Therapeutic Potential of ω-3 Polyunsaturated Fatty Acids in Human Autoimmune Diseases

The recognition of ω-3 polyunsaturated acids (PUFAs) as essential fatty acids to normal growth and health was realized more than 80 years ago. However, the awareness of the long-term nutritional intake of ω-3 PUFAs in lowering the risk of a variety of chronic human diseases has grown exponentially only since the 1980s (1, 2). Despite the overwhelming epidemiological evidence, many attempts of using fish-oil supplementation to intervene human diseases have generated conflicting and often ambiguous outcomes; null or weak supporting conclusions were sometimes derived in the subsequent META analysis. Different dosages, as well as the sources of fish-oil, may have contributed to the conflicting outcomes of intervention carried out at different clinics. However, over the past decade, mounting evidence generated from genetic mouse models and clinical studies has shed new light on the functions and the underlying mechanisms of ω-3 PUFAs and their metabolites in the prevention and treatment of rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis, and type 1 diabetes. In this review, we have summarized the current understanding of the effects as well as the underlying mechanisms of ω-3 PUFAs on autoimmune diseases.

Mounting evidence from both human and animal studies has demonstrated that ω-3 PUFAs, primarily EPA and DHA, can suppress inflammation and have beneficial roles in a variety of human diseases, including autoimmune diseases, diabetes, cancers, Alzheimer's disease (AD), and stroke (3,(20)(21)(22)(23)(24)(25)(26). Fish oil is the oil derived from the tissues of oily ocean fish. The main ω-3 PUFAs in fish oil are EPA and DHA. Researchers often use fish oil as the source of ω-3 PUFAs in clinical interventions. Although the effects of ω-3 PUFAs on these diseases have been extensively investigated in the past few decades, a series of recent studies involving the usage of a transgenic genetic model, fat-1 (or mfat-1) transgenic mice, have shed new light into the mechanisms underlying the protective effects of ω-3 PUFAs. The fat-1 transgenic mice, initially generated in Kang's lab (27) [and later the mfat-1 mouse model independently designed by our group (28)] carry a globally expressed Caenorhabditis elegans fat-1 transgene that encodes an ω-3 fatty acid desaturase. The FAT-1 enzyme can convert ω-6 PUFAs to the corresponding ω-3 forms by adding a double bond to the ω-3 position, thus allowing endogenous production of ω-3 PUFAs while reducing the ω-6: ω-3 ratio in tissues without special dietary adjustment (27,28). The fat-1 transgenic model allows researchers to bypass the traditional lengthy dietary approach of feeding fish-oil to the animals, and have been widely applied to the studies related to autoimmune diseases, tumorigenesis, metabolic, and cardiovascular diseases, as well as neurological diseases (24,27,29,30).
In this review, we choose to focus on the current understanding of the effects of ω-3 fatty acids on such debilitating diseases as rheumatoid arthritis (RA) (31), systemic lupus erythematosus (SLE) (32), type 1 diabetes (T1D) (33), and multiple sclerosis (MS) (34). Although there have been a large body of clinical research articles related to the application of ω-3 PUFAs in different indications, perhaps it should come with no surprise that not all published studies reached the same conclusions. The reasons behind such discrepancy are still unclear although different dose, source, and duration of ω-3 PUFAs treatment may have all played roles in the clinical outcomes. Unfortunately, many of these studies did not report the blood concentrations of EPA/DHA of the enrolled patients, making it difficult to evaluate if the enrolled patients for their studies have indeed gained sufficient EPA/DHA. Thus, for the benefit of readers, we have offered several formatted tables (by no means complete, but certainly representative) detailing the number of patients, dose, duration, and outcomes of each trial (Tables 1-4).

