Environmental triggers

Asthma appears to be caused by environmental factors, such as allergens, irritants and infections, in genetically predisposed individuals. Once asthma has become manifest, essentially the same agents may trigger worsening of the disease and precipitate serious asthmatic attacks, as well as contribute to the development of chronic disease. A host of triggering as well as some protective factors are known, but the basic understanding of why and how asthma develops is still rudimentary. A major factor contributing to the development of allergy and asthma is the so-called Western lifestyle. This is illustrated by the 2- to 3-fold higher prevalence of asthma in former West Germany compared with East Germany, and Hong Kong compared with nearby cities in China⁽Reference Leung and Ho^52, Reference von Mutius, Martinez and Fritzsch⁵³⁾. Gradients in the prevalence of allergic disease corresponding to social gradients are also found within individual countries, and allergic diseases have been described as a price paid for wealth and a high standard of living. Environmental and lifestyle factors have changed in a broad sense over the last decades in industrialised countries, but it is not known which specific conditions in the more affluent, industrialised or urbanised lifestyle drive the development of asthma and allergy. Also, asthma is a major health problem among the poor populations living in deprived inner city districts and the homeless in USA, with a very high prevalence of severe asthma, and prevalence is also high in some other underprivileged regions e.g. in South America. Asthma therefore does not, in all places, fit the ‘hygiene hypothesis’ model of more allergic disease among the ‘clean and rich’ and less among the poor (see later).

Allergen exposure will trigger asthma once the disease has become manifest, as will a wide array of non-specific irritants in the occupational setting, indoor environmental factors (e.g. second-hand tobacco smoke, strong odours and perfume) and outdoor air pollution factors like fine particulate matter, ozone and oxides of nitrogen. Also, physical exercise and psychological stimuli may precipitate asthma⁽Reference Postma, Kerstjens, Ten Hacken, Albert, Spiro and Jett⁴⁴⁾.

For the causation of asthma, loss of protective environmental factors may be as important as asthma-provoking factors. The observation that the risk of asthma is reduced with increasing number of older siblings was explained by the Th1–Th2 pardigm of immune responses (see later) and the so-called hygiene hypothesis. In its original form, this hypothesis stated that younger siblings got more infections and got them earlier than the older siblings, and that the infections stimulated the development of Th1 immunity and counteracted the development of allergy-associated Th2 responses⁽Reference Strachan⁵⁴⁾. Although the focus has moved away from the hygiene hypothesis in the original form⁽Reference Umetsu^55–Reference Cullinan⁵⁷⁾, it is still considered that ‘something with micro-organisms’ is protective in relation to asthma and allergy⁽Reference von Mutius⁵⁸⁾. Observations that more or less indirectly support this include the inverse relationship between family size and asthma⁽Reference von Mutius, Martinez and Fritzsch⁵⁹⁾, the protective effect of early placement in day care settings⁽Reference Ball, Castro-Rodriguez and Griffith⁶⁰⁾, the protective effect of exposure to farm animals⁽Reference Riedler, Braun-Fahrlander and Eder⁶¹⁾, the protective effect of endotoxin exposure (allergy more than asthma)⁽Reference von Mutius⁵⁸⁾, the reduced prevalence of asthma among anthroposophic Steiner school attendants⁽Reference Floistrup, Swartz and Bergstrom⁶²⁾, the protective effect of extensive exposure to cats and dogs⁽Reference Platts-Mills, Vaughan and Squillace⁶³⁾, the association between intestinal flora and atopic manifestations⁽Reference Bjorksten, Naaber and Sepp⁶⁴⁾ and the association between early use of antibiotics and increased incidence of allergy⁽Reference McKeever, Lewis and Smith⁶⁵⁾. Other hypotheses of asthma causation include reduced intake of antioxidants from foods like fruit and vegetables⁽Reference Wong, Ko and Hui^66, Reference Chen, Hu and Seaton⁶⁷⁾ and an altered balance of fatty acids in the diet⁽Reference Mickleborough and Rundell⁶⁸⁾. Finally, obesity⁽Reference Beuther and Sutherland⁶⁹⁾ and physical inactivity⁽Reference Shaaban, Leynaert and Soussan⁷⁰⁾ are associated with asthma and bronchial hyper-reactivity. Whatever the explanation, asthma must be said to represent ‘an epidemic of dysregulated immunity’⁽Reference Umetsu, McIntire and Akbari⁷¹⁾.

For COPD, the major known environmental trigger is tobacco smoke. Also occupational chemicals and particulate matter may contribute to the development of the disease, as well as indoor and outdoor air pollution. Diet has been reported to be a modifying factor, with fruit and vegetable consumption having a protective effect⁽Reference Watson, Margetts and Howarth⁷²⁾.

Perpetuating factors

In allergic diseases, like allergic rhinoconjunctivitis and allergic asthma, repeated and chronic allergen exposure will keep the disease active and worsening. Typically, general hypersensitivity and hyper-reactivity also to non-specific stimuli develop. These will contribute to perpetuating the disease, and viral infections and environmental pollutants (e.g. tobacco smoke and diesel exhaust particulates) will worsen the disease and may precipitate acute attacks of severe asthma. Largely, the same non-specific stimuli as seen in asthma, but in particular tobacco smoke, will contribute to perpetuating and accelerating COPD.

Mechanisms and mediators

Alveolar macrophages in the lumen of the airways are first-line defence cells, capable not only of engulfing foreign material to destroy it, but also of secreting several signal molecules (cytokines) that serve to attract and activate other cells to create inflammation. Macrophages can remove residual material after phagocytosis in two ways. They can go through the bronchiolar and alveolar walls to enter the lymphatic system carrying fluid and cells to the numerous lymph nodes located along the trachea and bronchi, or move out along the airways helped by the mucociliary escalator mentioned earlier, to be swallowed into the stomach or spat out. Particulate matter that is not dissolved may remain in the lymph nodes for a long time.

Epithelial cells are themselves active participants in immunoregulation and inflammation by secreting a number of inflammatory and immunoregulatory molecules. Furthermore, specialised cells (goblet cells) secrete protective mucus to cover the epithelial surface, and so-called type 2 cells secrete a family of molecules collectively referred to as surfactant. The surfactant has a number of beneficial functions, among others to regulate immune responses and inflammation⁽Reference Kingma and Whitsett⁷³⁾.

In the submucosa, various cells of the specific and non-specific immune systems can be found; among them, mast cells are important also in allergic reactions. The blood vessels bring various immune cells to the lung submucosa and epithelium. These include various subgroups of T and B lymphocytes and natural killer cells, monocytes, neutrophils, eosinophils and other cells. These various cells, in addition to performing defence tasks by cell-to-cell contact, carry with them or produce on-site two groups of molecules, signal molecules and defence molecules (with somewhat overlapping functions). Signal molecules more or less specifically regulate functions in other cells. For example, CD4+T lymphocytes can secrete the cytokines IL-13, IL-4 and IL-9 to stimulate mucous production by epithelial cells, IL-13 and IL-9 to stimulate bronchial muscle contraction, IL-4 and IL-13 to stimulate antibody production by B lymphocytes, IL-3, IL-4, IL-5, IL-9, IL-13 and granulocyte–macrophage colony stimulating factor to attract and activate various other cells (sometimes in a very specific way), and IL-3, IL-4, IL-9 and IL-13 to stimulate the development of other cells. Accumulation and activation of cells is an important feature of the inflammatory response, and the cytokines mentioned will more or less contribute to such a response. Some other cytokines are particularly important for inflammation, like TNF-α, IL-6, and IL-8 and IL-17, while IL-10 tends to downregulate inflammation as well as immune responses – an important function to protect against self-destruction. Other defence molecules are designed to directly destroy the intruders: lysozyme; lactoferrin; complement; α-1 antitrypsin; cationic proteins; lysophospholipases; catalases; non-specific esterases; others. The various granulocytes and the mast cells have in their cytoplasm granules containing preformed mediators that are released in the ‘combat’ situation.

At the heart of the allergic reaction is the interaction between IgE molecules bound to specific receptors on the membrane of mast cells and their corresponding allergens. When the IgE molecules are cross-linked by allergen, the mast cell is triggered to release the potent inflammatory mediators contained in its cytoplasmic granules, and the allergic inflammatory response develops. This response has two phases, an early virtually immediate reaction and a late response developing after some 6 to 8 h. Mast cells are the key cells in the early response, while eosinophils are the predominant cell in the late response. Eosinophils are thought to play a central role in allergic rhinitis as well as asthma⁽Reference Scadding, Mitchell, Albert, Spiro and Jett^41, Reference Van Wetering, Hiemstra, Rabe, Albert, Spiro and Jett⁷⁴⁾. Asthma historically changed from being looked upon as a disease of the smooth musculature of the bronchi to being a Th2-dominated chronic inflammatory disease, with the eosinophil as the typical infiltrating cell. Other cells that regulate Th2 responses, asthma and allergy are NKT cells⁽Reference Akbari, Stock and Meyer⁷⁵⁾, and the various types of regulatory T-cells (Treg cells) that have the capacity to inhibit the effector function of Th2 and Th1 cells⁽Reference Erin, Leaker and Nicholson⁷⁶⁾. Increased levels of the Th2 cytokines IL-4, IL-5, IL-9 and IL-13 have been demonstrated in the asthmatic airway⁽Reference Ray and Cohn⁷⁷⁾. The Th2-driven inflammation has two arms, one via B-cells activated by IL-4 to produce IgE, which triggers the mast cell-mediated allergic inflammation, and the other via IL-4-, but mainly via IL-13-mediated direct effects on epithelium and bronchial smooth muscle⁽Reference Barrios, Kheradmand and Batts⁷⁸⁾. However, whereas the Th1/Th2 concept revolutionised immunological thinking when first proposed in 1987, it has been challenged over the past several years for several reasons⁽Reference Umetsu^56, Reference Steinman⁷⁹⁾. Both Th1 and Th2 cells are pro-inflammatory, and Th1 cells do not always downregulate Th2 cells, but may instead exacerbate Th2-mediated diseases⁽Reference Hansen, Berry and DeKruyff^80–Reference Heaton, Rowe and Turner⁸²⁾. It is of interest that the Th1 cytokine IFN-γ, now considered an aggravating factor in severe asthma, is also considered to be important in COPD⁽Reference Saetta, Di and Turato⁸³⁾. Recently, TNF-α has also been reported to play an important role in severe asthma⁽Reference van Oosterhout and Bloksma⁸⁴⁾. That Th1 cells play a role in asthma development is also illustrated by the finding that the Th1 transcription factor T-bet controls features of asthma. In the absence of T-bet (e.g. in T-bet-deficient mice), airway remodelling and airway hyper-reactivity develop spontaneously⁽Reference Finotto, Neurath and Glickman⁸⁵⁾. Additionally, it has been demonstrated that different asthmatic individuals display different ‘individualised’ cytokine patterns, caused probably both by different genetic factors and by differences in environmental exposure⁽Reference Heaton, Rowe and Turner⁸²⁾. Neural mechanisms are central in the asthmatic inflammation⁽Reference Nassenstein, Schulte-Herbruggen and Renz^86, Reference De Swert and Joos⁸⁷⁾, but may also have a capacity to downregulate inflammation as has been shown in gastrointestinal inflammation⁽Reference Tracey⁸⁸⁾.

