*2.3. Astaxanthin Impaired the Phenotypic Maturation of LPS-Induced DCs*

Maturation is the key step in the DC-mediated regulation of immune responses. To investigate whether astaxanthin modulated the DC maturation, the expression levels of MHCII and costimulatory molecules in DCs were analyzed by FCM. With LPS treatment alone, the expressions of MHCII, CD40, CD80, and CD86 were markedly upregulated, whereas they were down-regulated remarkably with the treatment of astaxanthin (Figure 4). These data suggested that astaxanthin diminished LPS-activated DC phenotypic maturation and compromised the immunostimulation of the activated DCs.

**Figure 4.** Astaxanthin suppressed the expression of phenotypic markers by LPS-stimulated DCs in vitro. After stimulation for 24 h with astaxanthin or plus 100 ng/mL LPS, the expressions of phenotypic markers on DCs, including MHCII (**A**,**E**), CD40 (**B**,**F**), CD80 (**C**,**G**), and CD86 (**D**,**H**), were analyzed 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.

#### *2.4. Astaxanthin Increased the Endocytosis Capability of LPS-Induced DCs*

In response to inflammatory stimuli, DCs trigger the process of maturation; downregulation of endocytosis is a hallmark of maturation [34]. To investigate whether astaxanthin modulated the endocytosis of DCs, the fluorescent marker dextran was used. As shown in Figure 5A,B, LPS alone significantly decreased the endocytosis capability of DCs compared to the untreated control, while astaxanthin enhanced the uptake of dextran in LPS-induced DCs. Moreover, confocal laser scanning microscopy (CLSM) images displayed the amount of Alexa Fluor 647-dextran existing in the body of LPS-induced DCs and was enhanced after the treatment of astaxanthin (Figure 5C). These results suggested that astaxanthin significantly increased the endocytosis capability of LPS-induced DCs.

**Figure 5.** Astaxanthin enhanced the endocytosis ability of DCs after LPS treatment in vitro. After stimulation for 24 h with astaxanthin or plus 100 ng/mL LPS, the treated DCs were incubated with 1 mg/mL FITC-Dextran (**A**,**B**) or Alexa Fluor 647-Dextran (**C**) at 37 ◦C for 30 min. After incubation, the cells were washed three times with cold PBS and analyzed by FCM (**A**) or were observed by using confocal laser scanning microscopy (CLSM). Parallel experiments were performed at 4 ◦C to determine the nonspecific binding. The data shown are the means ± s.d. of three replicates and are representative of three independent experiments. (**C**) Dextran (Alexa Fluor 647; red) and Nuclei (4 ,6-diamidino-2-phenylindole (DAPI); blue). The results are from one representative experiment of three performed. Bars: 10 μm. Statistical significance is assessed by one-way ANOVA analysis to compare the results between different groups. \*\* *p* < 0.01.
