Impact of fatty acid status on immune function of children in low‐income countries

Abstract

In vitro and animal studies point to numerous mechanisms by which fatty acids, especially long‐chain polyunsaturated fatty acids (LCPUFA), can modulate the innate and adaptive arms of the immune system. These data strongly suggest that improving the fatty acid supply of young children in low‐income countries might have immune benefits. Unfortunately, there have been virtually no studies of fatty acid/immune interactions in such settings. Clinical trial registers list over 150 randomized controlled trials (RCTs) involving PUFAs, only one in a low‐income setting (the Gambia). We summarize those results here. There was evidence for improved growth and nutritional status, but the primary end point of chronic environmental enteropathy showed no benefit, possibly because the infants were still substantially breastfed. In high‐income settings, there have been RCTs with fatty acids (usually LCPUFAs) in relation to 18 disease end points, for some of which there have been numerous trials (asthma, inflammatory bowel disease and rheumatoid arthritis). For these diseases, the evidence is judged reasonable for risk reduction for childhood asthma (but not in adults), as yielding possible benefit in Crohn's disease (insufficient evidence in ulcerative colitis) and for convincing evidence for rheumatoid arthritis at sufficient dose levels, though formal meta‐analyses are not yet available. This analysis suggests that fatty acid interventions could yield immune benefits in children in poor settings, especially in non‐breastfed children and in relation to inflammatory conditions such as persistent enteropathy. Benefits might include improved responses to enteric vaccines, which frequently perform poorly in low‐income settings, and these questions merit randomized trials.

Introduction

The effects on immune function of fatty acids (FAs), and particularly of the long‐chain polyunsaturated FAs (LCPUFAs), have been intensively researched over the past several decades, and numerous comprehensive reviews are available (Grimble 2001; Harbige 2003; Calder 2008, 2009, 2010; Gottrand 2008; Galli & Calder 2009; Ganapathy 2009). This paper will not seek to replicate these reviews. Instead, it will attempt to extract from the myriad available data the elements most relevant to the immune defences of young children brought up in the nutritionally deprived and highly infectious environment still typical of many low‐income countries. We will attempt to focus on what is known and to highlight the far greater number of unknowns that arise from the fact that this is a most under‐researched topic.

Our task is made easier by the accompanying papers reviewing the FA profiles of typical diets in less‐developed countries (LDCs) (Michaelsen et al. 2011), the physiological roles of the various FAs during early life (German 2011), the genetics of PUFA conversion (Glaser et al. 2011) and the transfer mechanisms across the placenta and via breast milk (Lauritzen & Carlson 2011).

We have chosen to focus on immune functions as they develop and mature through infancy and into childhood in the belief that these are the critical periods at which interventions are most likely to be effective.

Key messages

• • There are multiple putative pathways through which fatty acids, especially long‐chain polyunsaturated fatty acids (LCPUFAs), may modulate immune function.
• • Evidence for these actions is derived mostly from in vitro and small animal studies.
• • Randomized controlled trials in affluent populations, mostly aimed at ameliorating inflammatory conditions, have yielded fairly robust evidence for efficacy in a number of diseases.
• • Populations in low‐income countries, particularly post‐weaning children, are very likely to have deficient intakes of LCPUFAs, which might affect immune function.
• • There have been almost no fatty acid or LCPUFA trials in low‐income countries, and this represents an important research gap.

The survival challenge facing children in low‐income settings

Despite the numerous advances in medical knowledge, antibiotics, drug therapies and vaccines, infections are still by far the largest contributor to the high levels of infant and under‐five mortality seen in many LDCs because of shortfalls in health provision, hygiene and vector control. Nutritional deficiencies are estimated to account (directly and indirectly) for about 35% of these deaths (Black et al. 2008), and case‐specific fatality rates are very strongly related to measures of undernutrition across diseases such as diarrhoea, pneumonia and measles (Black et al. 2008).