A number of genes have been implicated in asthma, e.g. ADAM33⁽Reference Van Eerdewegh, Little and Dupuis⁸⁹⁾. It is estimated that more than a dozen polymorphic genes regulate features of asthma like the inflammatory response, IgE synthesis, cytokine and chemokine production, airway remodelling and airway function⁽Reference Fahy, Corry and Boushey⁹⁰⁾.

On the molecular level, oxidative stress appears to play an important role in asthma⁽Reference Lee, Park and Park^91, Reference Fujisawa⁹²⁾, and the endogenous antioxidant capacity of the lungs has been found to be defective in asthmatic patients⁽Reference Dworski⁹³⁾. Oxidative stress is caused by ROS. The inflammatory cells recruited into the asthmatic airways have a capacity to produce ROS as part of normal antimicrobial defence. Also, exogenous factors precipitating asthma, not only ozone and oxides of nitrogen, but also fine particulate matter, appear to cause oxidative stress⁽Reference Chan, Wang and Li^94, Reference Vinzents, Moller and Sorensen⁹⁵⁾. In animal models, factors that inhibit increased ROS generation have been found to reduce inflammation and hyper-reactivity, two important components of asthma⁽Reference Lee, Link and Baluk⁹⁶⁾.

While irreversible airflow limitation is a functional hallmark of COPD, the lesion is histopathologically characterised by inflammation and tissue destruction. Dominating inflammatory cells are neutrophils, macrophages and CD8+-T lymphocytes and IFN-γ is an important cytokine⁽Reference Saetta, Di and Turato⁸³⁾. Oxidative stress appears to be important in the inflammation and tissue damage in COPD. Environmental agents linked to COPD, like tobacco smoke and particulate matter, induce oxidative stress and inflammation. Increased levels of markers of oxidative stress have been found in exhaled breath condensates of COPD patients⁽Reference Kharitonov and Barnes^97–Reference Paredi, Kharitonov and Barnes⁹⁹⁾, and in a mouse model of tobacco smoke-induced COPD tissue, damage due to oxidative stress was observed. Corticosteroid resistance in COPD has also been related to oxidative stress. An imbalance between protease and anti-protease enzymes is thought to be of importance for the development of the emphysema component of COPD⁽Reference Shapiro¹⁰⁰⁾, and this is supported by findings in experimental models⁽Reference D'Armiento, Dalal and Okada¹⁰¹⁾.

Normal function of inflammatory processes to maintain homeostasis

Controlled inflammation as a result of various triggers is likely to occur in normal joints. Possible triggers are local mechanical stress and transient infection. Local mechanical stress leads to induction of cellular stress proteins, which can be targeted by stress protein-specific regulatory T-cells⁽Reference van Eden, van der Zee and Prakken¹⁰²⁾. Besides genetic factors and infection, there is some conflicting data on failing T-cell regulation as a critical factor in the aetiology of rheumatoid arthritis (RA)⁽Reference van Amelsfort, Jacobs and Bijlsma¹⁰³⁾.

Definition and description of rheumatoid arthritis

RA is a common autoimmune disease characterised by chronic inflammation of the synovium of diarthrodial joints⁽Reference Lee and Weinblatt¹⁰⁴⁾. It can lead to long-term joint damage, resulting in chronic pain, loss of function and disability. Primarily, the small joints of the extremities are affected, but as the disease progresses joints become involved. The chronic inflammatory process changes the cellular composition (cellular infiltration) and the gene expression profile of the synovial membrane, resulting in hyperplasia of the synovial membrane, which causes structural damage in cartilage, bone and ligaments. Extra-articular disease affecting a variety of organs is a significant factor in morbidity and mortality of RA⁽Reference Pincus and Callahan¹⁰⁵⁾. The severity of RA encompasses a wide spectrum, ranging from self-limiting disease to chronic progressive disease, causing varying degrees of joint destruction and extra-articular organ involvement.

Epidemiology of rheumatoid arthritis

RA occurs in 0·5–1·0 % of the population worldwide⁽Reference Silman and Pearson¹⁰⁶⁾. RA is about two to three times more common in women than men. No increased incidence of RA has been noted over recent decades. In fact, decreases were documented, although this might have been artificially caused by a changed clinical classification of diseases previously characterised as RA⁽Reference Uhlig and Kvien¹⁰⁷⁾. Juvenile idiopathic arthritis (JIA) is one of the most common autoimmune diseases in childhood⁽Reference Woo and Wedderburn¹⁰⁸⁾. Although JIA has strong histopathological similarities to RA, it forms a separate clinical entity. Whereas untreated RA generally is progressive, JIA consists of different subtypes with striking differences in both severity and outcome. One subtype is persistent oligoarticular JIA, characterised by a remitting and often spontaneously self-limiting course. As a result of this, persistent oligoarticular JIA is unique among all autoimmune diseases. The self-limitation of an autoimmune process is often seen in experimental animal models but hardly ever in human subjects and seems to suggest the active involvement of immunoregulatory processes, such as regulatory T-cells. Indeed, the frequency of CD4+CD25+ regulatory T-cells in the peripheral blood and in the synovial fluid of JIA patients has been found to correlate with the clinical course of the disease⁽Reference de Kleer, Wedderburn and Taams^109, Reference de Kleer, Kamphuis and Rijkers¹¹⁰⁾. Neither hygienic nor unhygienic living conditions are associated with the risk of developing juvenile RA⁽Reference Nielsen, Dorup and Herlin¹¹¹⁾. Although the causes of RA remain elusive, the general consensus is that factors contributing to its occurrence and course (clinical heterogeneity) are probably both genetic and environmental. The main risk factors for the disease include genetic susceptibility, sex and age, smoking and infectious agents. In addition, hormonal, dietary, socio-economic and ethnic factors seem to contribute⁽Reference van der Helm-van Mil, Wesoly and Huizinga¹¹²⁾. For RA, the main predisposing genetic factor is HLA-DR4. HLA-DR4 predisposes for a more severe progressive disease course. Besides HLA-DR4, other genetic factors have recently been discovered in RA, such as the genetic polymorphisms in the lymphoid protein tyrosine phosphatase (LYP), associated with both type 1 diabetes and RA⁽Reference Begovich, Carlton and Honigberg¹¹³⁾. As LYP is responsible for the tuning of T-cell activation, genetic variation of LYP will lead to variation in overall lymphocyte reactivity. The presence of anti-cyclic citrullinated peptides (CCP) antibodies (see later) seems to be an indicator for a higher grade of joint destruction and disease persistence. Smoking was found to be a risk factor for the development of anti-CCP antibodies. Anti-CCP positive RA may be a distinct disease entity that can react differently to treatment⁽Reference van der Helm-van Mil, Verpoort and Breedveld¹¹⁴⁾.

Pathophysiological mechanisms of rheumatoid arthritis

RA involves many elements of the immune response. The synovium (or synovial membrane) is normally a relatively acellular structure consisting of one or two layers of synoviocytes. In RA, the synovium becomes hypertrophic and oedematous. Angioneogenesis, recruitment of inflammatory cells due to production of chemokines, local retention and cell proliferation contribute to the accumulation of cells in the inflamed synovium. Locally expressed degradative enzymes digest extracellular matrix and destroy articular structures⁽Reference Firestein¹¹⁵⁾. The synovial membrane that extends to the cartilage and bone is known as pannus. It actively invades and destroys the periarticular bone and cartilage at the margin between synovium and bone. T-cells are actively involved in the pathogenesis of RA. Activated T-cells that are abundantly present in the inflamed joints of RA patients can stimulate other cells (e.g. B-cells, macrophages and fibroblast-like synoviocytes)⁽Reference Panayi, Lanchbury and Kingsley¹¹⁶⁾. These T-cells are found to participate in the complex network of cell- and mediator-driven events leading to inflammation and joint destruction. B-cells play several critical roles in the pathogenesis of RA. They are the source of autoantibodies being produced in RA and contribute to immune complex formation (rheumatoid factors reactive with the constant region of their autologous IgG molecules) and complement activation in the joints⁽Reference Weyand, Seyler and Goronzy¹¹⁷⁾. Multiple other autoantibodies have been found in RA, with recent interest focused on those directed at CCP⁽Reference Bridges¹¹⁸⁾. Antibodies that are directed to citrullinated protein⁽Reference Vossenaar and van Venrooij¹¹⁹⁾ detect CCP in many different proteins and are present in about 80 % of the RA patients. Several lines of evidence suggest that citrullinated antigens have direct involvement in the rheumatoid disease process. Anti-CCP antibodies precede the clinical development of synovitis by many years. B-cells are also very efficient antigen-presenting cells, and can contribute to T-cell activation. The important role of B-cells in the disease aetiology is supported by the recent success of B-cell depletion therapy using rituximab⁽Reference Edwards, Leandro and Cambridge¹²⁰⁾. The major effector cells in the pathogenesis of arthritis are synovial macrophages and fibroblasts. Activated macrophages are critical in RA, not only due to macrophage-derived cytokines (in particular TNF-α and IL-1) in the synovial compartments⁽Reference Firestein, varo-Gracia and Maki¹²¹⁾, but also because of their localisation at strategic sites within the destructive pannus tissue. The variety and extent of macrophage-derived cytokines in RA and their widespread effect indicate that macrophages are local and systemic amplifiers of disease severity and perpetuation⁽Reference Kinne, Brauer and Stuhlmuller¹²²⁾. Among the many cell types present in the rheumatoid joint, the fibroblast-like synovial cell is prominent. It is now accepted that these cells are directly responsible for cartilage destruction, and also drive inflammation⁽Reference Kontoyiannis and Kollias¹²³⁾. There is evidence for proliferation and expression of inflammatory cytokines and chemokines by fibroblast-like synovial cells in inflamed synovia. Various studies have shown the presence and activity of regulatory T-cells in RA, both in peripheral blood and in the synovial compartment during active disease⁽Reference van Amelsfort, Jacobs and Bijlsma^103, Reference de Kleer, Wedderburn and Taams¹⁰⁹⁾. Although in one of the RA studies anti-TNF interventions were shown to restore a compromised activity of regulatory T-cells⁽Reference Ehrenstein, Evans and Singh¹²⁴⁾, it seems that impaired T-cell regulation is a more prominent feature of multiple sclerosis and type 1 diabetes than RA.