ω-3 PUFAS AND RHEUMATOID ARTHRITIS
Rheumatoid arthritis (RA) is a chronic autoimmune disease manifested by swollen and painful joints, bone erosion, and functional impairment. The joint lesions are characterized by infiltration of T lymphocytes, macrophages, and B lymphocytes into the synovium, as well as by synovial inflammation induced by PUFAs metabolites, cytokines, and matrix metalloproteinases (58)(59)(60). The intervention of RA with ω-3 PUFAs has attracted increasing attention. One of the earliest studies about ω-3 PUFAs' impact on rheumatoid arthritis was reported in Lancet in 1985 (35) ( Table 1). In this pilot study, seventeen RA patients consumed a daily supplement of 1.8 g of EPA for 12 weeks. At the end of treatment, the EPA intervention significantly shortened morning stiffness time than the control group at 12 weeks and lessened the number of tender joints at 12 weeks from baseline. Several years following this study, Kremer et al. reported in 1990 that the number of tender joints significantly improved from baseline after both the low-and high-dose intervention with fish oil supplement (36) ( Table 1). In addition to such beneficial effects, an ω-6 PUFAs-derived metabolite, leukotriene B4 (LTB 4 ), generated by the neutrophils of the fish oil-treated patients, decreased significantly (36) ( Table 1). In a prospective study, sixty patients with active RA were enrolled in a randomized 12week trial, who were given either fish oil supplement, or fish oil supplement with primrose evening oil (enriched in GLA), or no intervention (37) ( Table 1). Daily supplementation of fish oil alone or in combination with primrose evening oil significantly improved the number of tender joints and visual analog scale (VAS) score. There was a sharp decline in the ratio of ω-6/ω-3 fatty acids in the plasma samples from the groups of subjects receiving fish oil, which was strongly linked to the clinical improvements. Beyer et al. reported in a nutrition study involving 78 RA patients, of which 58% had active RA, that consuming the recommended seafood intake was correlated with better RA disease outcome (38) ( Table 1). In a separate study, sixteen patients received a daily dose of 2.04 g EPA and 1.32 g DHA for 12 weeks, the results also showed a significant reduction in joint swelling index and duration of early morning stiffness Dietary fish oil supplementation is effective in suppressing joint swelling index and duration of early morning stiffness. Other clinical indices improved but did not reach statistical significance. The mean neutrophil leucotriene B4 production in vitro showed a reduction after 12 weeks of fish oil supplementation. Leucotriene B5 production rose to substantial quantities during fish oil treatment.
Lee et al. (40) 183 RA patients and 187 placebo-treated RA controls were included in this meta-analysis The authors surveyed RCTs that examined the effects of ω-3 PUFAs on clinical outcomes in RA patients using MCCTR and by performing manual searches. Meta-analysis of RCTs was performed using fixed and random effects models. as well as a decrease of LTB4 production by the neutrophils isolated from patients' blood (39) ( Table 1). A meta-analysis of 23 similar clinical studies revealed a fairly consistent finding that ω-3 PUFAs had a beneficial effect on joint swelling, pain and morning stiffness, and that there was a significant reduction in the required dose of steroidal anti-inflammatory drugs (40) ( Table 1). In an animal model study, fish-oil feeding in mice delayed the onset and reduced the incidence and severity of type II collagen-induced arthritis compared with the vegetable oil-fed group (61). In the susceptible DBA/1 mouse strain, daily intake of marine ω-3 PUFAs in the form of phospholipids delayed the onset of arthritis, decreased the severity, reduced paw swelling, and knee joint pathology in collagen-induced arthritis (62). Both EPA and DHA have also been shown to suppress Streptococcal-induced arthritis in rats, although EPA appeared to be more effective than DHA (63). As additional proof, endogenous production of ω-3 PUFAs in the fat-1 transgenic mice drastically attenuated arthritis as well as local and systemic levels of inflammatory cytokines following the establishment of RA, whereas the wild type control mice developed overt arthritis (64). Thus, in both animal models and patients, ω-3 PUFAs were able to decrease not only the incidence but also the severity of RA. Dietary supplementation with fish oil affects the mechanisms involved in inflammatory and atherosclerotic vascular disease in patients with lupus nephritis, including neutrophil leukotriene B4 release was reduced 78 and 42%, respectively, by the low and higher doses of fish oil. The higher fish oil dose induced a 38% decrease in triglyceride and a 39% reduction in VLDL cholesterol associated with a 28% rise in HDL, cholesterol. The fish oil had no effect on immune complex or anti-DNA antibody.
Westbergetal and Tarkowski (43) 17 patients with moderately active SLE A double-blind, crossover study on the effect of MaxEPA (contained 180 mg EPA and 120 mg DHA), using olive oil as the control substance. 8/17 on MaxEPA, 2/17 on the control substance for 6 months.
MaxEPA had beneficial effects on the disease but short-lived.
Das et al. (44) 10 patients with newly diagnosed SLE Patients were given orally 6 capsules of EPA/DHA (each contained EPA 27 mg, and DHA 24 mg) for 1 or 2 months. Some of these patients who had significant renal and/or other target organ involvement were given steroids initially for not more than l−2 months in addition to EPA/DHA. At the end of 1 or 2 months of therapy, steroids were withdrawn while patients continued to take EPA/DHA capsules.
