of 17 cytokines did not reach the detection level of Luminex assay. Among the eight measurable cytokines, the levels of IFN-γ, IL9, KC, MIG and RANTES did not change significantly between the groups of scrapie infected mice and non-infected controls. In contrast, the levels of IP10 and M-CSF in the brains of both 139A- and ME7-infected mice were significantly increased (Table 1). The brain level of IL13 in 139A-infected mice was lower, while no change was detected in ME7-infected mice compared to controls.
Cytokines Ctrl (pg/mL) 139A (pg/mL) P value (139A vs Ctrl) ME7 (pg/mL) P value (ME7 vs Ctrl) IFN-γ 83.47 ± 35.62 80.78 ± 23.20 0.467 77.09 ± 11.24 0.411 IL-13 302.39 ± 10.44 225.02 ± 36.57 0.023 333.15 ± 52.63± 0.232 IL-9 13, 812.09 ± 1413.15 13, 703.33 ± 468.71 0.461 15, 595.23 ± 2226.84 0.196 IP-10 135.50 ± 8.12 4093.07 ± 497.60 0.00017 7178.91 ± 3986.38 0.0334 KC 40.36 ± 25.43 37.02 ± 26.68 0.452 177.57 ± 191.70 0.186 M-CSF 35.66 ± 4.16 95.18 ± 32.13 0.030 112.38 ± 47.99 0.044 MIG 350.36 ± 28.14 333.37 ± 129.58 0.432 538.53 ± 168.61 0.097 RANTES 19.41 ± 3.84 51.29 ± 37.81 0.151 33.74 ± 9.17 0.056 Italic indicates P value less than 0.05
Table 1. Comparative analysis of the levels of 8 brain cytokines between 139A- or ME7-infected and age-matched healthy mice.
Further, 10% brain homogenates from three different strains of mice infected with scrapie agent S15, including S15-infected C57, Balb/c and CD1 mice, as well as that of the PS- and RES-inoculated mice were subjected to the above Luminex assay together with the corresponding agematched healthy mice as the controls. Each testing group contained three mice. The tested results were summarized in Table 2. In line with the data of 139A- and ME7-infected; mice, the brain levels of IP10 and M-CSF in all three strains of mice infected with agent S15 were significantly upregulated, whereas those of IFN-γ and IL13 remained unchanged compared to PS-inoculated mice. The level of KC was also increased in all three strains of mice infected with scrapie agent S15. The brain levels of IL9 and MIG also increased in the brains of S15-infected C57 and Balb/c mice compared to PS-inoculated brain mice, but was not significantly different compared to healthy controls. The brain levels of RANTES increased in S15-infected Balb/c mice compared to PS-inoculated ones. Those data indicate that the brain levels of some cytokines, particularly IP10 and M-CSF, are significantly upregulated at the final stage of various scrapie strains infected mice, independently of the mouse genetic background.
Cytokines Ctrl (pg/mL) PS (pg/mL) S15 (pg/mL) RES (pg/mL) P value (S15 vs PS) C57 strain of mice IFN-γ 83.47 ± 35.62 120.73 ± 52.66 59.52 ± 19.28 / 0.100 IL-13 302.39 ± 10.44 282.07 ± 53.79 379.20 ± 133.85 / 0.197 IL-9 13, 812.09 ± 1413.15 6977.68 ± 3332.25 18, 175.15 ± 1812.72 / 0.007 IP-10 135.50 ± 8.12 150.73 ± 26.06 3456.68 ± 1303.87 / 0.012 KC 40.36 ± 25.43 41.74 ± 7.79 138.72 ± 22.57 / 0.002 M-CSF 35.66 ± 4.16 40.54 ± 10.86 110.69 ± 19.39 / 0.006 MIG 350.36 ± 28.14 181.43 ± 146.76 804.60 ± 253.96 / 0.020 RANTES 19.41 ± 3.84 47.51 ± 21.49 88.27 ± 57.41 / 0.200 Balb/c strain of mice IFN-γ 141.71 ± 50.20 - 103.29 ± 26.13 / - IL-13 325.08 ± 0 188.78 ± 0 395.82 ± 65.77 / - IL-9 12, 792.58 ± 1399.03 5533.93 ± 1390.15 10, 428.63 ± 1872.54 / 0.021 IP-10 177.52 ± 126.69 102.15 ± 26.89 1202.63 ± 579.35 / 0.028 KC 57.36 ± 0 11.62 ± 0 106.22 ± 13.15 / 0.0002 M-CSF 20.28 ± 16.91 20.10 ± 11.86 54.45 ± 9.06 / 0.016 MIG 424.44 ± 71.28 57.69 ± 17.58 309.76 ± 61.16 / 0.002 RANTES 28.24 ± 24.93 7.66 ± 2.94 25.34 ± 4.14 / 0.008 CD1 strain of mice IFN-γ 54.27 ± 16.96 55.95 ± 14.78 22.92 ± 0 28.11 ± 16.35 - IL-13 270.83 ± 52.55 402.92 ± 51.32 197.12 ± 122.75 0±0 0.070 IL-9 9229.83 ± 1259.10 6389.72 ± 665.87 10, 234.79 ± 3470.92 10, 060.40 ± 5691.00 0.099 IP-10 92.21 ± 9.09 221.24 ± 7.56 979.01 ± 236.50 234.13 ± 181.22 0.005 KC 00 ± 0 28.86 ± 13.87 135.51 ± 62.24 44.30 ± 15.00 0.039 M-CSF 16.50 ± 4.93 40.94 ± 2.67 79.53 ± 25.08 26.27± 22.15 0.048 MIG 86.53 ± 21.34 117.17 ± 64.77 163.81 ± 115.01 230.88 ± 34.21 0.322 RANTES 21.84 ± 2.78 15.41 ± 0.646 13.79 ± 1.44 14.28 ± 3.90 0.111 P values in italic mean statistically significant
Table 2. Comparative analysis of the levels of 8 brain cytokines from three different strains of mice infected with scrapie agent S15.
