Great improvement of EAE clinical signs in HQGZWW high dose group
To determine the role of HQGZWW in the pathogenesis of EAE, we randomly divided EAE mice into four groups upon four treatments (distilled water, HQGZWW high dose, HQGZWW low dose or prednisone) and then measured the clinical signs using the EAE score assessed by daily checks and histopathology. [17, 18] As shown in Figure 1A, from day 0 to day 21st after MOG35–55 peptide immunization, the EAE score were measured. For the maximum EAE score, compare with distilled water group (3.98 ± 0.04), there were significant improvement of EAE score in both HQGZWW high dose and prednisone groups (2.52± 0.09 and 2.46 ± 0.04, respectively, P = 0.001), however only slight improvement in HQGZWW low dose group (3.35 ± 0.09, P = 0.616). Among different treatments, it was shown that HQGZWW high dose group had much better effective on EAE mice than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.569).
Compare to pathological examination of spinal cord from naïve mice (HE, Fig.1B and LFB, Fig. 1G), it revealed sever inflammation and demyelination in distilled water group (HE, Fig.1C and LFB, Fig. 1H) or HQGZWW low dose group (HE, Fig.1E and LFB, Fig. 1J); on the another hand, there is slight inflammation and demyelination in HQGZWW high dose group (HE, Fig.1D and LFB, Fig. 1I) or prednisone group (HE, Fig.1F and LFB, Fig. 1K). Our data suggested that HQGZWW had similar effect at high dose level to typical conventional medicine prednisone on aEAE, but no effect at low dose level.
MOG35–55 specificity of purified CD8+ and CD4+T Cells
Try to answer the above questions, unfractionated T cells were isolated by CD8 or CD4 kit. As shown in Figure 2, there were around 90% CD8+ CD3+ cells in the fraction of T cells in 4 groups after CD8 bead enrichment and around 90% CD4+CD3+ cells in the fraction of T cells after CD4 bead enrichment in 4 groups. These CD8+CD3+ and CD4+CD3+ cells were highly pure and appropriate for the following functional experiments.
The purified CD8+ and CD4+ T cells were examined for antigen-specific functions by a proliferation assay. The representative results shown in Figure 3 indicated that the MOG35–55 peptide had a strong stimulatory effect on both CD8+ and CD4+ T cells. Although the purified CD8+ T cells had a generally lower response to MOG35-55 than CD4+ T cells, it was apparent that the response of CD8+ T cells was not always dependent on CD4 + T cells. By the way, upon to the highest MOG35-55 concentration (20mg/mL) as shown in Figure 3A, compare with distilled water group (21711.33 ±1589.42 cpm), there is lower antigen-specific responses of CD8+ T cells in HQGZWW high dose and prednisone groups (10510.67 ±1189.87 cpm and 9575.33 ±1064.00 cpm, respectively, P = 0.001), but not in when they are compared with HQGZWW low dose group (19665.33 ±1553.61 cpm, P = 0.104). Among different treatments, it was shown that antigen-specific responses of CD8+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.427).
As shown in Figure 3B, It is shown that similar pattern for CD4+ T cells proliferation, compare with distilled water group (64557.67 ± 2547.57 cpm), there were significant decrease of EAE score in both HQGZWW high dose, prednisone group and HQGZWW low dose groups (21281.67 ± 1739.08 cpm, 20104.00 ± 1364.82 cpm, and 50799.33 ± 2449.17 cpm, respectively, P = 0.001). Among different treatments, it was shown that antigen-specific responses of CD8+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.509). To sum up, the antigen-response of CD8+ or CD4+ T cells in four groups were consistent with the EAE score and histology result in Figure1.
Cytokine profiles of MOG35–55-specific CD8+ T and CD4+ T Cells
To determine the cytokine profiles of activated CD4+ and CD8+ autoreactive T cells, we used ELISAs to measure cytokine levels in the culture supernatants of activated CD4+ and CD8+ T cells at 24 hours post-stimulation. As shown in Figure 4A, compare with distilled water group (816.31 ± 42.88 pg/ml), there is much lower IFN-g secretion of CD8+ T cells supernatant in HQGZWW high dose and prednisone groups (531.66 ± 25.54 pg/ml and 484 ± 36.52 pg/ml, respectively, P = 0.001), and also lower in HQGZWW low dose group (718.33 ± 29.81 pg/ml, P = 0.008). Among different treatments, it was shown that less IFN-g secretion of CD8+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.127).