Oral supplementation of EPA and DHA induced prolonged remission of SLE without any side-effects.

Duffy et al. (45) 52 patients with SLE
A double-blind, double placebo-controlled factorial trial for 24 weeks. One group received 3 g MaxEPA (contained 180 mg EPA and 120 mg DHA) and 3 mg copper, another 3 g MaxEPA and placebo copper, another 3 mg copper and placebo fish oil, and the fourth group received both placebo capsules.
Dietary supplementation with fish oil is beneficial in modifying symptomatic disease activity. There was a significant decline in SLAM-R score from 6.12 to 4.69 (p < 0.05) in those subjects taking fish oil compared to placebo. No significant effect on SLAM-R was observed in subjects taking copper. The mechanisms underlying ω-3 PUFAs-initiated regulation of immunity in RA can be several. Most studies focused on molecular mediators, including cytokines, metabolites, and reactive oxygen species; others have interrogated the roles of immune cells such as T cells and antigen presenting cells. In the study by Espersen et al. (41) (Table 1), the level of interleukin-1β (IL-1β) in plasma was significantly suppressed in the patients who consumed fish oil by week 6 of the trial, and a further significant decrease was observed a few weeks later. Even in healthy volunteers, a clinical study involving supplementation of fish oil has demonstrated reduction in tumor necrosis factor-alpha (TNF-α), IL-1β, and interleukin-6 (IL-6) in endotoxin-stimulated monocytes cells (65), although the findings from a few other studies did not corroborate these results (66)(67)(68). Recently, interleukin-17 (IL-17) has been proposed to be a key cytokine in the onset and development of RA (69,70). Studies using fish oil supplement found that DHA and EPA have anti-inflammation benefit by reducing the population of interferon-γ (IFN-γ) and IL-17-producing CD4 + T cells in humans and animals (71)(72)(73)(74). Thus, suppression of inflammatory cytokines is likely an important contributing factor to the amelioration of clinical signs and symptoms in RA patients. In cultured cells, studies also reported that EPA and DHA could inhibit the production of TNF-α, IL-1β, and IL-6, the classic panel of pro-inflammatory cytokines (75)(76)(77)(78). The results derived from these clinical and cellular studies were also corroborated by similar findings in other animal models (24,30). For example, the fat-1 transgenic mice showed strong anti-inflammatory effects that rendered tissues strongly resistant to cytokines (IL-1β and TNF-α)-induced cell death (24,30). The inflammatory metabolites, prostaglandin E2 (PGE 2 ) and LTB 4 , were sharply decreased in the tissues of the fat-1 mice. Endogenous production of ω-3 PUFAs also markedly attenuated cytokine-induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-related kinase 1/2 (ERK1/2) (24, 30).
Our recent study and many other studies revealed that incubation of EPA and DHA with isolated human T cells could inhibit the proliferation of human T cells and their production of IL-2 (79)(80)(81)(82)(83). Similar findings were also made in the T-cells isolated from mice (80). In vivo, a dietary gain of ω-3 PUFAs has been shown to correct the imbalance of Th1 and Th2 ratios in such Th1-mediated autoimmune disease models as RA and EAE (84)(85)(86). Thus, both the proliferation and differentiation of T cells could be regulated by ω-3 PUFAs. EPA and DHA may have also influenced the function of antigen presenting cells. Studies derived from cultured cells have shown that the expression of major histocompatibility complex (MHC) II and antigen presentation via MHC II could be significantly reduced following the exposure to EPA and DHA (87)(88)(89). These findings are also supported by the observations in the mice fed fish-oil (90) or in humans with EPA/DHA supplementation (91). Taken together, these investigations supported the concept that ω-3 PUFAs can decrease levels of inflammatory cytokines, modulate T-cell differentiation, reduce antigen presentation via MHC II, and importantly correct a range of pathological conditions of RA.

