With growing unhealthy lifestyles as well as the exposure to stress and environmental extremes, impacts on the immune system have drawn great attention [1
]. The immune system is a network of cells, tissues, and organs, protecting humans from viruses, bacteria, fungi, the growth of cancer cells, etc. Several drugs and chemicals have been developed as immunostimulants to modulate human immunity; however, further investigations of other approaches are required due to their high cost and inevitable side effects [2
]. Among all choices, nutritional interventions have raised great expectations for their effectiveness and wide applications to enhance innate and adaptive immunities as well as resistance to diseases. Various active ingredients derived from natural foods have been demonstrated to be safe and effective in immunity enhancement. Particularly, bioactive peptides derived from food proteins have been shown to be promising in immune modulation for their low-molecular weights, highly digestible and absorbable features, and immunomodulatory, antimicrobial, antioxidative, antithrombotic, antihypertensive, and cholesterol-lowering activity [3
]. Since the biological activity of peptides is strongly associated with the protein origin (specific amino acid sequences and bioavailability), whereas immunomodulatory effects of plant-derived peptides have been less explored [6
], the effects of plant-derived peptides on immunity improvement are well worth exploring.
Oat (often referred to Avena sativa
L.) as an excellent whole grain choice, has been widely used for centuries for its various nutrients and health benefits [7
]. The composition and health benefit of naked oat (Avena nuda
L.), one of the ancient crops originating from China, still requires further investigation, even though the historical references on its detailed applications can be traced back to 1000 years before common era [10
]. In the past decades, studies have demonstrated that naked oat contains sizable nutrients even superior to common oats, such as the highest content of proteins (approximately 16%) among all grains, unsaturated fatty acids (approximately 7%), carbohydrates (approximately 62%), and soluble fiber (approximately 5%) [11
]. Notably, oat protein is not only outstanding in total content, but also in its balanced amino acid composition and high-level of lysine, which most grains lack. On the other hand, the booming demand of oat products and oil has resulted in a great quantity of oat extraction residues, which are abundant in nutritional proteins (mostly albumin and globulin) with a protein efficiency ratio of more than 2.0 [13
]. Except for their nutritional properties, studies on the health effects of oat proteins or peptides are still limited, only involving hyperglycemia, hypertension and dyslipidemia regulations, anti-fatigue effects, and celiac disease [15
]. Worth mentioning, these studies mainly focused on oat proteins or polypeptides, which have distinctly different absorption mechanisms compared to oat oligopeptides [20
]. Oat oligopeptides are more digestible and absorbable than polypeptides with much lower molecular weights and higher bioavailability. Even immunoregulatory effects of food-derived peptides can be of great importance, and naked oat-derived oligopeptides can be a promising immunomodulator superior to both oat proteins and polypeptides, the immunoregulatory effects of oat oligopeptides have not been observed. We aimed to investigate the immunomodulatory effects of oat (Avena nuda
L.) oligopeptides (OOPs) by intragastric administration in BALB/c mice with deionized water treatment as a control, and then explored the possible mechanisms.
Oats are one of the most widely recommended grains with the highest protein concentration among cereals. A number of studies have focused on oat β-glucan, while few have focused on oat proteins let alone oat oligopeptides. Oat protein is known for its positive effects on celiac disease, blood glucose, and cholesterol metabolism, and for its antioxidant and anti-fatigue effects [16
]. On the other hand, food-derived peptides have been considered as one of the most effective alternatives to drugs as immunomodulators [27
]. Therefore, OOPs, mainly composed of small molecule oligopeptides derived from oat (Avena nuda
L.) bran, can also be potential immunomodulators to enhance adaptive and innate immunities which form the immune system in vertebrates. Adaptive immunity is highly specific to antigens, including cell-mediated and humoral (antibody-mediated) immunity. Innate immunity provides non-specific protection, acting as the front line of defense through anatomical, physiological, cellular, and inflammatory components. Among immune cells such as dendritic cells, macrophages, NK cells, and neutrophils [22
], macrophages and polymorphonuclear leukocytes specialize in phagocytosis, while NK cells defend against virus-infected and tumor cells. Since the mechanism through which food-derived bioactive peptides regulate the immune system remains uncertain, we evaluated the immunomodulatory effects of OOPs via four aspects including macrophage phagocytosis, NK cell activity, cell-mediated immunity, and humoral immunity with seven assays. The results of our study showed that OOPs treatments could improve adaptive immunity through improving delayed-type hypersensitivity, ConA-stimulated splenic lymphocyte proliferation, spleen IgM responses to SRBC, and HC50
levels, hence enhancing cell-mediated and humoral immunities, respectively. As for innate immunity, we found that OOPs could enhance the phagocytic capacity of macrophages and NK cell activity.
