*2.7. Astaxanthin Protected the LPS-Induced Immune Dysfunction of DCs via Activation of HO-1/Nrf2 Axis*

To investigate whether astaxanthin modulated the DC maturation by the HO-1/Nrf2 pathway, the expression levels of HO-1 and Nrf2 on DCs were analyzed by FCM. As shown in Figure 8A–D, treatment of LPS-induced DCs with astaxanthin, HO-1, and Nrf2 were significantly upregulated, compared with the LPS-only group. Next, to study whether HO-1 played an important role in the suppression of DC maturation, the cytokine release (TNF-α and IL-10) (Figure 8I,J) and phenotypic markers (CD80 and CD86) (Figure 8E–H) were detected. The results showed that the effects of astaxanthin in the LPS-induced DCs were diminished when DCs were pretreated with SnPP (a HO-1 inhibitor) (Figure 8E–J). However, CoPP (a HO-1 inducer) aggravated the inhibitory effect of astaxanthin in the LPS-induced DCs (Figure 8E–J). Therefore, the Nrf2/HO-1 pathway played an important role in the inhibition of LPS-induced DCs maturation by astaxanthin.

**Figure 8.** Astaxanthin enhanced the expression of HO-1 and Nrf2 protein in the LPS-induced DCs. (**A**–**D**) DCs were incubated by astaxanthin or plus 100 ng/mL LPS for 24 h. FCM analysis of HO-1 and Nrf2 expression. Data shown are the means ± s.d. of three replicates and are representative of three independent experiments. (**K**) Experimental setting to study DC maturation, DCs were treated with 10 μM astaxanthin or plus 100 ng/mL LPS in the presence or absence of SnPP (25 μM) or CoPP (50 μM) for 24 h. (**E**–**H**) The expressions of CD80 (**E**,**G**) and CD86 (**F**,**H**) were detected by FCM. (**I**,**J**) TNF-α and IL-10 released from supernatants were detected by ELISA. The 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* < 0.05; \*\* *p* < 0.01. SN: supernatant.

#### *2.8. Astaxanthin Protected LPS-Induced Sepsis in Mice*

The overwhelming production of pro-inflammatory cytokines and mediators results in tissue damage or lethality. To determine the effects of astaxanthin on the LPS-induced septic lethal rate and production of cytokines in LPS-challenged mice, firstly, the biosafety of astaxanthin was evaluated in mice. As shown in Figure 1B, the body weight of mice was not changed in the astaxanthin group compared with the control group, even if the dose used was up to 300 mg/kg. Next, the changes in body weight and survival rates were monitored after LPS injection for 3 days or 40 h, respectively. As shown in Figure 9A, LPS administration markedly increased the loss of body weight in mice. However, the astaxanthin recovered the change of body weight in the LPS-challenged mice. Moreover, the astaxanthin decreased the mortality of the LPS-treated mice (Figure 9B). Next, the levels of cytokines in mice serum were detected by ELISA. The results showed that administration of astaxanthin significantly decreased the production of TNF-α, IL-6, and IL-10 (Figure 9C–E). Taken together, these data demonstrated that astaxanthin effectively protected LPS-induced sepsis in mice.

**Figure 9.** Astaxanthin recovered the changes in body weight and decreased the cytokines secretion in LPS-challenged mice. (**A**,**B**) The data represent the body weight changes and survival rates of each group (*n* = 10/group). (**C**–**E**) The level of cytokines in plasma was measured by ELISA. 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.

#### **3. Discussion**

Here, we explored the immunosuppressive properties of astaxanthin on the activation and maturation of DCs for the first time. Our data indicated that astaxanthin reduced the expression of activation markers (CD69), LPS-induced pro-inflammatory (TNF-α and IL-6), and anti-inflammatory (IL-10) cytokines by DCs; reversed the morphological changes of LPS-activated DCs; decreased the LPS-induced expression of phenotypic markers by DCs, including MHCII, CD40, CD80, and CD86; promoted the endocytosis levels in LPStreated DCs; and hindered the LPS-induced migration of DCs via downregulating CCR7 expression. Furthermore, astaxanthin abrogated allogeneic T cell proliferation by LPSinduced DCs. Finally, astaxanthin enhanced the survival rate of LPS-challenged mice and inhibited the production of inflammatory cytokines in serum, suggesting that astaxanthin can strongly protect LPS-induced sepsis (Figure 10).These results powerfully implied that astaxanthin may have a potential application in the treatment of sepsis.