Atopic eczema

Fifteen per cent of children will have eczema at some time during the first 12 years of their life⁽Reference Williams¹²⁵⁾. The clinical manifestations usually appear within the first year of life and often within a few weeks of birth. The rash of eczema is characteristically distributed (flexures) over ill-defined areas of intensely itchy redness containing an infiltrate of T lymphocytes and eosinophils. There is a clear genetic predisposition but expression of the phenotype is determined by environmental factors (among those suggested are lack of bacterial exposure providing an immune stimulus, intra-uterine exposure to allergens, poor fetal nutritional status). A major component in the pathogenesis of atopic eczema is the involvement of the immune system through a combination of immediate type (IgE-mediated) and T-cell-mediated immune hypersensitivities to environmental aeroallergens and to food allergens. Although the different types of hypersensitivity are demonstrable by prick test, intradermal injection or epicutaneous patch test challenge with a range of allergens, proving that the causal relevance of specific allergies is difficult and unreliable. The main effective therapies for atopic dermatis include topical steroids and systemic immuno-suppressives.

A fundamental component of the atopic state appears to be a dysregulation of the immune system such that T lymphocytes responding to atopic allergens differentiate towards the Th2 phenotype⁽Reference Krutmann and Grewe^126, Reference Thepen, Langeveld-Wildschut and Bihari¹²⁷⁾. This may represent a failure of maturation of the fetal immune system, which is maintained during pregnancy with a bias towards Th2 responses⁽Reference Saito¹²⁸⁾. Signals from DC are thought to determine the differentiation pathway of Th cells. Microbial components including lipopolysaccharides signalling through innate receptors such as toll-like receptors are critical in this, as reflected by the hygiene hypothesis⁽Reference Strachan^54, Reference von Mutius, Braun-Fahrlander and Schierl^129–Reference Romagnani¹³¹⁾. This proposes that early life exposure to microbes accelerates the maturation of the immune system away from the fetal Th2 bias and towards the adult Th1-biased differentiation pathway. Monocytes from atopic eczema sufferers release increased quantities of PGE2, which can drive T-cell differentiation towards the Th2 phenotype⁽Reference Chan, Kim and Henderson^132, Reference Chan, Henderson and Li¹³³⁾.

Psoriasis

Two to three per cent of the population suffer from psoriasis; 10–15 % of these have it severely and require management by dermatologists. Fifty per cent of these will have significant joint symptoms and psoriatic arthritis. There is a genetic susceptibility and a number of genetic loci have been identified that may contribute to this susceptibility. There are associations with hyperlipidaemia and CHD⁽Reference Ena, Madeddu and Glorioso^134, Reference Rocha-Pereira, Santos-Silva and Rebelo¹³⁵⁾ as well as CD⁽Reference Bernstein, Wajda and Blanchard¹³⁶⁾. Various factors are involved in the expression of the psoriatic phenotype: streptococcal infections; psychological stress; physical trauma to the skin. The rash of psoriasis is characterised by plaques of red scaly skin characteristically distributed over bony prominences. The pathophysiology involves an interaction between the immune system and the skin. There is an infiltrate of T lymphocytes (both CD4+ and CD8+) into the dermis, formation of clusters of neutrophils in the epidermis, involvement of two or three layers of the epidermis in proliferation and a greatly accelerated but incomplete differentiation. There are also proliferation and altered structure of the dermal capillaries, which become tortuous and dilated. Activation of the innate immune system by streptococcal products and, most likely, as yet unidentified factors, induces release of cytokines including IFN-α and -γ. The cellular source is unclear but could be plasmacytoid DC or other DC. This activates keratinocytes to proliferate and produce angiogenic factors that induce proliferation of dermal microvessels. The fundamental abnormality has not been identified, but the central feature is a failure of mechanisms of resolution of inflammation. The main treatment modalities include agents that modulate both keratinocyte proliferation and activation of T-cells. These include topical agents and systemic drugs. While many therapies have diverse effects on immune cells, cytokine production and/or cellular proliferation, it is unclear what the critical therapeutic target is. Retinoids, vitamin D analogues and glucocorticoids all have significant therapeutic efficacy and have in common action via the superfamily of ligand-activated nuclear transcription factors. The possibility is that nutritional intervention is suggested by the evidence of the involvement of eicosanoid mediators in psoriasis. Thus, there are reports that inhibition of leukotriene B4 (LTB4) with benoxaprofen was effective against psoriasis⁽Reference Thepen, Langeveld-Wildschut and Bihari^127, Reference Kragballe and Herlin¹³⁸⁾.

Atherogenesis

Atherosclerosis, or ‘hardening of arteries’ is the major cause of CVD. It is most often due to the formation of atheroma. Endothelial dysfunction is thought to presage atherosclerosis, and is characterised by altered endothelial function, enhanced adhesion molecule expression, increased leucocyte adhesion and impaired endothelium-dependent vasodilator responses⁽Reference Penn, Rangaswamy and Saidel^140–Reference Kupatt, Zahler and Seligmann¹⁴³⁾. Although the endothelium appears to continue to elaborate NO, its biological activity appears to be compromised in the early phases of atherogenesis⁽Reference Ohara, Peterson and Harrison¹⁴⁴⁾. The latter is likely to be due in part to the interaction of NO with other molecular species such as the superoxide radical. These interactions neutralise the protective effects of NO and generate products, such as peroxynitrite that may be cytotoxic and pro-inflammatory. These changes in the properties of the endothelium have given rise to the concept of endothelial dysfunction. Endothelial dysfunction is associated with enhanced thrombosis and impaired fibrinolysis, and a number of risk factors appear to contribute to endothelial injury, including smoking, hypertension and hyperlipidaemia. The emergence of the ‘lipid oxidation’ hypothesis⁽Reference Steinberg, Parthasarathy and Carew¹⁴⁵⁾ provided yet another mechanism of endothelial injury, and an explanation for the formation of lipid-laden, macrophage-derived foam cells. Uptake of cholesterol by cells is normally mediated by the LDL receptor, and in the presence of high cellular levels of cholesterol, the LDL receptor is downregulated, limiting cholesterol accumulation. However, the uptake of oxidatively modified LDL is mediated by ‘scavenger receptors’ and this mechanism is not regulated by cellular cholesterol content. The putative protective effect of antioxidants, such as vitamin E, that inhibit LDL oxidation is explained by this hypothesis, although it is important to note that these effects remain contentious⁽Reference Miller, Pastor-Barriuso and Dalal^146, Reference Bjelakovic, Nikolova and Gluud¹⁴⁷⁾.

Leucocytes become attached to the dysfunctional endothelium and subsequently accumulate within the subendothelial space⁽Reference Masuda and Ross^148, Reference Masuda and Ross¹⁴⁹⁾. Macrophages are converted to lipid-laden foam cells within the artery wall, giving rise to a lesion termed the fatty streak. The initial adhesion event is mediated by pairs of adhesion molecules. The endothelial adhesion molecules have been shown to be upregulated early in atherogenesis.

The progression of the atherosclerotic plaque is complex process, and in its early stages of development may be reversed⁽Reference Stary^150, Reference Stary¹⁵¹⁾. The conversion of the fatty streak into a fibrous plaque necessitates the recruitment and proliferation of vascular smooth muscle cells⁽¹⁵²⁾. This process is driven by the synergistic interplay of several growth factors.

Fatal myocardial infarction is usually associated with thrombi and plaque fissuring. Unstable plaques are thought to be particularly prone to fissuring, and these are characterised by a large lipid pool, thin fibrous cap and large numbers of inflammatory cells⁽Reference Lendon, Davies and Born¹⁵³⁾. Activated macrophages within the plaque are a rich source of matrix metalloproteinases that have the ability to degrade extracellular matrix⁽Reference George¹⁵⁴⁾. They therefore have the potential to destabilise the plaque, leading to localised regions of de-endothelialisation that may subsequently lead to focal thrombosis and plaque rupture. In the later stages of its evolution, a plaque may be stabilised. Although such lesions may be sufficiently prominent to cause luminal narrowing, and hence angina, or claudication, they are less likely to rupture, and are therefore considered relatively safe.

The role of chronic inflammation in atherogenesis

Atherosclerosis bears many hallmarks of a chronic inflammatory disease, and every stage of its evolution is characterised by monocyte/macrophage and T-lymphocyte infiltration⁽Reference Katsuda, Boyd and Fligner¹⁵⁵⁾. The possible stimuli to this inflammatory process include oxidised LDL, homocysteine, free radicals generated from cigarette smoking, and infectious micro-organisms, such as Chlamydia pneumoniae. The T-cell infiltrates are predominantly CD4+ cells and clones derived from human lesions have been found to react to antigens derived from oxidised LDL, heat-shock proteins and micro-organisms⁽Reference Hansson¹⁵⁶⁾. The cytokine milieu within atherosclerotic lesions is thought to promote a Th1-dominated response associated with macrophage activation and the elaboration of IFN-γ and IL-1β⁽Reference Hansson¹⁵⁶⁾. Hence, if the original insult is not adequately neutralised, inflammation may persist, causing the local and systemic release of growth factors and cytokines. These can, in turn, lead to intimal thickening by stimulating smooth muscle cell migration, proliferation and extracellular matrix elaboration.

Inflammatory biomarkers and atherosclerosis

There has been an increasing interest in the use of inflammatory markers to estimate the risks of acute events in patients with established coronary disease. In part, the predictive value of these markers may be related to their ability to identify patients with vulnerable plaques, which are rich in activated leucocytes. CRP, IL-6 and circulating leucocyte count are elevated in patients with myocardial infarction⁽Reference Rifai and Ridker¹⁵⁷⁾. There have also been reports of a positive relationship between serum CRP and subclinical atherosclerosis⁽Reference Hulthe, Wikstrand and Fagerberg¹⁵⁸⁾, although analysis may be confounded because risk factors such as smoking habit, indices of adiposity, blood pressure, TAG and HDL are also associated with CRP concentrations. Adipose tissue may be an additional source of inflammatory cytokines that could stimulate CRP production⁽Reference Mohamed-Ali, Coppack and Stanner¹⁵⁹⁾. Other inflammatory conditions, such as RA and systemic lupus erythematosus, are associated with impaired endothelial function and increased coronary risk⁽Reference Vaudo, Marchesi and Gerli^160, Reference Bruce, Urowitz and Gladman¹⁶¹⁾.

Epidemiology of CVD

The Framingham Heart and Seven Countries Studies were key to identifying cigarette smoking, high serum cholesterol, physical inactivity, high blood pressure, the menopause and psychological factors as contributors to coronary disease. The importance of multiple, low-level risk factors has become clearly evident⁽Reference Kannel, D'Agostino and Sullivan¹⁶²⁾. Death rates from CVD have been falling in many developed countries, including most countries of Western Europe, since the early 1970s⁽Reference Unal, Critchley and Capewell^163–166) and much of this reduction appears to be due to successful implementation of primary intervention due to dietary change, statin treatment, antihypertensive treatment and fall in smoking habit. The rate of reduction in CVD mortality differs among countries. For example, the fall in the UK is slower than that in Australia, Canada and Sweden. However, death rates from CVD have risen rapidly in many Eastern European countries. Poland is an exception to this because rates have fallen in association with a fall in the prevalence of smoking, reduced saturated fat and increased PUFA and fruit consumption⁽Reference Zatonski and Willett¹⁶⁷⁾. Besides national differences, there are very marked regional differences within countries^{(166, 168)}, which may reflect socio-economic differences. The incidence of CHD varies with race and ethnicity⁽Reference Frayn, Stanner and Stanner¹⁶⁹⁾. In the USA, black men and women have a higher risk of CHD than white men and women, respectively, while Hispanic groups have lower CHD rates. South Asians living in the UK have approximately 50 % higher rates of premature CHD death, and the difference is widening as rates do not appear to be falling as fast among this group as for the UK as a whole. This may relate to a higher prevalence of diabetes in this group.