To confirm the changes of cytokines evaluated by Luminex assay, the protein levels of IP10, M-CSF and KC in the experimental mice brains were measured by commercial ELISA kits. Almost all infected mice showed significantly higher levels of brain IP10, including 139A-, ME7-infected mice, as well as S15-infected C57, CD1 and Balb/c mice, whereas S15-infected Balb/c mice were not statistically different (Fig. 1A, 1D). The ELISA levels of KC were increased in brains of 139A- and ME7-infected mice, three strains of S15-infected mice and RES-inoculated CD1 mice. No statistical difference was found in 139A-infected mice compared to the non-infected controls, as well as in S15-infected C57 and Balb/c mice compared to PS-inoculated mice (Fig. 1B, 1E). The brain M-CSF levels were statistically increased in S15-infected C57 and CD1 mice. The brain M-CSF levels in 139A-, ME7-infected mice and S15-infected Balb/c mice were slightly increased compared to the non-infected controls and the PS-inoculated mice but without statistical difference (Fig. 1C, 1F). Overall, Luminex assay and ELISA were in agreement.
Figure 1. ELISA assays for detecting the levels of IP10, KC and M-CSF in the brains of various scrapie infected mouse models. A–C. ELISA for brain IP10 (A), KC (B) and M-CSF (C) levels in 139A- (red circle) and ME7-infected (blue square) mouse models versus agematched healthy controls (black triangle). D–E. ELISA for brain IP10 (D), KC (E) and M-CSF (F) levels in C57 (black triangle), Balb/c (red circle) and CD1 mice (blue square) infected with scrapie agent S15. Data are present as Mean ± SEM. Different cytokines are indicated on the top. IP10: interferon gamma-induced protein 10, KC: keratinocyte chemoattractant, M-CSF: macrophage colony stimulating factor. *P < 0.05 and **P < 0.01. Each experiment replicates three times.
To assess any possible changes of brain transcriptional levels of IP10, KC, and M-CSF, total RNA of the brain tissues from the scrapie infected mice and age-matched healthy controls were prepared. After cDNAs were synthesized, the specific transcript of each cytokine was evaluated by the corresponding qRT-PCR. As shown in Fig. 2A, the brain levels of specific mRNAs of IP10, KC and M-CSF in all scrapie infected mice were significantly higher than those of the healthy controls. Further, the transcriptional levels of IP10 in the brain tissues of 139A- and ME7-infected C57 mice were analyzed with a digital PCR technique. The mRNAs of those two kinds of scrapie infected mice were markedly higher than those of the normal controls (Fig. 2B). It indicates that the transcriptional levels of IP10, KC and M-CSF in the brains of scrapie infected experimental mice were significantly upregulated at the final stage of disease.
Figure 2. Comparative analysis of the brain transcriptional levels of IP10, KC and M-CSF in various scrapie infected mouse models with Real-time PCR (A) and Digital PCR (B). A The transcriptional levels of IP10, KC and M-CSF specific mRNA were determined relative to that of the individual β-actin. The relative intensity of the transcripts of IP10, KC and M-CSF gene in 139A-, and ME7-infected mice, as well as S15-infected Balb/c mice were relative to that of controls (C57 or balb/c healthy mice) that was set to 1. B The brain IP10 DNA copy number of 139A-, ME7-infected mice and non-infected controls. Nucleic acid free water used as the negative control. Various groups are indicated in X-axis. Y-axis shows the brain IP10 copy number which is expressed as log concentration (cp/μL-log) (left) or droplet fluorescence intensity in the blue acquisition channel (right). Blue droplets represent the IP10 positive copies and solid line represents the cut-off value. Data are representative of three independent experiments. *P < 0.05 and **P < 0.01.
