Laurence S. Harbige

Fatty Acids, the Immune Response, and Autoimmunity: A Question of n-6 Essentiality and the Balance Between n-6 and n-3

The essentiality of n−6 polyunsaturated fatty acids (PUFA) is described in relation to a thymus/thymocyte accretion of arachidonic acid (20∶4n−6, AA) in early development, and the high requirement of lymphoid and other cells of the immune system for AA and linoleic acid (18∶2n−6, LA) for membrane phospholipids. Low n−6 PUFA intakes enhance whereas high intakes decrease certain immune functions. Evidence from in vitro and in vivo studies for a role of AA metabolites in immune cell development and functions shows that they can limit or regulate cellular immune reactions and can induce deviation toward a T helper (Th)2-like immune response. In contrast to the effects of the oxidative metabolites of AA, the longer-chain n−6 PUFA produced by γ-linolenic acid (18∶3n−6, GLA) feeding decreases the Th2 cytokine and immunoglobulin (Ig)G1 antibody response. The n−6 PUFA, GLA, dihomo-γ-linolenic acid (20∶3n−6, DHLA) and AA, and certain oxidative metabolites of AA can also induce T-regulatory cell activity, e.g., transforming growth factor (IGF)-β-producing T cells; GLA feeding studies also demonstrate reduced proinflammatory interleukin (IL)-1 and tumor necrosis factor (TNF)-α production. Low intakes of long-chain n−3 fatty acids (fish oils) enhance certain immune functions, whereas high intakes are inhibitory on a wide range of functions, e.g., antigen presentation, adhesion molecule expression, Th1 and th2 responses, proinflammatory cytokine and eicosanoid production, and they induce lymphocyte apoptosis. Vitamin E has a demonstrable critical role in long-chain n−3 PUFA interactions with immune functions, often reversing the effects of fish oil. The effect of dietary fatty acids on animal autoimmune disease models depends on both the autoimmune model and the amount and type of fatty acids fed. Diets low in fat, essential fatty acid deficient (EFAD), or high in long-chain n−3 PUFA from fish oils increase survival and reduce disease severity in spontaneous autoantibody-mediated disease, whereas high-fat LA-rich diets increase disease severity. In experimentally induced T cell-mediated autoimmune disease, EFAD diets or diets supplemented with long-chain n−3 PUFA augment disease, whereas n−6 PUFA prevent or reduce the severity. In contrast, in both T cell- and antibody-mediated autoimmune disease, the desaturated/elongated metabolites of LA are protective. PUFA of both the n−6 and n−3 families are clinically useful in human autoimmune-inflammatory disorders, but the precise mechanisms by which these fatty acids exert their clinical effects are not well understood. Finally, the view that all n−6 PUFA are proinflammatory requires revision, in part, and their essential regulatory and developmental role in the immune system warrants appreciation.

Nutrition and Immunity with Emphasis on Infection and Autoimmune Disease

Nutrition and nutritional status can have profound effects on immune functions, resistance to infection and autoimmunity in man and other animals. Nutrients enhance or depress immune function depending on the nutrient and level of its intake. Protein-energy malnutrition and vitamin A deficiency are strongly associated with impaired immunity and infectious disease. The essential role vitamin A plays in infection and maintenance of mucosal surfaces has long been known. Recent evidence shows that T-cell subpopulations, cytokines and antibody subclasses are all affected by vitamin A. In animal studies supplementation with vitamin E protects against infection and is linked to stimulatory effects on the immune system. In man vitamin E and other anti-oxidants increase the number of CD4+ cells. Dietary lipids and zinc have a substantial impact on autoimmunity from protective to potentiation of immunopathological processes in animals. There is considerable potential to modify human autoimmune disease by manipulation of lipid nutrition. Deficiency of pyridoxine induces atrophy of lymphoid organs, marked reduction in lymphocyte numbers, impairs antibody responses and IL-2 production. Dietary copper is important in the prevention of infection in some animal species and T-cell function is defective under deficiency states due to an inability to produce IL-2. Selenium has been linked to viral infection, enhanced T-cell functions and TNFß induced increase in natural killer cell activity. Understanding the molecular and cellular immunological mechanisms involved in nutrient-immune interactions will increase our applications for nutrition of the immune system in health and in disease.

Dietary n-6 and n-3 fatty acids in immunity and autoimmune disease.