Interventions

The landmark statin trials have demonstrated that cholesterol lowering reduces cardiovascular mortality and morbidity⁽Reference Baigent, Keech and Kearney¹⁷⁰⁾. The early benefits observed in some of the primary prevention trials have suggested that statins may be exerting effects other than those attributable to cholesterol lowering alone, so-called ‘pleiotropic effects’ including effects on inflammation, although this remains a matter of considerable debate. The role of low-dose aspirin treatment in the prevention of coronary disease is also well supported by trial data⁽Reference Baigent, Sudlow and Collins¹⁷¹⁾. However, the mechanism for the protective effect may relate to the anti-platelet effects of aspirin, rather than an effect on inflammation. Although trials of antibiotic treatment have generally not shown a benefit in preventing coronary disease⁽Reference Taylor-Robinson and Boman¹⁷²⁾, it has been argued that diet regimens used to date may not eliminate the carriage of organisms by peripheral monocytes. There have been few studies describing the effects of dietary constituents on inflammation in healthy subjects or patients with CHD. Fish oil supplements, providing very long-chain n-3 PUFA (VLC n-3 PUFA) alter the composition of inflammatory cells resulting in decreased production of inflammatory eicosanoids and cytokines⁽Reference Calder¹⁷³⁾, alter cellular composition of human plaques, making them appear more stable⁽Reference Thies, Garry and Yaqoob¹⁷⁴⁾, and reduce mortality from sudden death in secondary prevention^(175, Reference Marchioli, Barzi and Bomba¹⁷⁶⁾. Meta-analyses support lowered cardiovascular mortality with increased VLC n-3 PUFA intake⁽Reference Bucher, Hengstler and Schindler^177, Reference Studer, Briel and Leimenstoll¹⁷⁸⁾.

Adipose-derived mediators

The prevalence of obesity (defined as a BMI >30 kg/mReference Elewaut, DiDonato and Kim²⁾ is rising throughout the world, and obesity is occurring at a younger age. In the US and most Western countries, obesity has an incidence rate above 20 %. This is a major public health issue because of the direct effect of obesity on the risk of developing type 2 diabetes, CVD, hypertension, stroke, abnormal blood lipids, arthritis, asthma and cancer⁽¹⁷⁹⁾. Until very recently, the role of adipose tissue has been thought to be passive and adipocytes were considered as little more than fat stores. However, it is now clear that this is far from the case and that adipose tissue is an important endocrine organ⁽Reference Hutley and Prins¹⁸⁰⁾. There is increased understanding that, depending on the nutritional state, the profile of hormones released from adipocytes can change from being beneficial to being detrimental⁽Reference Fantuzzi¹⁸¹⁾. The adipocyte plays a major role in the inflammatory response and releases a cocktail of inflammatory mediators and signalling molecules⁽Reference Halaas, Gajiwala and Maffei^182, Reference Montague, Farooqi and Whitehead¹⁸³⁾.

Four findings ignited the interest in the adipocyte as an endocrine organ. Firstly, the discovery of leptin in 1995 changed the way the adipocyte was viewed; the discovery that leptin has a role in signalling the status of adipose tissue to the brain was the first description of a feedback loop involved in body weight control⁽Reference Halaas, Gajiwala and Maffei¹⁸²⁾. Although its role in human subjects is more complicated, the importance of leptin as a hormone involved in energy balance has been exemplified by the case studies of families with leptin deficiency; the children are obese and lose weight with leptin therapy⁽Reference Montague, Farooqi and Whitehead¹⁸³⁾. As human subjects gain weight, they seem to develop leptin resistance. This may be mediated by suppressor of cytokine signalling, which increases in the hypothalamus in tandem with leptin. This blocks the central effect of leptin⁽Reference Greenberg and Obin¹⁸⁴⁾. Leptin has important effects on peripheral metabolism, which have been demonstrated through lipodystrophy models. Leptin has been shown to have a role to play in insulin sensitivity and TAG clearance from the circulation. This observation in evolutionary terms may have allowed weight gain in times of plenty without gross metabolic change.

Secondly, the discovery that adipocytes release TNF-α also changed the view of adipocytes⁽Reference Hotamisligil, Peraldi and Budavari¹⁸⁵⁾. In fact, mRNA expression studies show that adipocytes can synthesise TNF-α and several other cytokines, notably IL-1β and IL-6 among an ever-increasing list. Circulating TNF-α and IL-6 concentrations are mildly elevated in obesity. Most studies suggest increased TNF-α mRNA expression or secretion in vitro in adipose tissue from obese subjects. The factors regulating cytokine release within adipose tissue appear to include not only typical inflammatory stimuli such as lipopolysaccaride, but also the size of the fat cells per se and catecholamines. The effects of cytokines within adipose tissue include some actions that might be characterised as metabolic. TNF-α and IL-6 inhibit lipoprotein lipase, and TNF-α additionally stimulates hormone-sensitive lipase. TNF-α also downregulates insulin-stimulated glucose uptake via effects on GLUT-4, insulin receptor autophosphorylation and insulin receptor substrate-1. All these effects will oppose lipid accumulation within adipocyte⁽Reference Coppack¹⁸⁶⁾. Other effects appear more trophic, and include the induction of apoptosis, regulation of cell size and induction of de-differentiation (the latter involving reduced PPAR-γ). Cytokines are important stimulators and repressors of other cytokines. In addition, cytokines appear to modulate other regulatory systems. Examples of the latter include effects on leptin secretion (probably stimulation followed by inhibition) and reduction in β3-adrenoceptor expression. There seems to be no clear agreement as to which cytokines derived from adipose tissue act as remote regulators, i.e. hormones. Leptin, which is structurally a cytokine, is also a hormone. IL-6 appears to be released systemically by adipose tissue, but TNF-α is probably not. Both leptin and IL-6 appear to act on the hypothalamus, IL-6 acts on the liver, while leptin may have actions on the pancreas⁽Reference Coppack¹⁸⁶⁾.

Thirdly, the discovery of adiponectin, a protein which is specifically and very highly expressed in adipose tissue⁽Reference Gil-Campos, Cañete and Gil¹⁸⁷⁾. This hormone enhances insulin sensitivity in muscle and liver and increases fatty acid oxidation in several tissues, including muscle fibres. It also decreases serum fatty acid, glucose and TAG concentrations: if normal, lean mice are given injections of adiponectin in conjunction with a meal high in fat and sugar, the normal postprandial increases in plasma glucose and TAG concentrations are smaller as the result of an increased rate of clearance from the blood rather than a reduced rate of absorption from the gut. By contrast, if insulin-resistant mice are treated with physiologic concentrations of adiponectin, glucose tolerance is improved and insulin resistance is reduced. In human subjects, plasma adiponectin concentrations fall with increasing obesity and this effect is greater in men than women. Reduced adiponectin concentrations correlate with insulin resistance and hyperinsulinaemia. In addition, several polymorphisms of the adiponectin gene (APM1, mapped to chromosome 3q27) have been identified that are associated with reduced plasma adiponectin concentration and that increase the risk of type 2 diabetes, insulin resistance or the metabolic syndrome. Interestingly, adiponectin appears to be implicated in the development of atherosclerosis. Adiponectin concentrations are reduced in patients with CHD, and adiponectin inhibits TNF-α-induced expression of adhesion molecules and the transformation of macrophages to foam cells, both of which are key components of atherogenesis. In mice deficient in apo E (and thus susceptible to atherosclerosis), treatment with human adiponectin inhibits lesion formation in the aortic sinus by 30 % compared with that in untreated control animals⁽Reference Fantuzzi^181, Reference Greenberg and Obin¹⁸⁴⁾. The central importance of adiponectin is exemplified by the observation that it inhibits NF-κB and reduces the proinflammatory pathway.

Fourth is the observation that adipose tissue is not homogeneous. Macrophage numbers in adipose tissue increase with obesity, where they apparently function to scavenge moribund adipocytes, which increase dramatically with obesity. Macrophages are responsible for most of the cytokine production in adipose tissue, especially in obesity. In fact, adipose tissue macrophages are responsible for perhaps as much as 50 % of adipose tissue TNF-α production⁽Reference Greenberg and Obin¹⁸⁴⁾. In obesity, adipose tissue contains an increased number of resident macrophages so that, in some circumstances, macrophages can constitute up to 40 % of the cell population within an adipose tissue depot.

An integrated view of inflammation and obesity

The issues surrounding weight gain and inflammation cannot be seen in isolation, but need to be viewed alongside theories about variation in adipocyte differentiation, appetite regulation and control of appetite. Recent evidence has suggested that the formation of hypertrophic fat cells in environments of energy excess may be due to a genetically determined differentiation limit of stem cells to adipocytes or metabolic feedback controlling differentiation or more likely a mixture of the two. If there are limited numbers of adipocytes in an environment of energy excess, this will cause excessive lipid filling in the adipocyte and lead to hypertrophic adipocytes that are pro-inflammatory. The key issue at the beginning of the inflammatory process seems to be an excess energy intake. Many of the processes that follow excess energy intake may acutely be an advantage, but an environment of chronic excess energy intake and decreased energy output will work against human health. As the adipocyte stores TAG, there seems to be an increase in cytokine release. Of particular importance is the suppression of adiponectin and the increase in TNF-α. It is possible that TNF-α can decrease proliferation and differentiation of adipocytes. The control mechanisms for adipocyte proliferation and differentiation are complex, but examination of the transcriptional and extracellular signals necessary for stem cell differentiation into a preadipocyte, and from the preadipocyte into a mature fat cell, is being elucidated. Regulation of adipocyte proliferation and differentiation involves growth arrest and the coordinate activation/inactivation of nuclear transcription factors that regulate the expression of genes necessary for lipid storage and insulin sensitivity. ADD/SREB-1, C/EBP-a, -b and -d and PPAR-γ are a few of the transcription factors known to be involved. These transcription factors are regulated in response to extracellular signals, such as PG, cytokines, and hormones including corticosteroids and insulin⁽Reference Hausman, DiGirolamo and Bartness¹⁸⁸⁾. Defects in any one of these steps are potentially important in the failure of proliferation or differentiation of adipocytes. This will limit the number of adipocytes coping with the excess energy intake. This will have several effects.

1. (i) Effects on recruitement of monocytes into adipose tissue. As the lipid content of adipose tissue increases, the adipocytes increase leptin output and this has the effect of increasing expression of adhesion molecules such as ICAM-1 and platelet-endothelial cell adhesion molecule-1 in endothelial cells inducing adhesion and transmigration of monocyte/macrophages. The transmigration of macrophages is further enhanced by monocyte chemoattractant protein-1. The result is a tissue that produces the low-grade systemic inflammation seen in obesity⁽Reference Neels and Olefsky¹⁸⁹⁾.