As shown in Figure 4D, compare with distilled water group (1474.07 ± 90.43 pg/ml), there is much lower IFN-g secretion of CD4+ T cells supernatant in HQGZWW high dose and prednisone groups (722.66 ± 64.50 pg/ml and 646 ±64.50 pg/ml, respectively, P = 0.001), and also lower in HQGZWW low dose group (1323.12 ± 79.50 pg/ml, P = 0.04). Among different treatments, it was shown that less IFN-g secretion of CD8+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.249).
On the another hand, as shown in Figure 4B, compare with distilled water group (220.66 ±56.51 pg/ml), there is much higher IL-4 secretion of CD8+ T cells supernatant in HQGZWW high dose and prednisone groups (762.33 ±78.21 pg/ml and 954.06 ±75.07 pg/ml, respectively, P = 0.001), but not in HQGZWW low dose group (293 ±49.03 pg/ml, P = 0.215). Among different treatments, it was shown that more IL-4 secretion of CD8+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.07).
As shown in Figure 4E, compare with distilled water group (324.33 ±27.50 pg/ml), there is much higher IL-4 secretion of CD4+ T cells supernatant in HQGZWW high dose and prednisone groups (620.33 ±38.07 pg/ml and 707.66 ±35.59 pg/ml, respectively, P = 0.001), but not in HQGZWW low dose group (355.13 ±35.59 pg/ml, P = 0.349). Among different treatments, it was shown that more IL-4 secretion of CD4+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.221).
Another important Th2 cytokine, IL-10, was also detected. As shown in Figure 4C, compare with distilled water group (830.33 ± 68.24 pg/ml), there is much higher IL-10 secretion of CD8+ T cells supernatant in HQGZWW high dose and prednisone groups (2048.66 ±114.02 pg/ml and 2250.01 ±67.55 pg/ml, respectively, P = 0.001), but not in HQGZWW low dose group (1056.13 ±114.89 pg/ml, P = 0.237). Among different treatments, it was shown that more IL-10 secretion of CD8+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.12).
As shown in Figure 4F, compare with distilled water group (853.66 ± 48.26 pg/ml), there is much higher IL-10 secretion of CD4+ T cells supernatant in HQGZWW high dose and prednisone groups (2242.33 ± 156.35 pg/ml and 2380.21 ± 131.73 pg/ml, respectively, P = 0.001), but not in HQGZWW low dose group (1055.33± 66.01 pg/ml, P = 0.247). Among different treatments, it was shown that more IL-4 secretion of CD4+ T cells in HQGZWW high dose group than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.271).
In summary, the CD4+ and CD8+ T cell supernatants contained lower levels of IFN-g and higher levels of IL-4 and IL-10 in HQGZWW high dose and prednisone groups compared with HQGZWW low dose and distilled water groups. Our data suggested that there is Th2 cytokine profile of CD4+ and CD8+ T cells in HQGZWW high dose and prednisone groups, and it was consistent with the EAE score, histological studies in Figure1, and proliferation assay in Figure 3. By the way, in Figure 4, compared to CD4+ T cells, there is lower IFN-g and similar IL-4 and IL-10 secretion in CD8+ autoreactive T cells of 4 groups; and it is also consistent with our previous published data in EAE and EAU studies. [17, 19]
Adoptive transfer of MOG35–55 -specific CD8+ T Cells to naïve mice induces tEAE
To determine the role of MOG35–55-specific CD8+ T cells in the pathogenesis of EAE, we induced the disease in wild-type B6 naïve mice by adoptive transfer of MOG35–55-specific T cells from B6 aEAE mice of four groups upon four treatments (distilled water, HQGZWW high dose, HQGZWW low dose or prednisone) and then measured the clinical signs using the EAE score assessed by daily checks and histopathology. [17]
As shown in Figure 1A, from day 0 to day 21st after MOG35–55 adoptively transferring, the EAE score were measured. For the maximum EAE score, compare with distilled water group (2.95± 0.09), there were significant improvement of EAE score in both HQGZWW high dose and prednisone groups (2.10± 0.10 and 2.03 ± 0.04, respectively, P = 0.001), however only slight improvement in HQGZWW low dose group (2.91 ± 0.07, P = 0.742). Among different treatments, it was shown that HQGZWW high dose group had much better effective on EAE mice than HQGZWW low dose group (P = 0.001), and similar to prednisone group (P = 0.631).
It revealed moderate inflammation and demyelination in distilled water group (HE, Fig.1B and LFB, Fig. 1F) or HQGZWW low dose group (HE, Fig.1D and LFB, Fig. 1H); on the another hand, there is slight inflammation and demyelination in HQGZWW high dose group (HE, Fig.1C and LFB, Fig. 1G) or prednisone group (HE, Fig.1E and LFB, Fig. 1I). Our data suggested that HQGZWW had similar effect at high dose level to typical conventional medicine prednisone on tEAE, but no effect at low dose level.