Clearly there is much evidence to show that under well-controlled laboratory and dietary conditions fatty acid intake can have profound effects on animal models of autoimmune disease. Studies in human autoimmune disease have been less dramatic; however, human trials have been subject to uncontrolled dietary and genetic backgrounds, infection and other environmental influences, and basic trial designs have been inadequate. The impact of dietary fatty acids on animal autoimmune disease models appears to depend on the animal model and the type and amount of fatty acids fed. Diets low in fat, essential fatty acid-deficient, or high in n-3 fatty acids from fish oils increase the survival and reduce disease severity in spontaneous autoantibody-mediated disease, whilst linoleic acid-rich diets appear to increase disease severity. In experimentally-induced T-cell-mediated autoimmune disease, essential fatty acid-deficient diets or diets supplemented with n-3 fatty acids appear to augment disease, whereas n-6 fatty acids prevent or reduce the severity. In contrast, in both T-cell and antibody-mediated auto-immune disease the desaturated and elongated metabolites of linoleic acid are protective. Suppression of autoantibody and T lymphocyte proliferation, apoptosis of autoreactive lymphocytes, and reduced pro-inflammatory cytokine production by high-dose fish oils are all likely mechanisms by which n-3 fatty acids ameliorate autoimmune disease. However, these could be undesirable long-term effects of high-dose fish oil which may compromise host immunity. The protective mechanism(s) of n-6 fatty acids in T-cell- mediated autoimmune disease are less clear, but may include dihomo-gamma-linolenic acid- and arachidonic acid-sensitive immunoregulatory circuits such as Th1 responses, TGF beta 1-mediated effects and Th3-like responses. It is often claimed that n-6 fatty acids promote autoimmune and inflammatory disease based on results obtained with linoleic acid only. It should be appreciated that linoleic acid does not reflect the functions of dihomo-gamma-linolenic and arachidonic acid, and that the endogenous rate of conversion of linoleic to arachidonic acid is slow (Hassam et al. 1975, 1977; Phylactos et al. 1994; Harbige et al. 1995). In addition to effects of dietary fatty acids on immunoregulation, inflammation as a consequence of immune activation in autoimmune disease may also be an important mechanism of action whereby dietary fatty acids modulate disease activity. In conclusion, regulation of gene expression, signal transduction pathways, production of eicosanoids and cytokines, and the action of antioxidant enzymes are all mechanisms by which dietary n-6 and n-3 fatty acids may exert effects on the immune system and autoimmune disease. Probably the most significant of these mechanisms in relation to our current understanding of immunoregulation and inflammation would appear to be via fatty acid effects on cytokines. The amount, type and balance of dietary fatty acids and associated antioxidant nutrients appear to impact on the immune system to produce immune-deviation or immunosuppressive effects, and to reduce immune-mediated inflammation which will in turn affect the susceptibility to, or severity of, autoimmune disease.

The importance of energy and nutrient supply in human brain evolution.

Current evolutionary theories do not adequately address the question of how the human brain evolved to be larger and more sophisticated than that of other primates. The human brain/body weight ratio is 4–5 times higher than in primates and, relative to the rest of the body, requires up to 10 times as much energy as in other land-based mammals. Human brain evolution must therefore have required a stable food supply providing a reliable source of both high dietary energy and a cluster of ‘brain-specific’ nutrients over a long period of time. These nutrient and energy requirements are available in the marine and shore-based food chain but are difficult if not impossible to obtain in the terrestrial food chain. We suggest that marine and estuarine ecosystems provided hominids with the appropriate stimulus to develop a relatively large brain. This occurred in conjunction with the evolution of other uniquely human features, particularly relative hairlessness, bipedalism and abundant neonatal subcutaneous fat. Invertebrates, molluscs, small or slow-moving fish, and marine algae would have provided a stable, abundant supply of energy, long chain polyunsaturates and other nutrients essential for the brain and would have done so with comparatively little mammalian competition. The land-water interface would thus have allowed the hominid brain to develop sufficient neurological complexity to enable sophisticated tool and behaviour patterns to evolve in humans as a· natural sequel to such a biochemical and environmental stimulus.