The neonate is born into such a dangerous world with a largely naive immune system. If it is unlucky, it may already be carrying one of the infections that can cross the placenta or be acquired at birth, but in most cases, it will arrive uninfected and with an essentially aseptic gut. In the early hours, days and months, it is protected by its innate immunity, by maternal antibodies and (in an ideal world) by the many anti‐microbial factors provided by breastfeeding. But within minutes of birth, it must start the processes of acquiring the micro‐organisms that will colonize its gut and start training its immune system to discriminate between friend and foe at both the cellular level (symbionts vs. pathogens) and the molecular level (self and food antigens vs. toxins and molecules signalling the presence of a pathogen). Despite the protection of being swaddled close to its mother and receiving nutrients directly from her, such infants will inevitably be exposed from their very first breath to a wide array of micro‐organisms – some of which could be potentially fatal. Appreciating the immense complexities of these developing processes is a first step in trying to untangle a possible modulating role for FAs. The transition from this typical LDC situation to the more sterile environment of the first world has also been a strong driver of FA immune function research as changes in FA intakes have had a hypothesized role in the aetiology of asthma and allergies (Raviv & Smith 2010) – immuno‐regulatory diseases that become more common as hygiene improves.

FAs and immune function – a very basic summary

The human immune system has two arms: innate and adaptive. Innate immunity includes barrier defences, the complement system, numerous pathogen recognition receptors, multiple chemical signalling systems, natural killer cells, macrophages, neutrophils, mast cells, eosinophils, basophils and dendritic cells. It is phylogenetically more ancient than the adaptive mechanisms, but this should not be taken to imply simplicity; each of these processes represents an enormously complex biological system that needs to work in concert with the others. Innate immunity has the advantage that it responds to generic markers of potential pathogens that may never have been encountered by a neonate. Its chief disadvantage is that it is not perfect; for instance, maintaining a perfect epithelial barrier integrity over the numerous square metres of the intestinal and respiratory tracts is simply not possible, and numerous micro‐organisms have evolved ways to evade innate immune defences. Some have evolved to actually colonize immune defence cells; Mycobacteria tuberculosis, for instance, invades and replicates within the phagolysosome of macrophages. In contrast, adaptive immunity relies on prior exposure to an organism and develops targeted strategies of pathogen elimination involving T‐ and B‐cells, and specific antibodies. Its advantage is that its greater specificity is usually successful in avoiding or ameliorating the effects of a second exposure to a pathogen. Its limitations include the fact that it requires the infant to survive the first exposure (through the assistance of maternal antibodies and innate defences) and that it sometimes mistakes self antigens for foreign antigens and sets up a damaging autoimmune response.

Each of these cells and processes listed above, and the far greater number not listed, could potentially be modulated by FAs. Table 1 lists some generic mechanisms through which interactions may occur and for which there is some experimental evidence [see the numerous reviews available (e.g. Calder 2008, 2009, 2010; Galli & Calder 2009; Ganapathy 2009)]. It must be stressed that much of this evidence is quasi‐theoretical because it has been obtained from in vitro culture systems for which, among the many limitations, the issue of judging physiological ‘dose’ levels of FAs is very challenging. It is more than likely that some of these putative effects are of no significance in whole body mammalian systems, though some have been re‐validated in small animal studies.

Table 1.

Generic mechanisms by which fatty acids (FAs) may influence immune function

Immune component	Variables known to be affected by FA	Citation
Epithelial barriers	Cell–cell interactions Maintenance of patency Vulnerability to peroxidative damage	Calder et al. (2009) Calder et al. (2009) Roig‐Pérez et al. (2010)
Antigen presentation	Effects on MHC expression Altered dendritic cell function	Hughes et al. (1996) Sanderson et al. (1997) Shaikh & Edidin (2007) Weatherill et al. (2005)
Innate cell defences	Cell membrane structure and function Cell signalling	Katagiri et al. (2001) Razzaq et al. (2004) Leite et al. (2005) Yaqoob & Calder (2007)
Tolerance development	Immuno‐tolerance modulated (differentially) by n‐3 and n‐6 PUFAs	Harbige & Fisher (2001)
Adaptive cell defences	Cell membrane structure and function Intracellular and extracellular signalling Lipid rafts and T‐cell signalling	Katagiri et al. (2001) Horejsí (2003) 1998, 2001 Fan et al. (2003) Yaqoob & Calder (2007)
Cytokine‐mediated signalling	Pro‐ vs. anti‐inflammatory effects	Galli & Calder (2009)
Lipid mediators	Prostaglandins, leukotrienes, lipoxins, resolvins, protectins, etc.	Tilley et al. (2001) Vachier et al. (2002) Calder (2006) Serhan et al. (2004) Galli & Calder (2009)

MHC, major histocompatability complex; PUFA, polyunsaturated fatty acid.

