*2.5. Astaxanthin Inhibited the Migration Capability of LPS-Induced DCs*

DCs that are stimulated with inflammatory mediators can mature and migrate from nonlymphoid regions to lymphoid organs for initiating T cell-mediated immune responses. This migratory step is closely related to the CCR7 expression of DCs [35]. To investigate whether astaxanthin modulated the DC migration, the expression levels of CCR7 in DCs were analyzed by FCM. With LPS treatment alone, CCR7 expression was significantly increased, whereas they remarkably declined after the treatment of astaxanthin (Figure 6A,B). Moreover, chemotaxis assay in transwell chambers was used to examine the DC migration on the basis of attraction of mature DCs for CCL19 or CCL21. The migration of LPS-induced DCs was remarkably inhibited after the treatment of astaxanthin in response to CCL19 (Figure 6C,D). These results suggested that astaxanthin significantly inhibited the migration capability of LPS-induced DCs.

**Figure 6.** Astaxanthin decreased the LPS-induced CCR7 expression and DC's migration ability in vitro. DCs were incubated by astaxanthin or plus 100 ng/mL LPS for 24 h. (**A**,**B**) FCM analysis of CCR7 expression. Data shown are the means ± s.d. of three replicates and are representative of three independent experiments. (**C**,**D**) DCs from astaxanthin (10 μM) alone, LPS (100 ng/mL) alone, astaxanthin (10 μM) plus LPS (100 ng/mL) groups were seeded into the upper wells of a 24-well transwell chamber, and CCL19 (200 ng/mL) was included in lower chamber. After 4 h, the number of DCs that were transferred from the upper to the lower wells was counted by FCM. The spontaneous migration of cells (absence of CCL19) was also shown. Data shown are the means ± s.d. of three replicates and are representative of three independent experiments. Statistical significance is assessed by one-way ANOVA analysis to compare the results between different groups. \*\* *p* < 0.01.

### *2.6. Astaxanthin Impaired the Allostimulatory Capacity of LPS-Induced DCs*

Mature DCs are potent stimulators of allogeneic T cell proliferation in the mixed lymphocyte reaction (MLR) [36]. To determine the effects of astaxanthin on the ability of LPS-induced DCs to stimulate the MLR, DCs were collected and incubated with allogeneic CD4+ T cells. As shown in Figure 7, LPS-induced DCs stimulated proliferative responses more effectively than untreated DCs, while astaxanthin-treated DCs impaired proliferative responses derived from the LPS stimulation at all ratios of DC: T cell tests. These results suggested that astaxanthin strongly impaired the allostimulatory capacity of LPS-induced DCs.

**Figure 7.** Astaxanthin decreased LPS-induced DCs to increase the proliferation of allogeneic T cells. After incubation with astaxanthin or plus 100 ng/mL LPS for 24 h, the collected DCs were used in two graded cell numbers (DC/T-cell ratios: 1:1 (**A**,**C**) and 1:5 (**B**,**D**)) to stimulate CFSE-labeled naive CD4+ allogeneic T cells (5 <sup>×</sup> 105 responder cells per well). After 5 days, proliferation was detected by FCM. Data shown are the means ± s.d. of three replicates and are representative of three independent experiments. Statistical significance is assessed by one-way ANOVA analysis to compare the results between different groups. \*\* *p* < 0.01.
