**1. Introduction**

The immune system, as a tight and dynamic regulatory network, maintains an immune homeostasis, which keeps a balance between the response to heterogenic antigens and tolerance to self-antigens [1]. However, in some diseases, such as sepsis, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), and inflammatory bowel disease (IBD), this immune homeostasis is broken [2]. Sepsis is a highly heterogeneous clinical syndrome that mainly results from the dysregulated inflammatory response to infection, which continues to cause considerable morbidity and accounts for 5.3 million deaths per year in high income countries [3]. Recently, the incidence of sepsis is progressively increased and sepsis-related mortality cases remain at a high level in China [4]. The host immune response induced by sepsis is a complex and dynamic process. After infection, the conserved motifs of pathogens, termed the pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS, cell wall component of gram-negative bacteria) or lipoteichoic acid (cell wall component of gram-positive bacteria), are recognized by the pattern recognition receptors (PRRs) expressed by immune cells, and an overwhelming innate immune response is triggered in septic patients [5,6]. Under physiological conditions, the immune activation contributes to eliminating pathogens and clearing infected cells. However, when driven by sepsis, the immune homeostasis

**Citation:** Yin, Y.; Xu, N.; Shi, Y.; Zhou, B.; Sun, D.; Ma, B.; Xu, Z.; Yang, J.; Li, C. Astaxanthin Protects Dendritic Cells from Lipopolysaccharide-Induced Immune Dysfunction. *Mar. Drugs* **2021**, *19*, 346. https://doi.org/10.3390/ md19060346

Academic Editors: Donatella Degl'Innocenti and Marzia Vasarri

Received: 21 May 2021 Accepted: 15 June 2021 Published: 17 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

appears imbalanced and initiates a life-threatening "cytokine storm". Currently, no drugs have been approved specifically for the treatment of sepsis, and clinical trials of potential therapies have failed to reduce mortality; therefore, new approaches are needed. Immunemodulatory intervention is the main potential therapeutic strategy against sepsis [7]. For instance, single cytokine, or a combination of multiple cytokines, including G-CSF, GM-CSF, IFN-γ, IL-3, IL-7, and IL-15, were introduced into sepsis therapy, according to a diseasespecific progression and patient immune responses [8]. Some immunosuppressive agents, such as bursopentin [9], curcumin [10], and oleuropein [11], provided protection against inflammatory injury in the LPS-induced sepsis models.

Dendritic cells (DCs), as the most important potent antigen-presenting cells, link the innate and adaptive immune response. The maturation/activation of DCs is followed by the transformations of phenotype and function, improving their migration ability to draining the lymph node, resulting in the activation of downstream T lymphocyte cells [12]. In fact, DCs reside in all the tissues of the host mainly in an antigen-capturing state and maintain immune tolerance by migrating to the lymph nodes for presenting self-antigens to lymphocytes in a tolerogenic manner [13]. Therefore, DCs also balance the immune homeostasis in the host, and guide the skewing of the downstream immune response [14]. Notably, the abnormalities of DC homeostasis are implicated in sepsis. The differentiation level of monocytes into DCs is improved during sepsis [15]. The expression levels of surface molecules related to the DC function are changed [16]. Considering the critical role of DCs in the immune regulation in sepsis, the modification of the DC system is becoming an increasingly important target for sepsis therapy [15]. Modificatory DCs by adenovirus/IL-10 transduction maintained an immature state with low expressions of IL-12, CD86, and MHCII, and the survival rate of septic mice remarkably increased [17,18]. Bursopentin inhibited the LPS-induced phenotypic and the functional maturation of DCs [9,19]. These studies indicated that compartmental modification of DC function can alter the sepsisinduced immune response.

Astaxanthin, 3,3 -dihydroxy-β,β -carotene-4,4 -dione, is a naturally occurring red carotenoid pigment classified as a xanthophyll, found in microalgae and seafood such as salmon, trout, and shrimp [20,21]. The lipid-soluble carotenoid, with a polar–nonpolar– polar structure, is able to help astaxanthin easily pass through and fix into the double layers of the cell membrane. Moreover, free radicals inside and outside of the cell membrane can be scavenged by the polar structure of astaxanthin, and radicals located in the cell membrane can be captured by its polyene chain [22]. Therefore, astaxanthin has a strong antioxidant property, and is regarded as a potential candidate agent against many diseases [23–26]. Recent studies have shown that astaxanthin had a variety of pharmacological effects against inflammatory injury [27–29]. Astaxanthin provided a neuroprotection against diabetes-induced sickness behavior through inhibiting inflammation [30]. Astaxanthin also can attenuate monosodium urate crystal-induced arthritis by suppressing the level of pro-inflammatory cytokines [31]. Moreover, astaxanthin was shown to suppress LPS-induced inflammatory factors increase, MAPK phosphorylation, and NF-kB activation in vivo [32]. These studies demonstrated to us that astaxanthin have a great potential as a therapeutic agent of sepsis by an anti-inflammatory strategy.

In this study, we attempted to characterize the effects of astaxanthin on the immune activation and functional properties of the LPS-induced DCs for potential sepsis therapy. Our data suggested that astaxanthin protected DCs from LPS-induced immune dysfunction, which might be a simple, inexpensive, and highly effective anti-inflammatory strategy via regulating DC activity in sepsis.

#### **2. Results**

#### *2.1. Astaxanthin Inhibited LPS-Induced Cytokine Production by DCs*

Firstly, the biosafety of astaxanthin was evaluated in the murine DCs. The cells were treated with astaxanthin and the cell viability was analyzed by the CCK-8 assay. The results revealed that the cellular viability was not changed until 24 h after treatment

with astaxanthin up to 50 μM (Figure 1A). Next, we examined the expression of CD69, which is a critical activation marker of DCs. After exposure to LPS (100 ng/mL) for 24 h, the expression of CD69 was upregulated, whereas they were significantly inhibited with treatment of astaxanthin (Figure 2A,B). In addition, we tested whether astaxanthin affected the production of cytokines in LPS-induced DCs. Significantly, pro-inflammatory cytokines (TNF-α and IL-6) were downregulated by astaxanthin in a dose-dependent manner (Figure 2C,D). Surprisingly, the secretion of IL-10 was not increased (Figure 2E), implying that the suppressive effect of astaxanthin probably was not mediated through antiinflammatory cytokine. These results indicated that astaxanthin attenuated the cytokines secreted by LPS-induced DCs.

**Figure 1.** Biosafety evaluation of astaxanthin in vitro and in vivo. (**A**) The cytotoxicity of astaxanthin with different doses was performed in the DCs by using the CCK-8 assay. (**B**) Astaxanthin with different concentrations was given orally for five days every 24 h; the data represent the change of body weight in each group (*n* = 10/group). The data shown are the means ± s.d. of three replicates and are representative of three independent experiments. Statistical significance is assessed by unpaired Student's two-sided *t*-test to compare astaxanthin (0 μM).

**Figure 2.** Astaxanthin suppressed the secretion of cytokines from LPS-stimulated DCs. DCs were incubated with the astaxanthin or plus 100 ng/mL LPS for 24 h. (**A**,**B**) The expression of activation marker CD69 on DCs was analyzed by FCM. (**C**–**E**) Supernatants were collected and TNF-α, IL-6, and IL-10 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* < 0.01.