How can we sift through these numerous theoretical effects to determine which, if any, have a significant and potentially modifiable impact on immune function in children in LDCs? The usual answer would be to interrogate clinical trials that test aggregate effects of changes in FA supply on functional and clinical outcomes. However, as explained below, there have been virtually no such trials. In the absence of these, a very poor proxy can be obtained by reviewing the numerous trials conducted on clinical end points of interest in affluent populations. Calder (2006) lists 18 such disease end points, for some of which there have been numerous trials (viz. asthma, inflammatory bowel disease and rheumatoid arthritis). Evidence of benefit for most of the end points studied is absent or at best equivocal. For those most studied (usually with LCPUFA supplements), the evidence is judged reasonable for childhood asthma (but not in adults), as yielding possible benefit in Crohn's disease (insufficient evidence in ulcerative colitis) and for convincing evidence for rheumatoid arthritis at sufficient dose levels (Galli & Calder 2009), though formal meta‐analyses could not be found. All of these end points were in relation to chronic disease outcomes in people consuming high intakes of fats and possibly excessive intakes of n‐6 FAs.

This is a disappointing outcome given the wide range of possible immuno‐modulatory effects of FAs and that the outcomes most studied have a large inflammatory component for which there is a strong theoretical basis to expect effects. It cautions against the over‐optimistic extrapolation of theoretical processes of disease mediation into clinical efficacy, but there remains much to be done before reaching nihilistic conclusions.

This may be especially so in low‐income settings where very low levels of total fat intake and low levels of dietary diversity can limit the intake of preformed LCPUFAs and of the precursors of the n‐6 and n‐3 series PUFAs (Michaelsen 2011). For example, Fig. 1 shows estimates of α‐linolenic and docosahexaenoic acid (DHA) in young rural Gambian children as they progress from early exclusive breastfeeding, through partial weaning as complementary foods are introduced at 3–4 months of age, to total weaning at 18–24 months (Prentice & Paul 2000). These calculations showed that breast milk provided a more than adequate source according to the Food and Agriculture Organization recommended intakes but that the very poor supply from complementary and childhood foods leads to a situation of profoundly inadequate intakes in the absence of breast milk. Under such circumstances, there would be a greater rationale for testing possible health‐protecting and therapeutic effects of LCPUFAs, and it is therefore surprising that more trials have not been undertaken.

Figure 1.

Intakes of the n‐3 fatty acids α‐linolenic acid (18:3; upper) and DHA (22:6; lower) in Gambian children from birth to 36 months. From Prentice & Paul (2000). DHA, docosahexaenoic acid; FAO, Food and Agriculture Organization.

Local interactions between FA depots and immune tissues

The work of Caroline Pond has revealed that mammalian lymph nodes are usually in close proximity to adipose depots, and there is evidence of functional links between the two (Mattacks et al. 2004; Pond 2005). Lymph node lymphocytes and local dendritic cells obtain their FAs from local adipocytes. Dendritic cells that permeate the adipose tissue stimulate lipolysis especially after local immune stimulation, and inflammation alters dendritic cell FA composition as well as that of lymph node‐containing adipose tissue, thus counteracting the effects of dietary lipids and emancipating the immune system from fluctuations in lipid supply and type (Pond 2005). Such effects might be particularly important in children suffering a nutritional challenge at the same time as an infectious one, as is so commonly the case in developing countries. Furthermore, the issue of why illness provokes appetite suppression (often to a profound degree) is not well resolved, and, although highly speculative, it is possible that mobilization of endogenous FAs pre‐stored with a favourable immuno‐stimulatory profile might be a contributory factor. This would be an interesting thesis amenable to study.

These paracrine interactions between adipose and lymphoid tissues are also stimulated by n‐6 and attenuated by the n‐3 series fats.