2. (ii) Effects on apoptosis and TNF-α. In mature white adipocytes, TNF-α is reported to regulate cell size, the suggestion being that as the cells get bigger they produce more TNF-α, which in turn initiates changes to limit adipocyte size or to induce apoptosis. This is possibly coupled with increasing local hypoxia. The net result is recruitment of greater numbers of macrophages that surround the apoptotic cell⁽Reference Coppack^186, Reference Neels and Olefsky¹⁸⁹⁾.

3. (iii) Autocrine effects on adipokines. The increase in TNF-α has been shown to have several autocrine effects including reduced expression of the GLUT4 glucose transporter, increased hormone-sensitive lipase activity leading to increased lipolysis, decreased lipoprotein lipase activity, reduced insulin signalling and decreased C/EBP and PPAR-γ activity, so creating an insulin insensitive adipocyte with a high output of NEFA⁽Reference Coppack¹⁸⁶⁾.

4. (iv) Effects on peripheral tissue. From the earlier adipocyte hypertrophy, increased insulin resistance and increased fatty acid output result and excess dietary fat may be shunted to the liver, skeletal muscle and the β-cell. Importantly, if fat oxidation cannot be increased to compensate for the increased influx of lipid within these tissues, then intracellular accumulation of lipids will occur. The oxidation of fat is mediated by activating AMP-activated protein kinase (AMPK). It is known that adiponectin activates AMPK as does leptin. So, in normal physiology, leptin and adiponectin signal transduction involves the phosphorylation of STAT pathways, but how this translates into the observed changes in lipid metabolism is not clear. One likely mechanism is via increased phosphorylation activation of AMPK, which phosphorylates acetyl CoA carboxylase and malonyl CoA decarboxylase. Phosphorylation inactivates carboxylase, but activates malonyl CoA decarboxylase. Because carboxylase catalyses malonyl CoA formation, and malonyl CoA decarboxylase catalyses its decarboxylation, the net effect of AMPK activation on these target enzymes is to lower malonyl CoA concentration. Malonyl CoA is the first committed step in lipogenesis and a powerful inhibitor of carnitine palmityl transferase-1-mediated fatty acid oxidation. The combination of an increase in fatty acid oxidation and a decrease in fatty acid synthesis could account for the reduction in the lipid content of a cell. At present, however, we do not understand how leptin activates AMPK. In the inflammatory state there is low adiponectin and leptin resistance and therefore low activity of AMPK, and reduced oxidation of fat and the storage of TAG in cells. It is likely that it is products generated from TAG such as unoxidised palmitoyl-CoA that lead to insulin resistance and cell apotosis. Also diacylglycerol has been shown to have effects on protein kinase C; this can in turn affect the insulin signal by causing serine phosphorylation rather than tyrosine phosphorylation on IRS-1 so reducing the insulin signal. This theory is supported by the recent observation that there is a direct relationship between hepatocyte and myocyte lipid content and insulin sensitivity. Interestingly, IL-6 can also affect steroid hormone conversion, changing the balance of sex hormones, which is well known to regulate adipose tissue distribution⁽Reference Unger^190–Reference McGarry¹⁹²⁾.

The release of other cytokines (e.g. plasminogen activation inhibitor 1 (PAI-1)) from the adipocyte can have dramatic effects on risk of disease. There is evidence for an association between increased PAI-1 levels and myocardial infarction. Interestingly, PAI-1 has been shown to predict myocardial events in univariate analysis, but the predictive power was not affected by adjustement for inflammatory parameters and disappeared after adjustment for BMI, TAG and HDL cholesterol. This suggests that the metabolic syndrome is a precursor to high plasma PAI-1 levels in patients prone to atherosclerosis⁽Reference Alessi, Lijnen and Bastelica¹⁹³⁾.

Effect of weight loss on inflammatory markers

The induction of a negative energy balance has profound effects on inflammatory markers in obesity: weight loss with diet and exercise has been shown to decrease circulating IL-6 and TNF-α concentrations in obese subjects⁽Reference Bougoulia, Triantos and Koliakos^194, Reference Cancello and Clement¹⁹⁵⁾. Serum CRP concentration fell 4 months after bariatric surgery but remained significantly elevated relative to non-obese controls⁽Reference Zagorski, Papa and Chung¹⁹⁶⁾. Very recently, there has been a description of a fall in proinflammatory cytokines and macrophage numbers in white adipose tissue following weight loss⁽Reference Cancello and Clement¹⁹⁵⁾. A recent study on obese children has demonstrated falls in plasma TNF-α and CRP concentrations with weight loss⁽Reference Reinehr, Stoffel-Wagner and Roth¹⁹⁷⁾. Interestingly, liposuction has very little effect on inflammatory markers⁽Reference Hansen, Torquati and Abumrad¹⁹⁸⁾. The success of lifestyle intervention has been demonstrated with the American and Finnish Diabetes Prevention trials, which both show long-term benefit in decreasing the risk of type 2 diabetes⁽Reference Knowler, Barrett-Connor and Fowler^199, Reference Tuomilehto, Lindstrom and Eriksson²⁰⁰⁾.

Description, sources, normal intakes and roles

The general structure of a fatty acid is a hydocarbon (acyl) chain of varying length with a carboxyl group at one end and a methyl group at the other. The carboxyl group is reactive and readily forms ester links with alcohol groups for example those on glycerol or cholesterol. The most abundant fatty acids have straight chains of an even number of carbon atoms. Fatty acids containing double bonds are referred to as unsaturated fatty acids; a fatty acid containing two or more double bonds is called a PUFA. Most common unsaturated fatty acids contain cis rather than trans double bonds. However, trans double bonds do occur in ruminant fats (e.g. cow's milk), in plant lipids and in some seed oils. In some PUFA, the double bonds are not separated by a methylene (–CH2–) group, but are conjugated. The systematic name for a fatty acid is derived from the number of carbon atoms in the acyl chain, the number of double bonds, the position of the double bonds (counting the carboxyl carbon as carbon 1) and their configuration (cis or trans). An alternative shorthand notation for fatty acids is frequently used. This relies upon identifying the number of carbon atoms in the chain, the number of double bonds and the position of the double bond closest to the methyl terminus of the acyl chain (the methyl carbon is known as the n-carbon). Thus, cis 9, cis 12-octadecadienoic acid is notated as 18 : 2n-6. In addition to these nomenclatures, fatty acids are often described by a common name⁽Reference Calder, Burdge, Nicolaou and Kokotos²⁰¹⁾. Human subjects cannot synthesise the simplest n-6 and n-3 PUFA (linoleic acid (18 : 2n-6) and α-linolenic acid (18 : 3n-3)) and so these are regarded as essential fatty acids.

Fatty acids in fats, oils and foodstuffs are mainly esterified to glycerol, as TAG, although some are present as esterified components of phospholipids, glycolipids and other lipids. With regard to inflammatory processes, most attention has been paid to the effects of n?6 and n-3 PUFA⁽Reference Calder^173, Reference Calder²⁰²⁾, although other fatty acid classes including saturated and trans fatty acids may also influence inflammatory processes. Important dietary sources of the n-6 PUFA linoleic acid include vegetable oils (e.g. maize, sunflower, safflower and soyabean oils) and products made from those oils (e.g. margarines). Oils like soyabean also contain α-linolenic acid; other good sources of this fatty acid include some nuts, flaxseeds and flaxseed (linseed) oil. Some seed oils contain moderate to high proportions of relatively unusual fatty acids (e.g. γ-linolenic acid (18 : 3n-6) in borage (starflower) and evening primrose oils). Meat is an important source of the VLC n-6 fatty acid, arachidonic acid (20 : 4n-6). Fish can be classified into lean fish that store lipid as TAG in the liver (e.g. cod) or ‘fatty’ (‘oily’) fish that store lipid as TAG in the flesh (e.g. mackerel, herring, salmon, tuna, sardines). The oil obtained from fatty fish flesh or lean fish livers is termed ‘fish oil’ and it has the distinctive characteristic of being rich in the VLC n-3 PUFA eicosapentaenoic acid (EPA; 20 : 5n-3) and DHA (22 : 6n-3).

There are large differences in fat intake among countries with average intakes among adults varying from < 20 g/d in some developing countries to >100 g/d in some developed countries. The mix of fatty acids consumed also varies in accordance with the fatty acid compositions of the fats and oils used in food preparation and of the foodstuffs eaten. Average fat consumption has changed over time and continues to do so. In many developing countries fat intake is increasing, while in developed countries fat intake has tended to decline over the last 40 years or so.

The type of fat consumed has also changed over time, meaning that the fatty acid composition of the human diet has changed. The latest figures for the UK indicate that adult males consume an average of 85 g fat/d and adult females 60 g/d (both approx. 35 % of dietary energy). The main PUFA in the diet is linoleic acid followed by α-linolenic acid. On average, adult men in the UK consume about 12 and 1·9 g linoleic and α-linolenic acids, respectively, per day. Adult women in the UK consume about 8·7 and 1·4 g linoleic and α-linolenic acids, respectively, per day. Hulshof et al. ⁽Reference Hulshof, van Erp-Baart and Anttolainen²⁰³⁾ provided details of intakes of fatty acids among 14 Western European countries, while Burdge & Calder⁽Reference Burdge and Calder²⁰⁴⁾ included some data from North America and Australia. Longer-chain PUFA are consumed in lower amounts than linoleic and α-linolenic acids. Estimates of the intake of arachidonic acid in Western populations vary between 50 and 300 mg/d for adults⁽Reference Calder, Burdge, Nicolaou and Kokotos²⁰¹⁾. In the absence of fatty fish or fish oil consumption, α-linolenic acid is by far the principal dietary n-3 PUFA. Average intake of the VLC n-3 PUFA in the UK and in other Western countries where oily fish consumption is not the norm is estimated at < 250 mg per day⁽Reference Calder, Burdge, Nicolaou and Kokotos²⁰¹⁾.

Fatty acids are transported in the bloodstream largely in esterified form as components of lipoproteins, although albumin-bound NEFA also circulate⁽Reference Frayn²⁰⁵⁾. Fatty acids have many diverse functions in cells; their principal roles are as energy sources and as membrane constituents⁽Reference Calder, Burdge, Nicolaou and Kokotos²⁰¹⁾. Many types of fatty acid can fill these roles. Certain fatty acids have additional, specific roles: arachidonic, dihomo-γ-linolenic (20 : 3n-6) and EPAs act as precursors for eicosanoid synthesis; myristic and palmitic acids may be covalently attached to certain proteins and this is believed to play an important role in the functioning of these proteins; fatty acids are able to influence intracellular signalling processes; fatty acids are able to influence transcription factor activity and gene expression. Through these different actions, fatty acids are able to influence cellular functions and so affect physiological responses, including inflammation.

