Review
Diet, commensals and the intestine as sources of pathogen-associated molecular patterns in atherosclerosis, type 2 diabetes and non-alcoholic fatty liver disease

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Abstract

Stimulation of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 has been shown to promote the development of a variety of diseases involving dysregulated metabolism, including atherosclerosis, type 2 diabetes and non-alcoholic fatty liver disease. However, the origin and nature of the agents responsible for stimulating TLR2 or TLR4 signalling in these conditions remain to be clearly identified. This review summarises the evidence supporting the proposal that ‘pathogen-associated molecular patterns’ (PAMPs) derived from dietary and commensal sources may contribute to the chronic inflammatory processes that underpin the development of these diseases via stimulation of TLR2 and TLR4. In particular, insights gained from recent studies employing TLR-transfectant based bioassays to quantify the abundance of PAMPs in foodstuffs and specific commensal compartments are discussed. Finally, the major mechanisms by which TLR-stimulants may gain access to the circulation to promote systemic low-grade inflammation are considered.

Introduction

Atherosclerosis, type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) together form a triad of diseases linked to dysregulated metabolism that are endemic in the countries of the industrialised world and increasing in prevalence as the remaining countries develop. Since several factors common to the Western lifestyle, such as sedentary behaviour, obesity and diets rich in saturated fats are shared risk factors for these diseases, it has been largely assumed that dysregulation of lipid and carbohydrate metabolism represents the principle mechanism underlying these pathologies [1]. However, recent evidence from a variety of approaches has suggested that the establishment of a state of systemic, low-grade inflammation may also play a key role in the development of these diseases [2].

Although inflammation is generally considered to be a localised reaction, it is now understood that a ‘systemic inflammatory response’ may be generated when inflammatory stimuli gain access to the circulation. This response is characterised by systemic increases in the levels of circulating inflammatory cytokines, soluble adhesion molecules and acute phase proteins, such as C-reactive protein (CRP) and serum amyloid A (SAA). These mediators have been most widely studied in the context of sepsis, where the systemic inflammatory response may, in its most severe form, contribute to the complications of shock and multiple organ failure. However, more benign inflammatory stimuli have also been found to elicit modest, but significant, increases in the concentrations of circulating inflammatory mediators, even in the absence of discernable external symptoms.

While previously considered to be of little relevance to metabolic disease, these modest increases in circulating inflammatory mediators have since been found to be strongly associated with the development of atherosclerosis, T2DM and NAFLD [2], [3]. Crucially, it has also become clear that many of these inflammatory factors serve not merely as markers of these diseases, but as critical mediators of their development. For example, genetic deletion or blockade of a variety of pro-inflammatory signalling mediators results in a significant reduction in diet-induced atherosclerosis, NAFLD and insulin resistance in murine models of these diseases [4], [5], [6].

Because atherosclerosis, NAFLD and T2DM frequently occur together within subjects [7], and since the risk factors for these diseases are similar [1], it has been suggested that a common inflammatory principle may underpin each of these diseases [3]. To date, however, the nature and the origins of these primary inflammatory stimuli remain to be clearly identified. This review will summarise the evidence that inflammatory stimulants derived from commensal bacteria, infectious organisms and dietary sources, may serve as common inflammatory mediators contributing to each of these diseases.

Section snippets

PAMPs as primary stimulants of inflammation

In 1989, Charles Janeway first put forward the idea that inflammation could be triggered by the detection of highly conserved molecules, termed ‘pathogen associated molecular patterns’ (PAMPs), which are expressed by microbes but not host cells. It is now understood that PAMP detection is mediated by invariant, germ-line encoded receptors of the innate immune system, such as the RIG-I-like receptors, NOD-like receptors and Toll-like receptors (TLRs), the latter of which form the largest part of

Evidence that PAMPs may promote atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the arteries which represents the underlying cause of the majority of cardiovascular diseases. Lesion formation is characterised by the recruitment of circulating monocytes into the subendothelial space where they differentiate into fat-laden macrophages termed “foam-cells”. In addition to host dietary and lipid parameters, inflammatory processes are now understood to underpin each stage of the development of atherosclerotic plaques, as

Evidence that PAMPs may promote insulin resistance

When the cells or tissues of an organism lose sensitivity to the hormone insulin (a state which is termed ‘insulin resistance’), the ability of the host to lower blood sugar levels can become impaired in a process that can eventually lead to the development of T2DM. Recent evidence suggests that both of these conditions may be promoted by the stimulation of TLR-signalling. For example, mice genetically deficient in TLR2 [23], [24], [25] or TLR4 [5], [26], [27] are protected against diet-induced