What are the implications of these findings for immune function in children in LDCs? They may imply that the composition of lymphoid‐associated adipose tissue is under regulatory control that enables it to harvest a desirable FA profile from circulating lipids in readiness for a specific immune response, thus giving some freedom from the constraints of day‐to‐day dietary supplies and allowing adequate function despite marginal supplies of the LCPUFAs. However, a corollary of this is that a neonate's endowment of FAs may have a crucial role in determining its early immune responses and that intervention trials seeking to influence immune function may need to be of long duration (and may need to start in pregnancy) because the turnover of body fat stores is generally slow (though it should be noted that the turnover of peri‐nodal adipose depots could be much faster).

FA intervention studies in low‐income countries

There have been astonishingly few attempts to study the biological effects of LCPUFAs by means of controlled supplementation studies in low‐income countries, and so far as we can tell, there have been no published trials addressing immune outcomes.

Given that growth in infancy and young childhood in LDCs is strongly linked to infectious load, intervention studies with growth as the primary outcome would be potentially informative, but even this simple outcome has not been studied. In affluent settings, meta‐analyses of n‐3 PUFA interventions in term babies have shown no benefit to growth (Makrides et al. 2005; Horvath et al. 2007; Simmer et al. 2008a; Rosenfeld et al. 2009), and those in preterm infants have shown mixed and inconclusive results (Simmer et al. 2008b). Elsewhere in this supplement, Makrides et al. (2011) conclude that marine oil supplementation in pregnancy increases mean birthweight by 50 g and mean birth length by 0.48cm, and that these effects may be mediated by extending the length of gestation by approximately 2.5 days because there was no significant reduction in the proportion of small‐for‐dates babies. All of these trials were conducted in first‐world countries.

It has been speculated that essential FA deficiency may play a role in the pathophysiology of protein–energy malnutrition (Smit et al. 2004), but only a single trial in 10 malnourished Pakistani infants has been reported (Smit et al. 2000), and the only outcome studied was the change in erythrocyte FA profiles (which were significantly altered by DHA supplementation).

A search on the term ‘PUFA’ in the ClinicalTrials.gov (143 trials) and International Standard randomised Controlled Trial Numbers (ISRCTN) (10 trials) registers reveals only a single PUFA trial in a developing country. This trial (ISRCTN66645725) has recently been completed by our group in rural Gambia (van der Merwe 2009). The aim of the trial was to examine whether daily high‐dose PUFA supplements could help prevent the gut damage (chronic environmental enteropathy) that occurs in almost all children raised in the typically unhygienic conditions of low‐income countries. The enteropathy is characterized by villous atrophy, crypt hyperplasia, inflammatory cell invasion of the lamina propria and damage to tight junctions, resulting in a leaky gut that allows translocation of bacteria and potentially toxic or allergenic substances (Lunn et al. 1991; Campbell et al. 2004). Enteropathy generally starts early in life, coinciding with the first exposures to complementary foods, and has been strongly implicated as a driver of growth faltering (Lunn et al. 1991; Goto et al. 2009). Intestinal biopsy studies have revealed that a key feature of this chronic enteropathy is the presence of a fulminant, unresolving inflammation with some parallels to other inflammatory bowel diseases (Sullivan 2002; Campbell et al. 2003). Paradoxically, the inflammation is seen even in severely malnourished children who might be expected to be immunosuppressed. The rationale for our trial was that the long‐chain n‐3 series PUFAs DHA and eicosapentaenoic acid (EPA) may help to prevent and/or resolve this chronic inflammation through both topical and systemic routes, and hence allow the gut to heal. We reasoned that the intervention should start at the time when the enteropathy usually gains its first footholds (i.e. at around 3 months post‐partum).

The trial randomized 183 children at 3 months to receive daily capsules with 2 g of purified fish oil containing 200 mg DHA and 300 mg EPA, or 2 g of olive oil as placebo, for 24 weeks (i.e. until 9 months of age). Ninety‐four per cent (172 infants) completed the trial. Primary outcomes were growth and gut integrity (assessed by the lactulose–mannitol dual‐sugar permeability test). Secondary outcomes were plasma FA status, cognitive development (assessed by the Willatt's two‐step means–end problem‐solving test at 12 months and attention assessment), intestinal and systemic inflammation, and morbidities assessed by active surveillance. As anticipated, the intervention significantly altered the infants' plasma phospholipid FA profiles with increases in DHA (4.87 vs. 4.44% of total FA, P < 0.001) and EPA (2.13 vs. 1.34% of total FA, P < 0.001) with no change in the n‐6 series arachidonic acid. The trial revealed no excess of adverse events in either group so the intervention was judged safe.