ω-3 PUFAS AND SYSTEMIC LUPUS ERYTHEMATOSUS
In addition to the investigations of ω-3 PUFAs in the treatment for RA, there have also been a number of studies in animal models and clinical trials assessing the effects of dietary supplementation of ω-3 PUFAs in other autoimmune diseases, such as SLE (42,92). SLE is a common autoimmune disorder with diverse clinical manifestations including inflammation, blood vessel abnormalities, and immune-complex deposition, which are all associated with autoantibodies against cellular components (93,94). Since the earliest clinical trial in 1989 ( Table 2), there have been seven major published clinical studies focusing on the relationship between ω-3 PUFAs and SLE (42-48) ( Table 2). All but one of the clinical studies (48) ( Table 2) reported beneficial effects, including the improvement in endothelial function, disease activity, or inflammatory markers following the implementation of ω-3 PUFAs in SLE patients. In all of the studies with positive outcomes, more than 12 weeks of intervention appeared to be necessary. In a recent randomized placebo-controlled 6-month trial involving fifty SLE patients (47) ( Table 2), the patients who received ω-3 PUFAs intervention (2.25 g EPA and 2.25 g DHA) showed remarkable improvement in their scores derived from Physician Global Assessment (PGA) and RAND 36-Item Short Form Health Survey (RAND SF-36, Version 1.0). The circulating levels of several inflammatory markers, including IL-13 and IL-12, were significantly decreased (47) ( Table 2). A clinical nutritional study of SLE patients found that dietary patterns low in ω-3 PUFAs and high in carbohydrates positively correlated with the severity of disease activity, adverse serum lipids, and the presence of plaque (46) ( Table 2). A doubleblind, double placebo-controlled factorial trial in 52 patients with SLE (45) ( Table 2) reported a significant decline in SLAM-R score (revised Systemic Lupus Activity Measure) from 6.12 to 4.69 in the subjects receiving EPA/DHA compared to those on placebo.
In the study carried out by Das and colleagues (44) ( Table 2), daily oral supplementation of even moderate EPA and DHA (EPA 162 mg, DHA 144 mg) induced prolonged remission of SLE in ten patients. Furthermore, EPA and DHA also suppressed both T-cell proliferation and the production of IL-1, IL-2, as well as TNF-α in vitro and in vivo (44) ( Table 2). A comparative clinical study compared fatty acid (FA) compositions in the red blood cells (RBC) and plasma between the female SLE patients and age-matched females with a history of cardiovascular disease (CVD) or those SLE patients with no history of CVD (SLE+CVD and SLE-CVD) (5). The plasma levels of EPA and ω-3 index (EPA+DHA) were significantly lower in the SLE patients than the CVD controls. In RBC, the ratio of AA to EPA was also significantly higher in the SLE patients than in the controls. Thus, although there was no intervention with ω-3 PUFA supplement, these SLE patients clearly had altered plasma and RBC FA compositions favoring inflammation pathology (5). Following this study, Luca Navarini et al. analyzed fatty acids or metabolites in the plasma samples from SLE patients (95). RvD1, the main metabolic product of DHA, was found to be remarkably lower in the samples from SLE patients than those from the non-SLE controls (95), further supporting the potential of using ω-3 PUFAs and its metabolites in the clinical management of SLE. Mounting evidence built in SLE animal models strongly supports the preventative and therapeutic benefits of ω-3 PUFAs against SLE. Although several SLE models have been widely used in published animal studies, we decided to focus on the results derived from NZB/NZW F1 mice (herein after named B/W F1) because it is a classic spontaneous SLE model with close resemblance to the development of human SLE. Fernandes et al. found that a diet rich in long chain ω-3 fatty acids (10%) delayed the onset, attenuated the severity of autoimmune diseases, and prolonged the survival of female B/W F1 mice (96). Such amelioration of autoimmune disease was mainly attributed to the elevation of transforming growth factor β1 (TGF-β1) and IL-4 as well as the reduction of pro-inflammatory cytokines IL-2 from the splenocytes of B/W F1 mice (96). Perhaps not surprisingly, in their following study, the analysis of fatty acids revealed significantly elevated EPA and DHA levels in the kidney tissue samples of the EPA/DHA-fed animals, and further confirmed the decreased expression of TGF-β1 played a key role in the development of SLE (97). In a separate independent study, the B/W F1 mice, after fed linseed-oil (containing 70% ω-3 PUFAs), showed lower titers of antibodies to DNA as well as cardiolipin and less severe kidney damage than the B/W F1 mice fed control diets (98). Consistent with these findings, Halade et al. found that the concentrated EPA/DHA (Lovaza R ) had a dose-dependent therapeutic effect on SLE progression: the diet containing 1% Lovaza extending maximal lifespan to 517 days; and the diet containing 4% Lovaza further prolonged both the median (502 d) and maximal (600 d) life span in B/W F1 mice compared to the same mice fed standard chow diet (301 d) (99). In addition, the diet containing 4% Lovaza significantly decreased anti-dsDNA antibodies, glomerulonephritis, and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) in the splenocytes. Notably, NF-κB activation and p65 nuclear translocation were also lowered by 4% Lovaza-containing diet when compared to that in the control diet. An independent study by Pestka et al. found that ω-3 PUFAs-enriched diets delayed the onset and markedly attenuated the severity of autoimmune glomerulonephritis, plasma autoantibodies, and proteinuria in the B/W F1 mice (100); importantly, such suppression of autoimmunity was found to correlate with a generalized reduction of CD4 + T cell-specific gene expression in the kidneys and/or spleens of the SLE mice (100). Thus, daily oral supplementation of ω-3 PUFAs has shown promising therapeutic effects, even at low dosages, by suppressing glomerulonephritis and extending life span in patients and SLE-prone animals, most likely via reducing inflammatory cytokines. These studies further demonstrated that ω-3 PUFAs could alleviate the severity and slow the progression of SLE by regulating.