Effects on inflammatory processes and mechanisms of action

Arachidonic acid, arachidonic acid-derived eicosanoids and inflammation

The key link between fatty acids and inflammation is that eicosanoids that act as mediators and regulators of inflammation are generated from 20-carbon PUFA. Because inflammatory cells typically contain a high proportion of the n-6 PUFA arachidonic acid and low proportions of other 20-carbon PUFA, arachidonic acid is usually the major substrate for eicosanoid synthesis. Eicosanoids, which include PG, thromboxanes, LT and other oxidised derivatives, are generated from arachidonic acid by reactions catalysed by cyclo-oxygenase (COX) and lipoxygenase (LOX) enzymes (Fig. 5). There are at least two COX enzymes and several LOX enzymes that are expressed in different cell types, according to different conditions and which between them produce a range of mediators involved in modulating the intensity and duration of inflammatory responses⁽Reference Lewis, Austen and Soberman^206, Reference Tilley, Coffman and Koller²⁰⁷⁾. These mediators have cell- and stimulus-specific sources and frequently have opposing effects. Thus, the overall physiological (or pathophysiological) outcome will depend upon the cells present, the nature of the stimulus, the timing of eicosanoid generation, the concentrations of different eicosanoids generated and the sensitivity of target cells and tissues to the eicosanoids generated.

Fig. 5 Eicosanoid biosynthesis from arachidonic acid. COX, cyclo-oxygenase; HETE, hydroxyeicosatetraenoic acid; HPETE, hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; LT, leukotriene; PG, prostaglandin; TX, thromboxane.

The amount of arachidonic acid in inflammatory cells can be increased by including it in the diet⁽Reference Calder¹⁷³⁾ and may also be influenced by the dietary intake of its precursor, linoleic acid, although the range of linoleic acid intake over which this relationship occurs has not been defined for human subjects. The role of arachidonic acid as a precursor for the synthesis of eicosanoids indicates the potential for dietary n-6 PUFA (linoleic or arachidonic acids) to influence inflammatory processes. However, this has been little investigated in human settings. Supplementation of the diet of healthy young male subjects with 1·5 g arachidonic acid/d for 7 weeks resulted in a marked increase in the production of PGE2 and LTB4 by endotoxin-stimulated mononuclear cells⁽Reference Kelley, Taylor and Nelson²⁰⁸⁾. However, production of TNF-α, IL-1β and IL-6 by the latter was not significantly altered. Thus, increased arachidonic acid intake may result in changes indicative of selectively increased inflammation or inflammatory responses in human subjects. Supplementation of the diet of healthy elderly subjects with arachidonic acid (0·7 g/d in addition to a habitual intake of about 0·15 g/d) for 12 weeks did not affect endotoxin-stimulated production of TNF-α, IL-1β or IL-6 by mononuclear cells, did not alter superoxide production by neutrophils or monocytes and did not alter plasma-soluble adhesion molecule concentrations⁽Reference Thies, Miles and Nebe-von-Caron²⁰⁹⁾.

Mechanisms of action

It is clear that one anti-inflammatory mechanism of action of VLC n-3 PUFA is antagonism of production of inflammatory eicosanoids from arachidonic acid coupled with the generation of less potent EPA-derived eicosanoids and, in some conditions, anti-inflammatory resolvins⁽Reference Arita, Yoshida and Hong²²⁵⁾. Altered eicosanoid profiles may have downstream effects since some eicosanoids regulate production of inflammatory cytokines. Thus, an n-3 PUFA-induced decrease in eicosanoid production might affect production of TNF-α, IL-1β, IL-6, etc. However, the effects of n-3 PUFA on inflammatory cytokine production and on some other inflammatory processes appear to be eicosanoid independent. One alternative candidate mechanism of action is altered activation of transcription factors involved in inducing transcription of inflammatory genes (e.g. NF-κB, PPAR-γ; see Calder⁽Reference Calder^226, Reference Calder²²⁷⁾), which may occur as a result of altered plasma membrane signalling processes⁽Reference Lee, Plakidas and Lee^228–Reference Weatherill, Lee and Zhao²³¹⁾.

Effects on clinical outcomes in inflammatory conditions

Chronic obstructive pulmonary disease

Two recent studies have investigated the effects of fairly low supplemental intake of EPA+DHA on inflammatory markers, as well as exercise capacity, in COPD⁽Reference Broekhuizen, Wouters and Creutzberg^241, Reference Matsuyama, Mitsuyama and Watanabe²⁴²⁾. Broekhuizen et al. ⁽Reference Broekhuizen, Wouters and Creutzberg²⁴¹⁾ reported improved exercise capacity in a cycling test but no effect on systemic levels of IL-6 and TNF-α following intake of a supplement containing 1·0 g/d EPA+DHA. Matsuyama et al. ⁽Reference Matsuyama, Mitsuyama and Watanabe²⁴²⁾ measured both systemic (serum) and sputum TNF-α and IL-8 levels in a 2-year intervention with a supplemental intake of 0·6 g/d EPA+DHA. The present study also failed to find differences in serum cytokine concentrations, but did report decreased sputum TNF-α and IL-8 levels in the n-3 PUFA group. This effect was accompanied by improved exercise capacity in a walk test.

Rheumatoid arthritis

Dietary fish oil shows improvements in animal models of RA (see Calder⁽Reference Calder¹⁷³⁾). Several studies report anti-inflammatory effects of fish oil in patients with RA, such as decreased LTB4 production by neutrophils and monocytes, decreased IL-1 production by monocytes, decreased plasma IL-1β concentrations, decreased serum CRP concentrations and normalisation of the neutrophil chemotactic response (see Calder⁽Reference Calder¹⁷³⁾ for references). A number of randomised, placebo-controlled, double-blind studies of fish oil in RA have been reported (see Calder⁽Reference Calder¹⁷³⁾ for details). The dose of VLC n-3 PUFA used in these trials was between 1·6 and 7·1 g/d and averaged about 3·5 g/d. Almost all of these trials showed some benefit of fish oil, including reduced duration of morning stiffness, reduced number of tender or swollen joints, reduced joint pain, reduced time to fatigue, increased grip strength and decreased use of non-steroidal anti-inflammatory drugs. One study reported greater efficacy of fish oil against a background diet designed to be low in arachidonic acid⁽Reference Adam, Beringer and Kless²⁴³⁾. Reviews of the trials of fish oil in RA have concluded that there is benefit and a meta-analysis concluded that dietary fish oil supplementation significantly reduces tender joint count and morning stiffness⁽Reference Fortin, Lew and Liang²⁴⁴⁾. A recent meta-analysis has concluded that VLC n-3 fatty acids may reduce requirements for corticosteroids⁽Reference MacLean, Mojica and Morton²³⁴⁾. Thus, there is reasonably strong evidence that VLC n-3 PUFA have some clinical benefits in RA.

Oxidative stress and inflammation

There is a strong interaction between oxidative stress and inflammation. The generation of oxidants (e.g. superoxide radicals, hydrogen peroxide), for example by granulocytes exposed to bacterial cell wall peptides or lipopolysaccharides, is part of the host inflammatory response. Oxidants can damage components of host cells, such as the PUFA in cell membranes. In turn, oxidants and oxidised cell components, acting through transcription factors such as NF-κB, induce production of inflammatory eicosanoids and cytokines (Fig. 4). Thus, one mechanism to diminish inflammatory mediator production may be to prevent oxidative stress. This is accomplished through enhancing antioxidant defence mechanisms. In order to monitor antioxidant defence, individual components of that defence can be measured (e.g. vitamin C, vitamin E, antioxidant enzyme activities). Additionally, the effectiveness of the non-enzymatic endogenous antioxidant network can be assessed measuring total antioxidant capacity (TAC), defined as the moles of oxidants neutralised by 1 litre of the tested sample⁽Reference Serafini and Del²⁵¹⁾. TAC considers the cumulative action of all the antioxidants present in the matrix (plasma, saliva, diet, etc.), thus providing an integrated parameter rather than the simple summary of measurable antioxidants, giving an insight into the delicate balance between antioxidants molecules. Endogenous TAC has been shown to be modulated by dietary ingestion of plant food⁽Reference Serafini, Bugianesi and Maiani²⁵²⁾ and to decline in subjects affected by CVD⁽Reference Vassalle, Petrozzi and Botto²⁵³⁾ and cancer⁽Reference Ching, Ingram and Hahnel²⁵⁴⁾. Population-based case-control studies have shown an inverse association of the TAC of the diet with risk of gastric cancer⁽Reference Serafini, Bellocco and Wolk²⁵⁵⁾ and overall mortality⁽Reference Agudo, Cabrera and Amiano²⁵⁶⁾. Despite these promising results, evidence about the role of the dietary or endogenous TAC and inflammation is lacking. Recently, TAC has been shown to be inversely and independently associated with plasma levels of CRP in 243 non-diabetic subjects⁽Reference Brighenti, Valtuena and Pellegrini²⁵⁷⁾. This observation highlights the interrelationship between oxidative stress and inflammation and suggests that improving TAC by dietary means may be a fruitful anti-inflammatory approach.

Vitamin C

Effects in inflammatory conditions

Lung inflammation

Evidence of AA oxidation has been seen in adult respiratory distress syndrome⁽Reference Cross, Forte and Stocker²⁷³⁾. Adults with mild asthma exhibit decreased AA in lung fluid and increased GSSG concentrations which, in the presence of normal plasma concentrations, are suggestive of oxidative stress in the airways⁽Reference Kelly, Mudway and Blomberg²⁸¹⁾. Cross-sectional studies showed an inverse relation between plasma vitamin C and lung inflammation⁽Reference Winklhofer-Roob, Ellemunter and Fruhwirth²⁸²⁾ and suggest that high plasma AA concentrations or vitamin C intakes have a positive impact on lung function⁽Reference Schwartz and Weiss^283, Reference Britton, Pavord and Richards²⁸⁴⁾. Compared with the highest quintile, serum vitamin C concentrations in the lowest quintile were associated with an increased risk of asthma in children and adolescents⁽Reference Harik-Khan, Muller and Wise²⁸⁵⁾. Low maternal vitamin C intake during pregnancy is associated with asthma in 5-year-old children⁽Reference Devereux, Turner and Craig²⁸⁶⁾. Asthmatic children showed reduced vitamin C concentrations in leucocytes (50 %) and plasma (35 %)⁽Reference Aderele, Ette and Oduwole²⁸⁷⁾. Small clinical studies showed that vitamin C supplements of 2 g/d exert protective effects on airway responsiveness to viral infections and allergens⁽Reference Bucca, Rolla and Farina²⁸⁸⁾, but have little effect on exercise-induced asthma⁽Reference Cohen, Neuman and Nahum²⁸⁹⁾, although one study did report attenuated exercise-induced bronchoconstriction in patients with asthma⁽Reference Tecklenburg, Mickleborough and Fly²⁹⁰⁾.