In general, the results were somewhat disappointing with little evidence of benefit in terms of gut permeability, systemic or intestinal inflammation, morbidity or weight growth. Linear growth showed evidence of benefit with a substantial effect size but with wide confidence intervals (CIs), and the difference was not significant (+0.79 Z s‐score; 95% CI −0.27 to 0.90, P = 0.084). Most of the measures showed a small trend towards benefit in the intervention group, but they were generally quite far from significance even with the reasonable sample size of 80+ per group. At 9 months, there was a significant increase in mid‐upper arm circumference (MUAC) in favour of the PUFA group. This could be explained as a multiple testing artefact, but when the groups were reassessed at 12 months (3 months after the end of intervention), the PUFA group showed significantly greater change in MUAC, triceps, biceps and subscapular skinfold thicknesses from the 3‐month baseline, indicating an increased adiposity.

The dose levels used in this trial were as high as we considered reasonable, following consultation with experts in the field and noting that high doses of PUFAs might have theoretical adverse side effects including immunosuppression, anticoagulation and lipid peroxidation. The daily supplements were administered under direct observation by field staff, compliance was very high and plasma FA profiles changed as expected. Therefore, the null results cannot be attributed to inadequate dose or non‐compliance. There is a high degree of variability in the lactulose–mannitol and fecal calprotectin assays (both methodological and biological), and hence, the result could be a type II error that has failed to detect a real effect. The persistent advantage in terms of adiposity at 12 months might support this view. Alternatively, it could be that the FA status of these breastfed infants at 3 months was already adequate, as suggested by the plasma analysis and the fact that these were not very greatly altered (likely because of the fact that all infants were at least partially breastfed), and that interventions for this age group are not required. Figure 1 suggests that later interventions, after complete weaning, might have a greater effect on PUFA status and growth, but by this stage, the chronic enteropathy that was the primary target of our trial is well entrenched.

Research gaps in relation to FAs and immunity

In addressing the research gaps in relation to FAs and immunity, we return to the issue of the immense complexity of the developing immune system and its interactions with a child's nutritional status. Figure 2 represents just a very basic overview of the variables. These can be conceptualized as a multidimensional knowledge matrix that needs to be filled before we would have a complete operator's manual that could guide the precise design of preventative and therapeutic interventions. It will quickly be appreciated that this matrix contains tens of thousands of individual cells, most of which are currently empty. For instance, we might wish to know how a single LCPUFA, say DHA, alters dendritic cell function in the inflamed gut of a young infant and how this affects its response to enteric vaccines and hence, for instance, the infant's susceptibility to diarrhoeal diseases. Any attempt to fill even this single cell in the overall knowledge matrix would require numerous separate studies in vitro, in animals and in humans, and would still not capture the myriad of potential interactions with numerous other variables including the child's genetic background. From this example, it can easily be appreciated that the full matrix will never be completed in such piecemeal fashion. An alternative approach is needed. This becomes the domain of the new systems biology approaches in which the application of modern high‐throughput and wide spectrum analytical ‘omics’ tools together with powerful bio‐informatic interrogative methods seem to promise a way forward.

Figure 2.

Schematic representation of some of the complexities involved in understanding nutrient–immune function relationships. APR, acute phase response; BCG, Bacillus Calmette‐Guérin; DTP, diptheria, tetanus and pertussis; PUFA, polyunsaturated fatty acid.

Elsewhere, we have argued that the design of new and effective nutritional interventions for children in low‐income countries would benefit from the redirection of some of the large funds spent on purely empirical trials towards more basic studies designed to fill crucial knowledge gaps regarding the mechanisms of action of nutrients. In the case of FAs, this argument might be reversed because there is a wealth of theoretical mechanisms of action and an astonishing absence of experimental (small‐sample proof‐of‐principle studies) or clinical trials in low‐income countries. Our failure to identify relevant trials would suggest that there is room for a series of carefully designed studies in populations with evidence either of FA deficiencies or of clinical syndromes putatively linked to defects in FA metabolism.

Conflicts of interest

LVDM is now employed by Danone Research, the Netherlands. AMP has research collaborations with Valid Nutrition.