*4.9. Allogeneic Mixed Lymphocyte Reaction Assay*

Male BALB/c mice, 6 weeks old, were from the Animal Research Center of Yangzhou University (Jiangsu, China). Responder T cells were purified from mice splenic lymphocytes using a CD4+ T cell isolation kit and labeled with CFSE according to the manufacturer's instructions. Next, these cells were cocultured in duplicate with DCs (DC/T cell ratios of 1:1 or 1:5) in 5% CO2 incubator at 37 ◦C for 5 days and detected by FCM.

#### *4.10. HO-1 and Nrf2 Protein Expression Assay*

The treated DCs were incubated with Alexa Fluor 647 HO-1, PE-Nrf2, or the respective isotypes for 30 min at 4 ◦C. The cells were analyzed using FCM.

#### *4.11. Body Weight Change Assay*

Six-week-old C57BL/6 mice were divided into five groups (*n* = 10/group). In the treatment group, the mice were given astaxanthin orally for 4 days every 24 h, and the doses of astaxanthin were 50, 100, and 200 mg/kg, respectively; 48 h after the firstly oral administration, the mice received LPS (10 mg/kg body weight) by intraperitoneal injection, body weight changes were monitored for 3 days.

#### *4.12. Survival Rate and Cytokine Assay*

48 h after 1st oral administration, the mice received LPS (20 mg/kg body weight) by intraperitoneal injection, survival rates were monitored for 40 h as described previously [56]. The mice were euthanized and blood was collected at 4 h after LPS injection, the levels of cytokines (TNF-α, IL-6, and IL-10) in plasma were measured by an ELISA kit according to the manufacturer's protocol.

#### *4.13. Statistical Analysis*

Results were expressed as the means ± SD. Statistical significance between the 2 groups was determined by unpaired Student's two-sided *t*-test. To compare multiple groups, one-way ANOVA with Tukey's post hoc test was performed by using SPSS 17.0. \* *p* < 0.05, \*\* *p* < 0.01.

#### **5. Conclusions**

In summary, our findings showed that astaxanthin inhibited the immune dysfunction of DCs induced by LPS via the activation of HO-1/Nrf2 axis in vitro, and enhanced the survival rate of LPS-challenged mice in vivo, which might be used as a potential candidate strategy for clinical sepsis.

**Author Contributions:** Conceptualization, Y.Y., C.L. and J.Y.; methodology, Y.Y., N.X., Y.S. and B.M.; software, Y.Y., B.Z. and D.S.; validation, Y.Y., C.L. and J.Y.; formal analysis, Y.Y., N.X., C.L. and J.Y.; investigation, Y.Y., N.X. and Z.X.; resources, Y.Y., C.L. and J.Y.; data curation, Y.Y., N.X.; writing—original draft preparation, Y.Y., C.L. and J.Y.; writing—review and editing, Y.Y., C.L. and J.Y.; visualization, Y.Y., N.X.; supervision, Y.Y., C.L. and J.Y.; project administration, Y.Y., C.L. and J.Y.; funding acquisition, Y.Y., C.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (31600113, 31800284), the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province (CX(20)3092), a project funded by the Priority Academic Program Development of Jiangsu Higher Education (PAPD), and the Open Project Program of Jiangsu Key Laboratory of Zoonosis (R1909).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of Jiangsu Laboratory Animal Welfare and Ethical, and approved by the Jiangsu Administrative Committee of Laboratory Animals (Permission number: SYXKSU-2007-0005).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