Toll-like receptor (TLR) 4 signaling, leading to secretion of inflammatory productions, has been considered as a critical pathway in sepsis pathophysiology. LPS from gramnegative bacteria interacted with TLR4 to cause phagocytic cells to robustly generate a variety of proinflammatory cytokines [37]. CD69, as a type II C-type lectin, is known as a very early activation marker, which is first upregulated upon primary activation [38,39]. In our study, we found that astaxanthin reduced the activation level of LPS-treated DCs by downregulating CD69 expression, suggesting that the immunosuppressive ability of astaxanthin was involved in the early inflammatory response. After DC activation, a mass of inflammatory cytokines was released. TNF-α, as a rapid proinflammatory cytokine, can strongly accelerate DC maturation [40]. Furthermore, TNF-α also can regulate other inflammatory cytokines, especially for IL-6 [41], implying that astaxanthin might suppress the secretion of TNF-α, and then result in the down-expression of IL-6 in DCs. At the late stage of sepsis, the anti-inflammatory state may appear, showing a high expression of

IL-10, which may result in a further impaired immune response with an increased risk of nosocomial infections [42]. Therefore, we evaluated the effects of astaxanthin treatment in LPS-induced IL-10 expression, and found that IL-10 was also decreased, and thereby, astaxanthin plays a remarkable inhibition role on both pro- and anti-inflammatory stages.

**Figure 10.** Schematic of the proposed mechanism for astaxanthin, rescuing the LPS-induced immune dysfunction of DCs and protecting LPS-induced sepsis in mice. (**A**) Astaxanthin firstly activated the Nrf2 signaling pathway, and then significantly upregulated HO-1 expression, which suppressed the immune functions of LPS-induced DCs, including activation markers (CD69), the cytokines release (TNF-α, IL-6, and IL-10), phenotypic marker (MHCII, CD40, CD80, and CD86) and migration marker (CCR7). (**B**) Astaxanthin decreased the production of TNF-α, IL-6, and IL-10 in serum, recovered the change in body weight and decreased the mortality of the LPS-treated mice.

DCs possess two major states, including immature DCs (iDCs) and mature DCs (mDCs). The iDCs have a strong antigen capture ability with lower expression of phenotypic markers. After antigen uptake, iDCs were transformed into mDCs, which have a strong ability to stimulate the proliferation and differentiation of T cells by upregulating the surface levels of MHCII and costimulatory molecules. Moreover, DCs can easily mature into inflammatory DCs, thereby sustaining a continuous activation of the adaptive immune response at inflammation sites [43]. However, iDCs were able to induce immune tolerance, and have therefore been introduced as a therapy for systemic lupus erythematosus (SLE) [44,45]. In our data, astaxanthin can effectively inhibit LPS-induced phenotypic markers of DCs, including MHCII, CD40, CD80, and CD86, suggesting that astaxanthin was able to prevent the transformation from iDCs into mDCs. In addition, LPS-induced DCs with astaxanthin treatment possessed a strong antigen capture ability, indicating that the DCs remain in an immature state. Furthermore, once DCs mature, the chemokine receptor CCR7 displays a high-upregulation, which will guide the DCs to migrate toward a draining lymph node, a T cell-rich area with a high expression of CCL19 and CCL21 (CCR7 ligands), for an expanded immune response [46]. Our data suggested that astaxanthin could probably block the connection between DCs and draining lymph nodes via down-regulating CCR7 expression, and lead to limit extensive immune responses. Even if contact happened, LPS-induced DCs with astaxanthin treatment were hardly promoted to a proliferation of allogeneic T cells in our allogeneic mixed lymphocyte reaction assay, which might be associated with the down-regulation of MHCII, costimulatory molecules, and cytokines.

Inflammation is the most common feature of many chronic diseases and complications. Previous studies have revealed that the transcription nuclear factor erythroid 2-related factor 2 (Nrf2) contributes to the anti-inflammatory process by orchestrating the recruitment of inflammatory cells and regulating gene expression through the antioxidant response element (ARE) [47]. Heme oxygenase-1 (HO-1) is the inducible isoform and rate-limiting enzyme that catalyzes the degradation of heme into carbon monoxide (CO) and free iron, and biliverdin to bilirubin [48]. Several studies have demonstrated that HO-1 and its metabolites have significant anti-inflammatory effects mediated by Nrf2 [49]. It has been reported that activation of Nrf2 prevents LPS-induced transcriptional upregulation of pro-inflammatory cytokines, including IL-6 and IL-1β [50]. Here, we have demonstrated that astaxanthin inhibited the maturation of LPS-induced DCs via the activation of the HO-1/Nrf2 axis. Interestingly, astaxanthin is a potential antioxidant, and the HO-1/Nrf2 axis is also a key known antioxidative pathway; whether astaxanthin utilizes its antioxidant property to activate the HO-1/Nrf2 pathway and then to initiate an anti-inflammatory response needs to be further investigated.

LPS and other PAMPs are related in the pathogenesis of sepsis and the activation of immune responses, resulting in tissue pathological injury and multiple organ failure [51]. Management of excessive inflammatory response is a key strategy for sepsis treatment [52]. In the present study, we performed a series of experiments to determine the anti-inflammatory activities of astaxanthin using LPS-challenged mice. Our results showed that administration of astaxanthin promoted the survival rate of LPS-challenged mice. Additionally, administration of astaxanthin reduced the levels of inflammatory cytokines in serum, including TNF-α, IL-6, and IL-10, which was in line with the result of DCs in vitro. These results implied that DC-targeted anti-inflammatory strategies have great potential in the treatment of sepsis.