ω-3 PUFAS AND TYPE 1 DIABETES
T1D is a polygenic and organ-specific autoimmune disease, in which a certain subclass of T lymphocytes is involved in executing autoimmune attacks that lead to the destruction of pancreatic β cells (101,102). Two recent large-scale clinical observational studies revealed clear preventative and therapeutic benefits against the development of T1D through early intervention with EPA/DHA-enriched agents to infants and children. Stene et al. initially reported in a large populationbased case-control study that dietary supplement of codliver oil and vitamin D in the first year of life significantly reduced the incidence of childhood-onset T1D (49) ( Table 3). In a separate longitudinal, observational study (part of the Diabetes Autoimmunity Study in the Young, DAISY), Norris and colleagues showed that EPA/DHA supplement starting from first year of life sharply lowered the incidence of islet autoimmunity and the titers of autoantibodies in children with high risk of T1D (50) ( Table 3). In an attempt to treat a childhood onset T1D, Ricordi's group successfully normalized blood glucose and sharply reduced the dose of injected insulin to just once a day after a year and half of oral EPA/DHA and vitamin D supplement in an 8-year-old child (51) ( Table 3). A multicenter, two-arm, randomized, double-blind pilot trial of DHA supplementation, beginning either in the last trimester of pregnancy (41 infants) or in the first 5 months after birth (57 infants) with a high genetic risk for T1D was conducted by Dr. Chase's group (52) ( Table 3). There were no statistically significant reductions in the blood levels of inflammatory cytokines, such as IL-1β, TNF-α or interleukin-12 subunit p40 (IL-12p40). However, high-sensitivity C-reactive protein (hsCRP) was significantly lower in breastfed DHA-treated infants compared to all formula-fed infants at age 12 months, suggesting that the nutritional matrix of breast milk, particularly the ones with increased DHA contents, has an anti-inflammatory effect (52) ( Table 3).
A series of recent genetic and clinical studies have shed new light on the mechanisms and the therapeutic potential of ω-3 PUFAs in suppressing autoimmunity as well as protecting and enhancing islet functions. In animal studies, Otani et al. reported that low ratio of ω-6/ω-3 at 3.0 in the maternal diet during gestation and lactation delayed the onset of diabetes in NOD mice offspring (103). Their further study indicated that diet with a low ω-6/ω-3 ratio started immediately after the onset of overt diabetes prolonged survival of type 1 diabetes in NOD mice (104). However, a study by a different group failed to see a significant delay in the onset of diabetes with the supplementation of fishoil (105). The author ascribed these results to the low power of statistical method and the possibility that EPA/DHA may have undergone oxidation after thawing from the −20 • C storage condition (105). However, other factors, including the dosage and the concentrations of the administered EPA/DHA, should not be excluded.
The recent research coming from our own group revealed that elevation of cellular ω-3 PUFAs via the transgenic expression of fat-1 almost completely blocked the pancreatic β-cells from the attack of pro-inflammatory cytokines while significantly enhancing insulin secretion (30). These effects were primarily mediated via the reduction of PGE 2 secretion from the islets and β-cells, suggesting that the control of inflammation through reduction of ω-6 PUFAs and their metabolites was the main theme in the regulation of β-cell functions (30). These findings derived from the fat-1 transgenic model was also consistent with the results from earlier nutritional studies that dietary gain of marine ω-3 PUFAs could restore palmitate acid-or LA-impaired insulin secretion (106,107).