Evidence for Cytokine Dysregulation in Multiple Sclerosis: Peripheral Blood Mononuclear Cell Production of Pro-inflammatory and Anti-inflammatory Cytokines During Relapse and Remission

We investigated circulating anti-inflammatory and pro-inflammatory cytokines, and their ex vivo PBMC production in the absence or presence of the neuroantigens myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) and T cell mitogen (PHA) in MS patients in relapse and remission, patients with other neurological disorders (OND) and normal healthy controls. MS patients in relapse exhibited significantly increased PBMC production of TNF-α spontaneously compared with MS remission and healthy controls and with MBP compared with MS remission. Patients in relapse had significantly increased spontaneous, PHA- and MBP-induced PBMC IL-1β production compared with remission MS, and was increased compared (PHA only) with OND and healthy controls. In relapse there was also significantly increased PBMC IFN-γ production (PHA only) compared with remission and a significantly lower production of biologically active TGF-β1 (PHA only) compared with remission MS and OND. In contrast, MS patients in remission produced significantly less spontaneous and MBP-induced TNF-α, spontaneous, PHA- and MBP-induced IL-1β and PHA-induced IFN-γ together with increased production of biologically active TGF-β1. MOG non-specifically increased PBMC TNF-α and IL-1β production in all groups. Pro-inflammatory cytokines in corresponding plasma samples were undetectable whilst the concentration of biologically active TGF-β1 was the reverse of ex vivo PBMC findings. The increase in biologically active TGF-β1 production ex vivo in OND patients, despite active disease, compared with the low level in the MS relapse may indicate a regulatory defect in MS. We conclude that the balance between biologically active TGF-β1 and the pro-inflammatory TNF-α, IL-1β and IFN-γ is dysregulated during MS relapse-remission and that normal counter-regulatory mechanisms during the relapse phase are defective.

Essential fatty acids alter the activity of manganese-superoxide dismutase in rat heart

The effects of oil-derived dietary essential fatty acids on the activities of mitchondrial Mn-SOD (manganese-superoxide dismutase) and cytosolic cupric zinc-superoxide dismutase (Cu/Zn-SOD) were investigated in rat heart. A control group of rats was fed a stock diet for 29 d, and a second group was fed on a fat-free diet. Three other groups were fed fat-free diets that were supplemented with (i) borage oil, which is rich in linoleic (18∶2n−6) and γ-linolenic (18∶3n−6) acids, (ii) fungal oil, which is rich in γ-linolenic, but low in linoleic acid, or (iii) evening primrose oil, which is rich in linoleic acid and low in γ-linolenic acid. An increase in the percentage composition of arachidonic acid (20∶4n−6) in both the choline and ethanolamine phospholipids, together with a decrease in linoleic acid in ethanolamine phospholipids, were found in heart membranes after feeding the rats with diets containing borage oil or fungal oil as compared to those fed the stock diet. The respective activities of Mn-SOD in rats fed the borage or fungal oil diets were also significantly higher than in rats fed the stock diet alone. No change in cytosolic Cn/Zn-SOD activity was observed. Dietary supply of linoleic acid-rich evening primrose oil resulted in an increased proportion of choline phospholipid linoleic acid without any changes in arachidonic acid content or in the activity of Mn-SOD. By contrast, a reduction in the activity of Mn-SOD was detected in rats fed a fat-free diet. These results show that the activity of heart mitochondrial Mn-SOD is influenced by dietary essential fatty acids, whereas the activity of cytosolic Cu/Zn-SOD remained unaffected.

Diet and gene expression : Delta-5 and delta-6 desaturases in healthy chinese and European subjects

Aim: To compare the composition of fatty acids (FAs) in diet, and the expression of delta-6 desaturase (D6D) and delta-5 desaturase (D5D) genes in peripheral blood mononuclear cells (PBMCs) between Chinese and Europeans. Methods: Three-day dietary records from 20 subjects from Beijing, China (n = 10) and Kent, UK (n = 10) were analysed. Expression of PBMC D6D and D5D genes of the subjects was determined using RT-PCR. Results: The dietary intake of Chinese subjects contained less saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), but more essential fatty acids (EFAs) than that of Europeans. Levels of expression of PBMC D6D and D5D genes of Chinese subjects were significantly lower than those of Europeans. A significant positive correlation was found between dietary intake of total SFAs and total MUFAs and expression of PBMC D6D and D5D genes, but a significant negative correlation between dietary intake of linoleic acid (LA) and α-linolenic acid (LNA) and the expression of PBMC D6D and D5D genes. Conclusion: Intake of high SFAs and MUFAs appears to increase expression of PBMC D6D and D5D genes, whilst high EFAs intake appears to decrease expression of PBMC D6D and D5D genes. A follow-up study of the expression of D6D and D5D genes in Chinese who live in European countries with high SFA and MUFA diets would be of interest.

