Crude Polysaccharides From Cistanche Deserticola Y.C. Ma As An Immunoregulator And An Adjuvant

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Abstract: Cistanche deserticola Y. C. Ma (CD) are ideal herbal remedies for health problems. In this study, modulator activities of CPCD on activating dendritic cells (DCs) and the adjuvant potential for the foot-and-mouth disease vaccine (FMDV) were evaluated. CPCD without cytotoxicity induced DC activation via upregulating the phenotypic markers and antigen uptake. TLR-2 or TLR-4 antibodies suppressed levels of CPCD-mediated CD40 and CD86 as well as IL-6 and IL-1β in DCs. CPCD induced the phosphorylation of MAPKs related molecules and NF-κB. When co-injected intramuscularly with FMDV in mice, CPCD increased FMDV-specific antibody, lymphocyte proliferation, effector T-cells. Specifically, CPCD elicited Th1 response, as indicated by the higher expressions of CD4+IFN-γ, CD8+ IFN-γ, and CTL response. CPCD enhanced the expressions of MHC-II, CD80, CD86, and CD40 in DCs and lowered Tregs frequency. Therefore, CPCD could effectively stimulate stronger humoral and cellular responses by modulating DC activation through TLR-2/TLR-4 related MAPKs and NF-κB pathways.

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1:Introduction

Most of the polysaccharides derived from medicinal herbs have attracted increasing interest in the field of adjuvants due to their low toxicity and the broad-spectrum immune booster (Chen & Huang, 2018; He et al., 2020). Many investigators have successfully used medicinal herbs as adjuvant candidates for many vaccines. Typical polysaccharides including Astragalus polysaccharide, Panax ginseng polysaccharide, Poria cocos polysaccharide, and lentinan have been widely used as adjuvants for the vaccines of several infectious diseases (P. Li & Wang, 2015; ReyLadino, Ross, Cripps, McManus, & Quinn, 2011; B. Sun et al., 2018). Cistanche deserticola Y. C. Ma (C. deserticola, “Rou Cong-Rong” in Chinese) has been traditionally used as a tonic herbal supplement for the treatment of diseases in Traditional Chinese Medicine (TCM) according to Chinese Pharmacopoeia for centuries (S. Li, Wu, & Le, 2021; Yong & Tu, 2009). In vivo and in vitro studies revealed that C. deserticola extracts exhibited many pharmacological functions including alleviating fatigue and anti-inflammatory, neuroprotective, antiviral, and immunomodulatory functions (Fu, Fan, Wang, & Gao, 2018; Tian Wang, 2012). Polysaccharides from C. deserticola, the active constituents of C. deserticola, have been proved to have broad-range biological activities including the modulation of immune response and antioxidative anti-inflammatory, and anti-fatigue functions (Dong, Yao, Fang, & Ding, 2007; W. Zhang et al., 2016). In 2018 and 2019, we demonstrated that crude polysaccharides of C. deserticola (CPCD) acted as an adjuvant for OVA antigen favoring Th1/Th2 immune responses. CPCD could induce DC activation through Toll-like receptor 4 (TLR-4) (Zhang et al., 2018). CPCD was also found to elicit Th1 immune responses, including IFN-γ secretion and IgG2a generation against influenza vaccine in mice (Zhao, Lian, Wang, Li, & Zhang, 2019). Therefore, it is necessary to uncover the underlying mechanism of CPCD-elicited immune response and the immune potentiating effect of CPCD on inactivated FMDV.

Foot-and-mouth disease (FMD) is an important viral disease in animals since it may lead to high economic losses and social consequences. Inactivated FMD vaccines (FMDV) are the most effective control measures of FMD. Current inactivated FMDV are safe but poorly immunogenic due to low antibody production, short duration of antibody persistence as well as poor immune responses in swine (Y. Cao, Lu, & Liu, 2016; Doel, 2003). Higher vaccine doses or increased treatment times, different injection routes (intramuscular versus intradermal vaccination), and accelerated dosing programs have been used as important strategies for improving the protective efficacy of vaccines. Increasing evidence has demonstrated that adjuvants can boost vaccine efficacies against infectious diseases (Steven G. Reed, Orr, & Fox, 2013; N. Zhang et al., 2010). Novel adjuvants and their incorporation into FMDV may increase immunogenicity and even improve the duration of protection (Y. Cao, 2014; Dar, Kalaivanan, Sied, Mamo, & Kondabattula, 2013). Currently, many efforts are being devoted to developing safe and effective adjuvants that can maximize immunogenicity with a tailored immune response (Pulendran & O’Hagan, 2021). Diverse adjuvants are required for vaccines. Compared with traditional adjuvants, polysaccharide adjuvants from TCM may be helpful to exert immune potentiating effects on FMDV by modulating lymphocytes, cytokines, and antibody levels and generating stronger cellular and humoral immune responses.