In our latest published report using the spontaneous type 1 diabetic NOD mice, an EPA/DHA-enriched diet and a fat-1 gene therapy not only delayed the onset but also reversed the autoimmunity and diabetes (80). Both methods of treatment, provided as either a preventative or a therapeutic modality, significantly suppressed Th1 and Th17 cell populations, reduced the secreted levels of IFN-γ and IL-17, increased the proportion of Th2 and regulatory T cells (Tregs). Thus, EPA/DHA-initiated global changes in CD4 + T-cells are likely the primary cellular mechanisms stalling the development of autoimmunity in T1D (80). Multiple molecular mechanisms may have contributed to the effects of ω-3 PUFAs on CD4 + T-cell differentiation. In particular, the metabolites of PUFAs played critical roles in the modulation of immune system. For example, arachidonic acid (an ω-6 PUFA)-derived eicosanoids, synthesized through the activities of such enzymes as lipoxygenase, cyclooxygenase, and cytochrome P450, often have a pro-inflammatory effect (108). In contrast, EPA/DHA-derived eicosanoids and docosanoids are less inflammatory than those from ω-6 PUFAs (16). In our analysis, different eicosanoids and docosanoids played distinct roles in CD4 + T-cell differentiation (80). Prostaglandin D3 (PGD3) (EPA-derived metabolite) had a strong inhibitory effect on Th1 and Th17 differentiation and increased the percentage of Th2 and Treg.17, 18-dihydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (17,, another EPA-derived metabolite, only decreased the population of Th17. One of the DHA-derived metabolites, resolvin D1 (RvD1), had a strong inhibitory effect on Th1 differentiation and promoted Th2 and Tregs differentiation. In contrast, the pro-inflammatory eicosanoids (synthesized from AA), such as 15-Hydroxyeicosatetraenoic acid (15-HETE) and 20-Hydroxyeicosatetraenoic acid (20-HETE), had exactly the opposite effects on naive CD4 + T-cell differentiation than those of EPA/DHA-derived metabolites (80).
Olefsky' group has identified a receptor for ω-3 PUFA, which is G-protein coupled receptor 120 (GPR120), that can mediate at least in part the anti-inflammation effect of ω-3 PUFAs (103,109). GPR120 couples to β-arrestin2 and inhibits TAK1 binding protein 1 (TAB1)-mediated activation of transforming growth factor-β activated kinase 1 (TAK1), providing a mechanism for inhibition of both the NF-κB related Toll-like receptor (TLR) signaling and TNF-α pro-inflammatory signaling (103,109) (see the proposed model in Figure 1). Deficiency of GPR120 in mice and humans produced symptoms very similar to those of wildtype mice fed LOW ω-3 PUFA diets, including reduced energy expenditure, obesity, insulin resistance, and an elevated state of inflammation (114).
Our recent studies further revealed that the mTOR has emerged as an important signaling switch for ω-3 PUFAregulated differentiation of CD4 + T helper cells (110). In particular, application of rapamycin has been shown to inhibit the proliferation of T-cells and the differentiation to Th1 even in the presence of TCR co-stimulatory molecules, such as CD28 (82). T-cell specific mutation of mTOR gene or rapamycin treatment could suppress differentiation to Th17 cell (115). In contrast, rapamycin or diminution of mTOR activity promoted naive CD4 + T cells differentiating into Tregs (115). EPA/DHA and ω-6 PUFAs (particularly AA) had the opposite effects on mTORC1 signaling pathway. EPA/DHA reduced phosphorylation of ribosomal protein S6, indicative of inhibition of mTORC1 activity, whereas AA increased S6 protein phosphorylation levels by activating mTORC1. Moreover, rapamycin, the inhibitor of mTOR, completely blocked the AA-induced Th1 differentiation. Thus, the totally opposite impact of ω-6-and ω-3 PUFAs on the regulation of mTOR complexes serves a molecular switch to dictate the differentiation fate of CD4 + T cells following the treatment of PUFAs (80) (for a proposed working model, see Figure 1).