Rheumatoid arthritis

Evidence of AA oxidation has been seen in RA⁽Reference Lunec and Blake²⁷²⁾. An intervention study with a Mediterranean diet, rich in antioxidant containing foods, in RA showed a negative correlation between plasma vitamin C levels and disease activity⁽Reference Hagfors, Leanderson and Skoldstam²⁹¹⁾.

CVD

Vitamin C and other single vitamin supplementation and CVD have been reviewed recently⁽Reference Traber²⁹²⁾. Associations between antioxidants and markers of endothelial function (sICAM-1; flow-mediated vasodilation) and inflammation (CRP, fibrinogen and white blood corpuscles count) were studied in subjects recruited from the general population. Plasma vitamin C and CRP concentrations were inversely related⁽Reference van Herpen-Broekmans, Klopping-Ketelaars and Bots²⁹³⁾. Recent randomised controlled trials have shown that vitamins C plus E improved arterial stiffness and endothelial function in patients with essential hypertension⁽Reference Plantinga, Ghiadoni and Magagna²⁹⁴⁾, vitamin C decreased sPselectin concentration and vitamins C plus E decreased sICAM-1 in patients with chronic degenerative aortic stenosis with or without concomitant coronary artery disease⁽Reference Tahir, Foley and Pate²⁹⁵⁾.

Vitamin E

Effects in inflammatory conditions

Carotenoids

Effects in inflammatory conditions

While β-carotene has been studied quite extensively, data are limited for other carotenoids. In Table 2, the effects of carotenoids as well as of vitamins C and E are listed. Biomarkers of inflammation have been included in a variety of studies and quite consistently either showed an inverse relation between status of different carotenoids and inflammation or reduction in elevated biomarkers in response to intervention using carotenoid-rich foods or supplements. Clinical end point studies are particularly scarce. Like other serum antioxidants such as vitamin C, plasma carotenoid concentrations decline during the acute-phase response to infection and injury in the presence of increased levels of biomarkers of inflammation⁽Reference Louw, Werbeck and Louw³⁴⁴⁾. Inverse relations between plasma β-carotene concentrations and biomarkers of inflammation have been shown in a re-evaluation study of the National Health and Nutrition Examination Survey III study⁽Reference Kritchevsky, Bush and Pahor³⁴⁵⁾, as well as in studies in critically ill patients⁽Reference Quasim, McMillan and Talwar³⁴⁶⁾. The underlying mechanisms are not known, but may involve a cellular carotenoid-binding protein⁽Reference Lakshman and Rao³⁴⁷⁾. Most importantly, however, β-carotene and retinol levels are restored after resolution of the inflammatory state in the absence of β-carotene and/or retinol supplements. Study of the effects of lycopene on murine bone marrow-derived DC, which are the most potent antigen-presenting cells, revealed that lycopene downregulates NF-κB as well as mitogen-activated kinases such as extracellular signal-regulated kinase 1/2, p38 and jun N-terminal kinase⁽Reference Kim, Kim and Ahn³⁴⁸⁾. Astaxanthin showed a dose-dependent anti-inflammatory effect in endotoxin-induced uveitis in rats by suppressing NO, PGE2 and TNF-α production⁽Reference Ohgami, Shiratori and Kotake³⁴⁹⁾. Mice fed lycopene showed significant inhibition of ear swelling induced by UV radiation⁽Reference Lee, Faulhaber and Hanson³⁵⁰⁾. In a RCT, high intake of carotenoid-rich fruits and vegetables reduced CRP concentrations⁽Reference Watzl, Kulling and Möseneder³⁵¹⁾. In another RCT, dietary intake of a tomato-based drink (providing 5·7 mg lycopene, 1 mg β-carotene, 3·7 mg phytoene and 2·7 mg phytofluene and 1·8 mg α-tocopherol per day), was associated with 34 % decrease in TNF-α in whole blood⁽Reference Riso, Visioli and Grande³⁵²⁾.

Table 2 Summary of studies investigating the anti-inflammatory actions of vitamins C and E and of carotenoids

LT, leukotriene; COX, cyclo-oxygenase; CEHC, carboxyethyl hydroxychroman; ERK, extracellular signal-regulated kinase; JNK, jun N-terminal kinase; iNOS, inducible NO synthase; RA, rheumatoid arthritis; ICAM, intercellular adhesion molecule; CRP, C-reactive protein; WBC, white blood cell count; NHANES, National Health and Nutrition Examination Survey; EPIC, European Prospective Investigation of Cancer; IBD, inflammatory bowel disease; IFN, interferon; MDA, malondialdehyde.

Inflammatory bowel diseases. Plasma concentrations of different carotenoids are decreased in patients with CD⁽Reference Wendland, Aghdassi and Tam^277, Reference Rumi, Szabo and Vincze³⁵³⁾. In a rat model of colitis, lycopene efficiently counteracted the inflammatory response and mucosal damage in iron-supplemented animals⁽Reference Reifen, Nur and Matas³⁵⁴⁾, which can be explained by the fact that transition metal iron can give rise to the formation of hydroxyl radical, the most reactive and cytotoxic ROS that has the capacity of oxidising biomolecules including PUFA, proteins and DNA bases and that lycopene acts as an antioxidant.

Asthma. Whole blood concentrations of total carotenoids and lycopene were decreased in patients with asthma compared with controls and correlated with sputum concentrations of carotenoids⁽Reference Wood, Garg and Blake³⁵⁵⁾.

Rheumatoid arthritis. An inverse association between serum β-cryptoxanthin levels and RA has been reported in women aged 55–69 years⁽Reference Cerhan, Saag and Merlino³⁵⁶⁾. A nested case-control study within the European Prospective Investigation of Cancer Incidence study focused on dietary intake and occurrence of new cases of inflammatory polyarthritis and showed that subjects with dietary intake of zeaxanthin and β-cryptoxanthin in the highest tertile were at lower risk of developing inflammatory polyarthritis compared with those in the lowest tertile. The effect of β-cryptoxanthin remained significant also after adjustments for total energy and protein intakes as well as for smoking⁽Reference Pattison, Symmons and Lunt³⁵⁷⁾. In another study, plasma β-carotene concentrations preceding diagnosis of RA by 2–15 years were lower in patients who developed RA compared with controls⁽Reference Comstock, Burke and Hoffman³⁵⁸⁾.

CVD. Association between antioxidants and markers of endothelial function (sICAM-1 and flow-mediated vasodilation) and inflammation (CRP, fibrinogen and white blood cell count) were studied in subjects recruited from the general population. Lutein and lycopene were inversely related to sICAM-1 and β-carotene was inversely related to white blood cell count and CRP⁽Reference van Herpen-Broekmans, Klopping-Ketelaars and Bots²⁹³⁾. In a nested case-control study within the Women's Health Study, CVD risk was associated with plasma lycopene concentrations such that women in the upper three quartiles of plasma lycopene concentrations had a 50 % risk reduction⁽Reference Sesso, Buring and Norkus³⁵⁹⁾. Similar results were not observed in men participating in the Physicians' Health Study⁽Reference Sesso, Buring and Norkus³⁶⁰⁾.

Obesity. Low carotenoid and elevated CRP concentrations are frequently observed in obesity, and are attributed to unfavourable dietary habits and low-grade inflammation⁽Reference Suzuki, Inoue and Hioki^361, Reference Wallstrom, Wirfalt and Lahmann³⁶²⁾. An inverse correlation between plasma carotenoid concentrations and BMI was reported in obese children and adolescents⁽Reference Winklhofer-Roob, Sargsyan and Maritschnegg³⁶³⁾.

Description, sources, normal intakes and roles

Polyphenols are secondary metabolites of plants involved in pigmentation, reproduction and protection against pathogens⁽Reference Manach, Scalbert and Morand³⁶⁴⁾. Presently there are more than 8000 known polyphenolic substances sharing a common chemical structure (hydroxyl group on aromatic ring) with different constituents. Flavonoids are the most abundant polyphenols present in the human diet and represents a subclass of molecules characterised with a C6–C3–C6 backbone structure⁽Reference Manach, Scalbert and Morand³⁶⁴⁾. Flavonoids can be divided into several classes according to different constituents such as flavanones, flavone, flavanols and flavonols. They can be found in almost all plant foods and, among the flavonols, myricetin, kaempferol and quercetin are the most representative, while catechins are the most abundant flavanols contained in tea leaves. Flavanones are mainly represented in the diet by taxifolin, naringinin and hesperitin. The main sources of flavanones are citrus fruits. Flavones, luteolin, wogonin and apigenin, are less common and identified in sweet red pepper and celery. In addition to these, other classes of flavonoids are present in the diet such as proanthocyanidins and their oligomers.

Dietary intake of flavonoids, calculated in 1976 in the US⁽Reference Kuhnau³⁶⁵⁾, was 1 g/d with 16 % as flavonols, flavones and flavanones; 20 % as catechins; 17 % as anthocyanins and 45 % as bisflavones. However, the high variability of polyphenol intake, the lack of homogeneous database and reliable biomarkers of exposure make it difficult to provide a reliable estimate of flavonoid intake in the different countries.

Over the last years, there have been significative advances in the understanding of the absorption and metabolism of flavonoids. In order to be absorbed, flavonoid glycosides, the most abundant form in food, are hydrolysed by intestinal enzymes or colon microflora. During the process of absorption, flavonoids are conjugated in the small intestine and later in the liver to increase their hydrophilicity and facilitate their elimination from the body in urine and bile. The circulating forms of flavonoids are mainly conjugated and evidence regarding their accumulation in specific target tissues is still lacking.

Flavonoids have been suggested to have anti-inflammatory activity through several action mechanisms involving the reduction in the concentration of prostanoids and leukotrienes through the inhibition of eicosanoid generating enzymes such as phospholipase A2, COX and LOX⁽Reference Baumann, von Bruchhausen and Wurm³⁶⁶⁾.