Evidence that PAMPs may promote NAFLD

The hepatocytes of the liver play a key role in maintaining host energy and lipid reserves in the form of intracellular stores of glycogen and lipid. In health, this intracellular lipid generally constitutes a low proportion of liver weight. However, the term ‘fatty liver disease’ is applied when ≥5% of hepatocytes are affected by steatosis. Such steatosis can occur either in response to alcohol (alcoholic liver disease), or in the absence of exposure to alcohol (NAFLD). Both diseases can

Evidence that PAMPs may modulate diet-induced obesity

An interesting recent proposal is that PAMPs may modulate the development of adipose tissues in murine models of obesity. This hypothesis is supported by the observation of Tsukumo et al. that mice lacking functional TLR4 were of significantly lower body weight than controls after 8 weeks of high fat feeding [27]. It was also reported that chronic LPS infusion increased whole body and adipose tissue weight gain compared to mice treated with saline alone [29]. However, not all studies of the

The gastrointestinal tract is a major source of PAMPs and a site of PAMP translocation

If systemic exposure to PAMPs may contribute to the development of metabolic diseases as suggested by the studies summarised above, a key question that remains to be addressed in this field is: from where are the majority of circulating PAMPs derived? It seems likely that three principle sources are responsible, namely: infections, commensals and the diet (Fig. 1).

Although it is difficult to quantify the contribution infectious organisms may make towards overall PAMP exposure in human subjects,

The oral microbiota

Evidence is accumulating to suggest that products of the oral microbiota may contribute to increased risk of developing atherosclerosis and T2DM. For example, murine models of periodontitis, in which mice are given oral inoculations with the periodontal pathogen Porphyromonas gingivalis, have revealed that such infections result in systemic activation of vascular inflammatory signalling, elevated serum endotoxin levels and accelerated atherosclerosis [51], [52]. Human population studies have

The small intestine

It is generally accepted that the commensal microflora of the small intestine is far more limited than that of the mouth or the large intestine. Specifically, the bacterial load in the small intestine increases from 100–4 cfu/ml in the duodenum and the jejunum to 100–5 cfu/ml in the proximal ileum, and reaches a maximum of 105–8 cfu/ml only in the most distal centimetres of the terminal ileum in a transition zone before the caecum [59]. Notably, the microbiota of the upper small bowel consists

The large intestine

By far the majority of the host commensal microbiota exists within the large intestine, with luminal contents containing between 1010 and 1012 bacteria per gramme in healthy subjects [59]. Application of the TLR-transfection assay to stool samples of healthy subjects has provided an estimate of the levels of PAMPs in the human large intestine (Fig. 2) [61]. Surprisingly, TLR4-stimulants were found to be present at much lower concentrations (1–25 μg E. coli LPS equivalents per gramme) than were

Dietary PAMPs

Having established that the small intestine is generally exposed to low levels of LPS and lipopeptide in health, with luminal concentrations rarely exceeding around 100 and 1000 ng/ml, respectively, we next aimed to quantify the abundance of PAMPs in a variety of foodstuffs. These studies led to the surprising discovery that although PAMPs are generally not detectable in fresh, whole foods, they can be abundant in a number of processed foods common to the Western diet. In particular, processed

Fructose as a modulator of intestinal PAMP concentrations

Another factor common to the Western diet that is receiving attention in terms of metabolic disease is the sweetening of products, particularly soft drinks, with fructose. It has been found that fructose may be poorly absorbed by some subjects, leading to an expansion of bacteria in the small intestine. Accordingly, increased portal endotoxin levels and hepatic steatosis are observed in mice consuming water sweetened with fructose, but not glucose [37], [68]. Both of these effects were reversed

Conclusion

Recent evidence gained from a variety of approaches suggests that the small intestine may represent a key site for the absorption of fat-soluble stimulants of TLR2 and TLR4 into the circulation, and that these agents may promote the development of atherosclerosis, insulin resistance and fatty-liver disease. Products of the oral microbiota, rather than enteric organisms, are likely to set the basal levels of these PAMPs in the small intestine of healthy subjects. However, the occasional

Acknowledgement

The author is supported by a University of Leicester Department of Cardiovascular Sciences Research Fellowship.

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