Interestingly, even in a chemically (Streptozotocin, STZ) induced diabetic model, the pancreatic enrichment of ω-3 PUFAs as a result of fat-1 expression could also prevent the development of diabetes (116). This dramatic effect was accompanied by the reduction of the expression of proinflammatory cytokine genes, the blockade of NF-κB activation, and a sharp suppression of proinflammatory PUFA-derived lipid mediators in the pancreas of the fat-1 mice (116). In contrast, the EPA-derived anti-inflammatory metabolites, lipoxin A4 (LXA4), and 18hydroxy-5Z,8Z,11Z,14Z,16E-eicosapentaenoic acid (18-HEPE), were highly elevated in the STZ-treated fat-1 mice (116). Taken together, these clinical and animal studies have provided the hope that intervention with ω-3 PUFAs can be successfully applied to the prevention, and even therapeutic treatment of T1D. Additional studies are required to validate the effect of administration of ω-3 fatty acids on the progression of T1D.
Taken together, the reduction of ω-6 PUFAs and elevation of ω-3 PUFA intake are critical to the prevention and treatment of type 1 diabetes, which should help direct the future clinical intervention with EPA/DHA supplementation.

ω-3 PUFAS AND MULTIPLE SCLEROSIS
MS is a chronic autoimmune inflammatory disease of the brain and spinal cord in which immune cell infiltration promotes inflammation, demyelination, and neuroaxonal degeneration, leading to the damage of neuronal signaling (117,118). At the initial stages of the disease, most patients experience reversible neurological dysfunction, which typically lasts several days or weeks. Over time, irreversible clinical, and cognitive deficits develop (119,120).
ω-3 PUFAs have attracted a great deal of attention as the potential agents in the clinical management of MS owing to their anti-inflammatory, antithrombotic, antioxidant, and immunomodulatory functions (118,(121)(122)(123). Indeed, largely inspired by Swank's observation that a higher incidence of MS occurred in Norwegians with diets of higher levels of animal fats and dairy products than those with diets of higher amounts of fish, several clinical studies have been conducted with the focus on dietary supplements like vitamin D, polyunsaturated fatty acids or other specific diets (124). A Norwegian intervention study of the patients with newly diagnosed MS reported that a 2-year treatment regimen with 0.9 g/day supplement of long chain marine fatty acids and vitamins showed a significant reduction in the mean annual exacerbation rate and the mean expanded disability status scale (EDSS) as compared to the pre-study values (53) ( Table 4). In a randomized, placebocontrolled clinical trial with EDSS score and inflammation as the primary outcomes, a significant improvement in EDSS compared with placebo was observed in the patients taking ω-3 fatty acid plus vitamin D supplements (54) ( Table 4). In a multicenter incident case-control study (involving 267 cases and 517 controls) conducted in four regions of Australia, high intake of ω-3 PUFAs was strongly associated with a decreased risk of a first clinical diagnosis of central nervous system (CNS) demyelination (55) ( Table 4). In a double-blind and randomized trial for the duration of 1 year, 27 relapsing-remitting MS (RRMS) patients were treated with the same doses of ω-3 PUFAs and olive oil (56) ( Table 4). Patients were randomly divided into two groups: the ω-3 group with dietary fat intake restricted to <15% of the daily calorie intake and the olive oil group with dietary fat intake to <30%. The results showed no significant effect on relapse rate or clinical severity (EDSS) scores following the 12-month intervention. However, the patients with relapsingremitting MS benefited from a low fat diet supplemented with ω-3 PUFAs as the mean change of relapse rate in the ω-3 group: −0.797 relapses/year (P = 0.021) vs. −0.69 (P = 0.044) in the olive oil control group (56) ( Table 4). However, FIGURE 1 | Proposed working model of ω-3 PUFAs induced inhibitory effect on inflammation and autoimmunity. Elevation of ω-3 PUFAs can stimulate one of its major receptors, GPR120. GPR120 couples to β-arrestin2 and inhibit TAB1-mediated activation of TAK1, providing a mechanism for inhibition of NF-κB related pro-inflammatory signaling pathways in autoimmune disease (103,109). ω-3 PUFAs and their metabolites suppress the activation of Th1 cells through inhibiting of mTORC1 (80,110,111), which in turn activates suppressor of cytokine signaling (SOCS) (112). ω-3 PUFAs can inhibit signal transducer and activator of transcription (STAT) 4 which is essential for the development of Th1 cells from naive CD4 + T cells (113). In another signaling pathway, ω-3 PUFAs can dictate the differentiation toward Th2 cells by activating 4E-BP1 through dephosphorylation and then stimulate STAT6 (80). not all clinical studies yielded positive outcomes. Torkildsen et al. evaluated the effect of ω-3 PUFAs on MS disease severity in a randomized, double-blind, and placebo-controlled trial (57) ( Table 4). They applied 1,350 mg of EPA plus 850 mg of DHA daily in the treatment group with placebo as the control group for 6 months. Magnetic resonance imaging (MRI) was applied to quantify the number of new T1-weighted gadoliniumenhancing lesions during the first 6 months. Relapse rate, disability progression, fatigue, and quality of life were measured after 9 and 24 months. There was no difference between the ω-3 PUFAs and placebo groups during the first 6 months in the number of new gadolinium-enhancing MRI lesions, as well as in disease activity after 6 months or 24 months (57) ( Table 4).