With further investigation, the immunomodulatory mechanism of OOPs may attribute to the regulation of lymphocyte function, cytokine secretion, and antibody production. To start with, since the key role of cell-mediated immunity is T lymphocytes (T cells, CD3+), consisting of helper T cells (Th cells, CD4+), cytotoxic T cells (Tc cells, CD8+), and regulatory T cells (Treg cells), subsets of T lymphocyte were analyzed. Our study found that the percentages of CD3+ and CD4+ T cells were significantly increased after OOPs treatment, indicating improvement in T cell quantity and proportion especially in Th cells, which can secrete cytokines and mediate cellular immune responses with antigen presenting cells (APCs), in accordance with our adaptive immunity test results.
To be specific, CD4+
T cells consist of T helper type 1 (Th1) cells, which induce cell-mediated immune responses via producing inflammatory cytokines of IL-2, IFN-γ, GM-CSF, and TNF-α, and T helper type 2 (Th2) cells, which induce humoral responses by secreting cytokines of IL-4, IL-5, and IL-10 [29
]. With further investigation in the cytokine profile, we found increases in Th1 secreted cytokines of IL-2, IFN-γ, GM-CSF, and TNF-α in the OOPs groups. The elevated levels of these cytokines led to the improvement of Th1 cell differentiation and growth, secretion of IL-1 and IL-6, elevated functions of B cells, NK cells, and macrophagocytes as well as the negative accommodation of Th2 cells [30
], partly explaining the positive effects of OOPs on the enhancement of cell-mediated immunity assessments including DTH and ConA-stimulated splenic lymphocyte proliferation, and contributing to the improvement of NK cell activity and macrophagocyte function. Moreover, elevated levels of IL-12 (mainly secreted by dendritic cells) in OOPs 1.0 and 2.0 g/kg BW groups implied the enhancement of APC function, which is a vital promoting factor in Th1 cell immune responses and an inhibitor of Th2. As for Th2 cell-secreted cytokines, we also found gradually elevated levels of IL-10, a major immunosuppressive factor, along with increasing doses of OOPs treatments. On the other hand, IL-2 can also improve Treg cell function, which is an inhibitor of CD4+
T cells. Due to the interactions between Th1, Th2 cells and the cytokines they produced, the inhibition of IL-10 and Treg cells in Th1 cell function and secretions of IL-2, IFN-γ, GM-CSF, and TNF-α were likely to result in an unclear dose-effect relation of Th1-secreted cytokines. Possibly along with the inhibition effects of IL-12 and IFN-γ in Th2 cells, the significant changes in IL-4 and IL-5 weren’t observed. In addition, increases in mononuclear macrophage-secreted cytokines such as IL-1α and IL-6 were also found in all four OOPs groups, leading to activated NK cells and enhanced macrophage function, which were in accordance with NK cell activity and macrophagocyte phagocytosis assay results. These results may also attribute to the improvement of macrophagocytes acting as APCs. By processing and presenting antigens to lymphocytes, macrophagocytes can assist T and B lymphocytes in recognizing antigens and regulating immune responses.
On the other hand, humoral immunity involves B lymphocytes (B cell) as a significant part of adaptive immune responses, producing antibodies, binding to antigens, and labeling them for macrophage destruction. Antibodies are the main effectors in humoral immune responses. When the host is under pathogen attack, circulatory IgM and antimicrobial peptides are constitutively expressed as low-cost defense effectors. IgA plays an important role in mucosal immunity by transcytosis across epithelial cells in frontline immunity [31
]. When these responses above are not sufficient, IgG as the richest and most long-lasting antibody in serum is induced, which is crucial in monocyte-macrophage phagocytosis to defend against infections [32
]. A previous study has shown that protein hydrolysates from rohu (Labeo rohita) egg (roe) significantly enhanced humoral immune responses (IgA) [33
]. We more notably observed that OOPs treatment induced major rises in serum IgA, IgM, and IgG concentrations, contributing to humoral immunity, macrophage phagocytosis, and NK cell activity improvements. These can be attributed to antibody functions of IgA, IgM, and IgG as well as the interaction between innate and adaptive immune systems. Combined with the results of cytokine secretions, improvements effects of OOPs on humoral immunity are more likely via increasing antibody production rather than cytokine secretion stimulation. In the future, specific effective peptide segments and sequences can be investigated, as well as the possible content changes after intestinal digestion to further elucidate the biochemical mechanisms of immune enhancement effects of OOPs. Meanwhile, the immunoregulation effects of OOPs under special conditions such as autoimmunity and immunosuppression are also worth exploring.