Breast milk levels of zinc and ω‐6 polyunsaturated fatty acids and growth of healthy Chinese infants

Aim: To examine the concentrations of zinc and ω‐6 polyunsaturated fatty acids (ω‐6 PUFAs) in breast milk, the impact of zinc on ω‐6 PUFA metabolism, and the growth rate of infants.

Immune Response of Healthy Adults to the Ingested Probiotic Lactobacillus casei Shirota

Daily ingestion of a probiotic drink containing Lactobacillus casei Shirota (LcS; 1.3 × 1010 live cells) by healthy adults for (1) 4‐week LcS, (2) 6‐week discontinuation of LcS and (3) a final 4 weeks of LcS was investigated. There was a significant increase in expression of the T cell activation marker CD3+CD69+ in ex vivo unstimulated blood cells at weeks 10 and 14, and there was a significant increase in the NK cell marker CD3+CD16/56+ in ex vivo unstimulated blood cells at weeks 4, 10 and 14. Expression of the NK cell activation marker CD16/56+CD69+ in ex vivo unstimulated blood cells was 62% higher at week 10 and 74% higher at week 14. Intracellular staining of IL‐4 in ex vivo unstimulated and PMA‐/ionomycin‐stimulated CD3+ β7+ integrin blood cells was significantly lower at weeks 10 and 14. Intracellular staining of IL‐12 in ex vivo unstimulated and LPS‐stimulated CD14+ blood cells was significantly lower at weeks 4, 10 and 14. Intracellular staining of TNF‐α in LPS‐stimulated CD14+ blood cells was significantly lower at weeks 4, 10 and 14. Mucosal salivary IFN‐γ, IgA1 and IgA2 concentrations were significantly higher at week 14, but LcS did not affect systemic circulating influenza A‐specific IgA or IgG and tetanus‐specific IgG antibody levels. In addition to the decrease in CD3+β7+ integrin cell IL‐4 and a reduced CD14+ cell pro‐inflammatory cytokine profile, at week 14 increased expression of activation markers on circulating T cells and NK cells and higher mucosal salivary IgA1 and IgA2 concentration indicated a secondary boosting effect of LcS.

A pilot study investigating the effects of Yakult fermented milk drink (L. casei Shirota) on salivary IFN-y, sIgA, IgA1 and IgA2 in healthy volunteers

This pilot study investigated the effects on salivary cytokine and sIgA before and after daily consumption of Lactobacillus casei Shirota (LcS) in nine healthy adult volunteers. Subjects were asked to consume two bottles each day of a fermented milk drink containing a total of 1.3 · 1010 live cells of LcS (Yakult), for 4 weeks. Volunteers were asked to avoid food and drink for at least 1 h before morning unstimulated saliva samples were collected over a 5 min period. Samples were collected at baseline, at week 1, 2 and 4 of LcS consumption, and 2 weeks after cessation of consumption. We found a significant transient increase in salivary IFN-g levels, but were unable to detect IL-12, and an increase in salivary sIgA, IgA1 and IgA2 secretion. Baseline salivary IFN-g was below the assay detection limits. However, at week 1, 4/9 subjects had detectable salivary IFN-g levels, 8/9 (P<0.01) at week 2 and 3/9 at week 4. None had detectable IFN-g levels at week 6. IFN-g concentrations ranged from 35.3 to 92.0 pg/ml. There was a significant (P<0.02) increase in salivary sIgA secretion rate at week 4 (Fig. A). At week 6, the sIgA secretion rate was greater than baseline. The IgA1 secretion rate was significantly greater than baseline at week 4 (P<0.05) and week 6 (P<0.02) (Fig. B). The IgA2 secretion was also significantly increased at week 4 (P<0.05) and week 6 (P<0.01) (Fig. B) and the IgA1:IgA2 ratio significantly greater at week 2 (P<0.01). This pilot study demonstrates that LcS can induce increased salivary IFN-g, sIgA, IgA1 and IgA2 secretion in healthy adults, which may improve mucosal immunity in the upper respiratory tract and provide health related benefits. A larger detailed study investigating cytokine networks, immunoglobulins and T cell responses in different immune system compartments in parallel with a placebo control group would define more rigorously and precisely the immunological effects of LcS (Yakult).