The activation of dendritic cells (DCs) has been proven to initiate adaptive immune responses and regulate Th1/Th2 immunity balance (Kapsenberg, 2003). The effect of new adjuvants on DCS needs to be elucidated. TCM polysaccharides could improve the expression of surface molecules on DCs, including MHC II, CD80, CD86, and CD40, and augment the level of pro-inflammatory cytokines, such as IL-6 and IL-1β (J. Li, Li, & Zhang, 2015). The immunomodulatory activities of TCM polysaccharides are achieved through directly binding membrane receptors on DCs (Jiang, Zhu, & Jiang, 2010). In other words, the immunoadjuvant activity of CPCD is associated with the immunomodulatory effects on DCs. Additionally, TLRs are sentinel receptors promoting the activation and recruitment of DCs and triggering B cell and T cell responses. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) pathways are critical downstream pathways of TLRs and activating these pathways can induce the production of proinflammatory cytokines including IL-6 and IL-1β (Akira & Takeda, 2004). Therefore, it is necessary to investigate TLR-related downstream pathways for the purpose of understanding immune cell responses to infection. In this study, the ICR mouse was usually chosen as a model animal in the laboratory for FMDV to determine a relationship between adjuvant and vaccine potency because of the inconvenient use of targeted animals, and CPCD as an FMDV adjuvant was investigated in vitro and in vivo to explore its role in DCs activation by TLR-2/TLR-4-related downstream MAPKs and NF-κB pathways. This study provides the basis for understanding the induction mechanisms of immunomodulatory activities by CPCD as well as the potential of CPCD as a polysaccharide adjuvant for FMDV in targeted animals.

2:Material and methods

C. deserticola (No. CD17042001) was provided the identification by Professor Jiang He. Inactivated FMD viral O-serotype 146S antigen and ISA-206 adjuvant were kindly offered by Tecon Biology Co., Ltd. (China). Fetal bovine serum (FBS) was from Biological Industries. RPMI 1640 medium was purchased from GIBCO (USA). polymyxin B (PMB), Lipopolysaccharide (LPS), 3-(4,5-dimethyl thiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) were Sigma product. Cell Counting Kit-8 (CCK-8) was from Biosharp (China). Murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was from PeproTech (USA). IL-6 and IL-1β kits were from Boster (China).

Golgistop, Cytofix/Cytoperm, Perm/Wash buffer, and antibodies against CD3, CD4, CD8, CD44, CD11c, MHC-II, CD80, CD86, CD40 were from BD Biosciences (USA). Nuclear and cytoplasmic extraction kits and BCA protein quantitation kits were from Cwbiotech (China). Anti-TLR-2 and anti- TLR-4 antibodies were purchased from Novus Biologicals (USA). MAPKs and NF-κB pathway kits were from Cell Signaling Technology (USA). Horseradish peroxidase-labeled anti-mouse/rabbit antibodies (IgG/IgG1/IgG2a) from Southern Biotech (USA). Tregs staining kit from eBiosciences (USA).

3:result

Neutral carbohydrate content was determined to be 50.53% ± 1.28% and acidic carbohydrate content was 17.81 ± 0.51%. The average molecular weight (Mw) of CPCD was calculated to be 46.77 kDa with retention time (Fig. 1A). The monosaccharide composition of CPCD reveals that the major sugar components of CPCD are Glc, Ara, Gal, Rha, GlaA, and Man in molar percentages of 33.17%, 28.98%, 16.39%, 10.97%, 8.05%, 2.42% (Fig. 1B). Fig. 1C shows the FT-IR spectrum of CPCD. The brands at 3383.64 cm− 1 were due to the hydroxyl group. The absorption bands of the alkyl group were observed at 2926.97 cm− 1 and 1417.99 cm− 1. The absorption bands of the carbonyl group were observed at 1635.56 cm− 1. The signals from 1257.06 to 1032.02 cm− 1 indicated the presence of pyranose rings. To further investigate whether MAPKs and NF-κB pathways were activated by CPCD treatment, DCs were stimulated with CPCD at a different time and the expressions of key proteins involved in the two pathways were examined. CPCD induced the phosphorylation of JNK, ERK, and p38 in a time-dependent manner and the phosphorylation levels of JNK, ERK, and p38 were the highest after 15–30 min (Fig. 5AD). In addition, the phosphorylation of cytoplasmic NF-κB p65, IκBα, IKKα/β, and nuclear NF-κB p65 also was induced in a time-dependent manner. The phosphorylation levels of cytoplasmic p65, IκB, and IKKα/β, and the proportion of p65 in the nucleus were the highest after different times: 15–30 min for p65, 30 min for IκB, 60 min for IKKα/β, and 20 min for the proportion of p65 in the nucleus (Fig. 5E-5 J).

This article is extracted from Journal of Functional Foods 87 (2021) 104800 journal homepage: www.elsevier.com/locate/jff