In this study, we fermented natural fish oil and compared the differences between FFO and NFO on the modulation of the immune system. Administration of FFO potently induced the generation of CD4+CD25+ Foxp3+ Treg in the spleen compared with NFO treatment. Increased TGF-β and Foxp3 at sites of inflammation was associated with elevated CD4+CD25+Foxp3+ Treg populations, which can down-regulate the progression of experimental immune disorders. The ingestion of FFO generated regulatory T cell populations.
AD mainly appears together with a variety of diseases such as eczema, asthma and allergic conjunctivitis. The symptoms of AD include pruritus, peripheral eosinophilia, epidermal hyperplasia and tissue remodeling, such as spongiosis.
In the present study, we used the DNCB-induced experimental AD mouse model to investigate the anti-inflammatory effect of FFO or NFO. IgE is an important target in the treatment of allergy, and signaling through FcεRI can induce histamine release from mast cells, which leads to potent induction of pruritus. Scratching behavior is a typical consequence of pruritus and aggravates the symptoms of dermatological diseases such as AD. Therefore, we tested whether FFO or NFO can reduce scratching behaviors, serum histamine, IgE hyperproduction and cutaneouse edema. The administration of FFO or NFO decreased scratching behavior, IgE, histamine levels and edema compared with the induction group (Figures and A). We predicted that the decreased IgE and histamine levels after FFO or NFO treatment would lead to alleviation of pruritus and cutaneous edema. H&E staining and IHC of the ear tissue confirmed that ingestion of FFO or NFO alleviated inflammatory cell infiltration (CD3+ cells) and expression of TSLP compared with the induction group (Figure B-E). Furthermore, the FFO treatment had stronger inhibitory effects than NFO treatment on most of these signs of allergic disease.
AD is generally characterized by increased numbers of activated CD4+
T cells, and enormous numbers of infiltrating CD4+
T cells in the dermis. In the majority of patients, AD is primarily associated with a dysfunction of the body’s immune. In particular, IL-4 plays a crucial role in allergic responses and the differentiation of Th2 cells from naïve T cells [35
]. Thus, we assessed whether FFO or NFO has anti-inflammatory effects on AD model, which is a Th2-mediated immune disorder. The induction group had extremely elevated IL-4 levels, which were decreased in the FFO and NFO groups. In addition, ingestion of FFO markedly reduced IL-13 and IFN-γ levels and increased the expression of specific Treg cells markers (TGF-β and Foxp3) compared with NFO group in DNCB-stimulated atopic dermatitis mice model (Figure ). Based on these results, we concluded that FFO and NFO have potent anti-inflammatory properties, although the activity of FFO was greater than that of NFO. Increased expression of TGF-β and Foxp3 by FFO treatment at the sites of inflammation could reflect the increased number of Foxp3+
The spleen plays an important role in regulating the immune system and contains a range of immune cells. Also, enlared spleen means a splenomegaly by abnormality of immune system function. We observed the morphologic features of the spleen in AD model. The induction group had markedly enlarged spleens; spleen size was reduced in the FFO and NFO-treated groups. Mice in the FFO group had smaller spleens than mice in the NFO group (Figure A). So, FFO reduced enlarged spleen by alleviating abnormality of immune system function. Th2 cells produce a variety of cytokines, such as IL-4, -5 and −13, and preferentially express GATA3, which is important for Th2 differentiation. Th1 cells produce IFN-γ and the transcription factor (T-bet) is critical for the differentiation of Th1 cells [36
]. We measured whether FFO and NFO ingestion regulate the expression of Th1/Th2 associated factors in experimental AD model. FFO and NFO decreased IFN-γ and IL-4 levels and the expression of T-bet and GATA3 (Figure B-D).
How does FFO suppress a variety of immune response in DNCB-stimulated experimental AD? Regulatory T cells (Tregs) play a key role in various immune responses, including Th2 cell-mediated diseases such as AD, and prevent or suppress differentiation, proliferation, and function of various immune cells including CD4+
T cells in a TGF-β/IL-10/cell contact-dependent manner. Since all Tregs express CD25 at the cell surface and produce the Foxp3 transcription factor, these proteins have been identified as markers of Tregs [37
]. We analyzed whether the administration of FFO affects the expression of factors related to Tregs differentiation. Although FFO treatment did not increase the Foxp3 level and there was no difference in the number of CD25+
Tregs compared to the NFO treatment group, the FFO group did show increased expression of the immune-suppressive cytokines TGF-β and IL-10 (Figure B-D). Interestingly, FFO administration more than doubled the expression of TGF-β and Foxp3 at the site of inflammation (Figure C). Therefore, we analyzed whether administration of FFO and NFO increases the expression of factors related to Tregs differentiation following stimulation. FFO increased Foxp3 level (14.2%) and the CD4+
Tregs population (16.0%) more than two-fold compared with NFO in CD4+
T cells stimulated with anti-CD3 and anti-CD28 (Figure E and F). In addition, we analyzed whether administration of FFO or NFO increases the number of CD4+
Tregs following stimulation. FFO treatment increased the CD4+
Tregs population (11.5%) more than two-fold compared with NFO treatment (5.5%) (Figure G). As a result, ingestion of FFO significantly increased Foxp3 expression and induced the differentiation of CD4+
Tregs from CD4+
T cells. These results indicate that FFO is more effective than NFO in disease suppression and inducing CD4+
Why does FFO show more potent anti-allergic effects than NFO? Fish oil contains a variety of substances, such as stearic acid, eicosatrienoic acid, linolenic acid, EPA and DHA [38
]. Especially, EPA and DHA are mainly effective for skin inflammation. Several previous studies have suggested that EPA and DHA diet alleviate ear oedema and inhibit the production of pro-inflammatory factors (IκB, MAPKs, TNF-α, IL-6 and COX-2) in experimental skin inflammation models [39
]. FFO contains more than double the concentration of EPA and DHA (Table ). In addition, fermentation would transmute the chemical structure of some constituents of fish oil to create new substances, which have immune-modulatory activities by regulating the development of CD4+
Fatty acid composition of natural fish oil and fermented fish oil
In summary, FFO had stronger inhibitory effects on various experimental AD symptoms than NFO by up-regulating the generation of Tregs. It is not yet clear why FFO has stronger immunomodulatory effects than NFO. So we are currently trying to identify the interrelationships between FFO, EPA, DHA and Tregs. Our results suggest that the anti-allergic effect of FFO is associated with the enrichment of CD4+CD25+Foxp3+ Treg at the site of inflammation and that fermented fish oil may be an effective treatment for the allergic symptoms of AD.