The brain slices of cortical regions of 139A-infected mice were further subjected to IHC assay with IP10, KC and M-CSF specific antibodies, respectively. Microscope examinations revealed large amount of fine IP10 positive signals in the brain slices of infected mice, which seemed to be distributed in the cytoplasm, especially in the region of thalamus (Fig. 3A). KC positive signals surrounded the cells in all selected brain regions. Although the amount of the positive cells between infected and normal mice seemed to be indistinguishable, the size of the KC-positive signals in the infected brains was larger than in the controls and distributed in the region of cortex, hippocampus, and thalamus (Fig. 3B). The signals of M-CSF in the slices of control brains were very fine-grained and distributed outside of the cells, whereas the brain slices of infected mice were stained as large astrocytic particles accompanying with numerous fine grains, thalamus being the most affected brain region (Fig. 3C). Quantitative analysis showed that positive staining levels of IP10, KC and M-CSF increased compared to non-infected controls. These data suggested distinct depositing patterns of those three cytokines in the brain tissues between scrapie infected mice and age-matched healthy controls.
Figure 3. IHC assays of the distributions of IP10 (A), KC (B) and M-CSF (C) in cortical regions of 139A-infected mouse models (100×). The white arrows indicate the positive stainings. The quantitative analysis of the staining of IP10, KC and M-CSF per area is presented on the right. IHC staining results of mice cortical regions are indicated on the left. Various uninfected brain regions are indicated on the top as negative controls. **P < 0.01 versus noninfected control. Each experiment replicates three times.
To understand the dynamic changes of IP10, KC and M-CSF in the brains of the mice during scrapie infection, the brain specimens of 139A- and ME7-infected mice collected at 86, 117, 147 and 183 dpi were selected for Luminex assays and ELISA (Fig. 4). Dynamic data found that IP10, KC and M-CSF in the group of age-matched healthy control remained at low level, while IP10 increased sustainably in both 139A- and ME7-infected mice along with the incubation period in both Luminex and ELISA tests. Significant differences in the brain samples were found at 117 dpi compared to controls, although brain IP10 in 139A-infected mice declined in 183 dpi but still maintained at high levels (Fig. 4A, 4D). The KC levels were significantly increased in the brain samples of ME7-infected mice at terminal stage of infection (183 dpi) in Luminex assays, as well as in the brain samples of ME7-infected mice of 147 and 183 dpi in ELISA (Fig. 4B, 4E). Meanwhile, both Luminex and ELISA tests for KC illustrated that the KC levels in the brains of 139A-infected mice were remained unchanged. The brain levels of M-CSF were increased significantly in the brain samples of 139A-infected mice at 147 dpi as well as in ME7-infected mice at 117 dpi in the Luminex assays. This increase was however not significant in ELISA (Fig. 4C, 4F). It indicates a gradual increase of a number of cytokines, particularly IP10, in the brain tissues of scrapie infected mice during the incubation period.
Figure 4. Dynamic analysis of IP10, KC and M-CSF in brains of various scrapie infected mouse models with Luminex assay (A–C) and ELISA (D–F). Red circle represents 139A-infected mouse models, blue square represents ME7-infected mouse models, and black triangle represents age-matched healthy controls. *P < 0.05 and **P < 0.01 versus non-infected control. Each experiment replicates three times.
To assess the possible alteration of IP10 in CSF, CSF samples from 45 probable sCJD patients and 48 non-PrD patients were subjected to a commercial ELISA kit for human IP10. The CSF IP10 level of each patient was measured. It revealed a broad distribution of IP10 content in the tested samples (Fig. 5), ranging from 0.74 pg/mL to 330.31 pg/mL. The median level of CSF IP10 in the group of sCJD cases (71.56 pg/mL) was slightly higher than those in the group of non-PrD patients (39.26 pg/mL). Statistical assay identified significant difference in the CSF IP10 levels between the two groups (P = 0.0162). More cases with higher CSF IP10 amount (> 71.56 pg/mL) were in the group of sCJD (22/45, 48.89%) than in that of nonPrD (9/48, 18.75%). Further analysis of the diagnostic results from nine patients in the non-PrD group with IP10 greater than 71.56 pg/mL found that their clinical diagnosis was very different, including two viral encephalitis, two epilepsy, two autoimmune encephalitis, one cognitive dysfunction, one bilateral thalamic lesions, and one disturbance of consciousness, indicating CSF IP10 increase is sCJD specific.