Anti-inflammatory actions of flavonoids

Quercetin was the first flavonoid shown to inhibit neutrophil phospholipase A2⁽Reference Lee, Matteliano and Middleton³⁶⁷⁾ with an IC50 of 57–100 μm. Flavanones such as hesperitin and naringenin were shown to be less effective inhibitors of snake venom phospholipase A2 compared with the flavonols quercetin, kaempferol and myricetin⁽Reference Welton, Tobias and Fiedler-Nagy³⁶⁸⁾. Biflavonoids such as amentoflavone, ginkgetin, ochnaflavone and iso-ginkgetin were shown to be good inhibitors, with an IC50 of about 10 μm for phospholipase A2 from several sources. Luteolin, 3,4-dihydroxy-flavone, galangin, apigenin and morin were the first flavonoids to be shown to inhibit COX⁽Reference Baumann, von Bruchhausen and Wurm³⁶⁶⁾. Quercetin and kaempferol are potent inhibitors of COX from rat peritoneal macrophages⁽Reference Welton, Tobias and Fiedler-Nagy³⁶⁸⁾. The flavones apigenin, luteolin, galangin, kaempferol and quercetin and the biflavonoids amentoflavone, broussochalcone A and kuraridin are potent COX-1 inhibitors, although with different potencies. Catechin weakly inhibited COX-2 but at very high concentration (100 μm)⁽Reference Noreen, Serrano and Perera³⁶⁹⁾. Flavonols such as kaemferol, quercetin, morin and myricetin were found to be better LOX inhibitors than flavones and with a preferential effect on 5-LOX compared with 12-LOX. Flavanones such as naringenin were not inhibitory against 5- and 12-LOX. Endothelial NOS was inhibited weakly by quercetin at very high concentration (IC50 220 μm)⁽Reference Chiesi and Schwaller³⁷⁰⁾ with a lack of effect on nNOS and iNOS. Several flavonoids (rutin, hepseridin, catechin and tricin) had no effect on the three forms of NOS. Using LPS- or cytokine-treated macrophages or macrophage cell lines for stimulated NO production, quercetin, apigenin and luteolin were found to inhibit NO production⁽Reference Kim, Murakami and Nakamura³⁷¹⁾, downregulating iNOS expression. Catechins and flavanones at concentrations below 100 μm did not affect NO production by LPS-treated RAW 264·7 cells⁽Reference Liang, Huang and Tsai³⁷²⁾. Glycosylated flavonoids, such as vitexin, had little effect on NO production with no effect at a concentration of 100 μm. Flavones such as apigenin, wogonin and luteolin were the most efficient inhibitors (IC50 10–20 μm). Furthermore, studies⁽Reference Kim, Cheon and Kim³⁷³⁾ showed that the inhibitory effect of apigenin, genistein and kaempferol on NO production is mediated by an effect on induction of iNOS expression. Taken together, these results suggest that some flavonoids are natural inhibitors of iNOS induction, but not inhibitors of iNOS activity. With regard to inflammatory cytokines, genistein was reported to inhibit IL-1β, IL-6 and TNF-α production by LPS-stimulated human blood monocytes⁽Reference Geng, Zhang and Lotz³⁷⁴⁾. Genistein and silybin were shown to inhibit TNF-α production from LPS-treated RAW cells⁽Reference Cho, Kim and Park³⁷⁵⁾. Flavonoids appear to inhibit the expression of inflammation-related enzymes/proteins partly by suppressing activation of NF-κB and AP-1, an effect potentially mediated by the inhibition of different protein kinases (e.g. mitogen-activated protein kinase; extracellular signal-regulated kinase 1/2) involved in signal transduction pathway.

Quercetin has been shown to affect iNOS expression by inhibiting p38 MAPK and TNF-α from LPS-induced RAW cells by inhibiting jun N-terminal kinase/SAPK and Ap-1 DNA binding⁽Reference Wadsworth, McDonald and Koop^376, Reference Wadsworth and Koop³⁷⁷⁾. TNF-α was also inhibited through inhibition of extracellular signal-regulated kinase 1/2 and p38 MAPK. Quercetin was shown also to affect NF-κB activation by extracellular signal-regulated kinase and p38 kinase inhibition⁽Reference Cho, Park and Kwon³⁷⁸⁾. The effect on NF-κB was shown also for genistein, apigenin, kaempferol and epigallocatechin 3-gallate. The evidence so far shows that flavonoids are able to inhibit the expression of inflammation-related enzymes/proteins partly by suppressing activation of NF-κB and AP-1, an effect potentially mediated by the inhibition of different protein kinases involved in the signal transduction pathway.

Regarding the effect of flavonoids on the expression of inflammatory markers and processes in vivo, scientific evidence is largely lacking. Quercetin inhibited release of TNF-α from carrageenan-induced air-pouch exudates in rats⁽Reference Morikawa, Nonaka and Narahara³⁷⁹⁾. Quercetin and rutin were found to suppress lethal endotoxic shock induced by LPS in mice⁽Reference Takahashi, Morikawa and Kato³⁸⁰⁾. Orally administered luteolin was showed to inhibit TNF-α production in LPS-treated mice⁽Reference Ueda, Yamazaki and Yamazaki³⁸¹⁾.

Human studies investigating the effect of flavonoids on markers of inflammation are scarce and most of them focus on the use of flavonoid-rich foods and not on pure molecules. One month of supplementation with 30 g/d ethanol as wine or as gin in healthy men was able to decrease plasma levels of IL-1α and fibrinogen⁽Reference Zern, Wood and Greene³⁸³⁾. However, expression of the several adhesion molecules on monocytes and T-lymphocytes was significantly decreased only after wine ingestion. Wine also decreased serum concentrations of sVCAM-1, sICAM-1 and CRP. In another study⁽Reference Zern, Wood and Greene³⁸³⁾, supplementation with a grape polyphenol extract containing anthocyanins, quercetin, myricetin, kaempferol and resveratrol (36 g/d for 4 week) produced a significant decrease in plasma TNF-α with no effect on CRP and IL-6 concentrations in both pre- and post-menopausal women. In both studies described earlier, the lack of measurement of polyphenol levels in body fluids does not allow any conclusion about the molecules responsible for the anti-inflammatory effect to be made. In an intervention study where plasma levels of flavonoids were measured, daily black tea supplementation (900 ml) for 4 weeks significantly increased plasma catechin levels (29 %) compared with baseline values but without any changes on CRP and TAC⁽Reference Widlansky, Duffy and Hamburg³⁸⁴⁾. Moreover, in a prospective study in non-diabetic women, the intake of flavonols and flavones was not significantly associated to plasma concentrations of CRP and IL-6 or with the development of type 2 diabetes⁽Reference Song, Manson and Buring³⁸⁵⁾. Recently, in the attempt to investigate diet-induced postprandial oxidative and inflammatory stress, acute ingestion of 50 ml of extra virgin olive oil has been shown to decrease plasma levels of inflammatory molecules (LTB4 and TXB2) together with an increase in serum TAC⁽Reference Bogani, Galli and Villa³⁸⁶⁾.

As overall, only about 1 % of the polyphenol that is consumed is absorbed, with large differences according to the different categories, the plasma concentration that is achieved after eating polyphenol rich food is about 1 μm⁽Reference Manach, Scalbert and Morand³⁶⁴⁾. The low concentration of flavonoids in biological fluids might present an obstacle in exerting the putative anti-inflammatory action in vivo. Human studies involving food items need to be linked with a chemical characterisation of the flavonoid composition of the food and with the assessment of specific biomarkers of single phenolics and antioxidant activity. Moreover, information obtained on the basis of acute ingestion studies might be extremely valuable for designing long-term intervention trials. On the basis of the existing evidence, a clear conclusion cannot be drawn and further human trials are needed to elucidate the role of flavonoids as anti-inflammatory agents in vivo.

Gut microflora

The intestinal epithelium of the gastrointestinal tract and its associated microflora are vital to the protection of the body⁽Reference Rolfe, Mackie, White and Isaacson^387, Reference Holzapfel, Goktepe, Juneja and Ahmedna³⁸⁸⁾. At birth, the gastrointestinal tracts of babies are sterile but then rapidly become colonised by micro-organisms from the mother's faecal and vaginal microbiota and the immediate environment. Consumption of oxygen within the gut by the first colonising organisms, facultative anaerobic bacteria, enables fully anaerobic bacteria to then colonise the lower gut. Bifidobacteria are more predominant in breast-fed babies, whereas a more complex population (similar to that of adults) develops in formula-fed babies, which comprises clostridia, bacteroides, enterobacteria, streptococci and a lower level of bifidobacteria⁽Reference Edwards and Parrett³⁸⁹⁾. SCFA produced by bifidobacteria help to protect the baby against infection. During the first year of life, the intestinal microbiota stabilises and gradually resembles that of an adult. Up to 1014 micro-organisms are contained in the adult human body – ten times more microbial cells than human – most of these micro-organisms are located in the gastrointestinal tract⁽Reference Holzapfel, Haberer and Snel³⁹⁰⁾. More than 1 kg of bacteria is present in the gut and faeces typically comprise 50 % bacteria, meaning that human subjects excrete 50–100 g of bacteria each day. Micro-organisms are at their lowest numbers ( < 103 CFU/g) in the stomach due to its low pH and fast transit time but reach levels as high as 1012 CFU/g in the colon, due to its much slower transit time, less acidic pH and low oxygen levels. Faecal isolates are generally representative of the microbiota of the distal colon. At least 500 different bacterial species have been cultured from faeces (including lactobacilli and bifidobacteria), yet just forty species account for 99 % of those that have been identified. The microbial inhabitants of the intestinal microbiota are still not fully identified due to the limitations of identification techniques and individual variations. New molecular typing methods are now enabling identification of further gut-associated species.

Studies with germ-free animals have demonstrated the importance of the gut microflora for the normal development of the gastrointestinal tract and the education and stimulation of the immune system⁽Reference Rhee, Sethupathi and Driks^391–Reference Yamanaka, Helgeland and Farstad³⁹³⁾. In the absence of an intestinal microflora, the intestinal immune system is underdeveloped and certain functional parameters, such as macrophage phagocytic ability and Ig production, are reduced⁽Reference Wostmann³⁹⁴⁾. The intestinal microflora may also be important in protection against atopic disease⁽Reference Ouwehand, Isolauri and Salminen^395–Reference Rautava, Ruuskanen and Ouwehand³⁹⁷⁾, as demonstrated by Sudo et al. ⁽Reference Sudo, Sawamura and Tanaka³⁹⁸⁾ who showed that oral tolerance could be restored in germ-free mice by reconstitution of the intestinal microflora with bifidobacteria at the neonatal stage. The intestinal microflora of infants with atopic disease may differ from healthy infants, with higher levels of clostridia and lower levels of bididobacteria in those with atopy⁽Reference Kalliomaki, Kirjavainen and Eerola³⁹⁶⁾.

The major function of the colonic microflora is the fermentation of dietary substances that have passed undigested through the small intestine and mucus produced within the gut. Saccharolytic fermentation produces SCFA such as acetate, propionate, butyrate, which are essential nutrients for the colonocytes. The acids have an additional protective benefit by lowering the pH of the intestinal lumen and by their antimicrobial nature. Butyric acid (produced by bifidobacteria) is particularly important because it is the preferred energy source of the epithelial cells in the colon and influences cell proliferation. It is thought to play a prominent role in reducing the risk of colon cancer. By contrast, some of the end products of proteolytic fermentation are carcinogenic⁽Reference Macfarlane, Macfarlane, Fuller and Perdigon³⁹⁹⁾. A healthy, or balanced, intestinal microflora is thought to be one that is predominantly saccharolytic and that comprises significant numbers of bifidobacteria and lactobacilli, such that their activities predominate over those that may be harmful (e.g. clostridia, Staphylococcus aureus, toxic E. coli, sulphate-reducers and certain Bacteroides species)⁽Reference Cummings, Antoine and Azpiroz^10, Reference Gibson and Roberfroid^400–Reference Fooks, Fuller and Gibson⁴⁰²⁾. Predominance of the latter groups of bacteria may predispose the host to a number of clinical disorders while increasing susceptibility to infections by transient enteropathogens⁽Reference Fooks, Fuller and Gibson⁴⁰²⁾.

Prebiotics

Probiotics

Effects in inflammatory conditions