It is still unclear about the discrepancy of outcomes in different trials although the dosage, the source and concentrations of EPA/DHA agents, the duration of the trial, and the methods of evaluation all could have led to the different results at the end of the trial.
Some of the positive clinical observations mentioned above prompted a series of cellular and animal research studies about the effects of ω-3 PUFAs on MS development. As described above, several different groups (including our own) have reported that ω-3 PUFAs could inhibit the activation of NF-κB via increasing the expression of PPARs (24,30,(125)(126)(127)(128), which in turn reduces the production of inflammatory markers. In a combinatorial treatment with DHA and alltrans-retinoic acid (a bioactive derivative of vitamin A), the expression of IL-17 and RORγt gene, which is vital for the initiation and progression of MS, was significantly attenuated in the PBMCs isolated from the patients with relapsingremitting multiple sclerosis (129). Addition of EPA and DHA in cultured cells significantly decreased the production and activity of matrix metalloproteinase-9 (MMP-9) and inhibited the migration of human T cell derived from the PBMCs from MS patients, which implied the reduction of infiltrating capability of T-cells (130).
Kong et al. first reported in 2011 that dietary ω-3 fatty acids could suppress EAE in an animal model (73). Their extensive work showed that pretreatment of bone marrow-derived dendritic cells (DC) with DHA could prevent LPS-induced DC maturation and weaken the stimulation of antigen-specific T cells (73). Consistent with these in vitro observations, the results from the animal model further demonstrated decreased numbers of IFNγ-and IL-17-producing CD4 + T cells in both spleen and CNS (73). In another independent study, the mice with myelin oligodendrocyte glycoprotein (MOG)-induced EAE showed a significant delay in onset and amelioration in severity by DHAenriched diet (131). In such context, the female mice were fed 4 weeks of either the control diet, or the diet containing 0.3 or 1.0% DHA in either phospholipid (PL) form or triacylglycerol (TAG) form pre-EAE induction and for 4 weeks post-EAE induction. The TAG-form DHA appeared to be effective in lower daily scores in the early phase of EAE during day 9-16 while PLform DHA was effective in decreasing daily scores in the final outcome during day 23-28 (131). Increasing EPA content in the diet appeared to have the benefit similar to that of DHA-enriched diet. Unoda et al. found that EPA-enriched diet significantly lowered clinical EAE scores, and remarkably inhibited the production of IFN-γ and IL-17 (132). Consistent with these findings in the nutritional intervention studies, modeling of MS with cuprizone in the fat-1 mice revealed that endogenous production of ω-3 PUFAs strongly promoted remyelination after a toxic injury to CNS oligodendrocytes via modulation of the immune system (133,134). Interestingly, some of the protective effects were mediated at least in part through EPAderived lipid metabolites, such as 18-HEPE by acting directly on oligodendrocytes and neurons (133,134). Taken together, the findings coming out of the clinical investigations and animal models provided the hope of applying high dose of EPA/DHA agents in the treatment of MS.

CONCLUSION
The promising findings coming from the cumulative research work over the last decade solidified the role of ω-3 PUFAs as a potential candidate to prevent or even treat such autoimmune diseases as type 1 diabetes, RA, SLE, MS. Undoubtedly, many of the beneficial effects of ω-3 PUFAs can be traced back to their anti-inflammation actions; however, other mechanisms, such as the regulation of mTOR activity may also be in place. Although still facing some technical issues, particularly clinical evaluation of efficacy and safety, the use of high dosages of EPA/DHA or fat-1 gene therapy to generate endogenousω-3 PUFAs have a huge potential in the clinical treatment of these debilitating diseases.