1. Introduction

Diet plays a role in the induction and progression of Chronic Low-Grade Inflammation (CLGI) which has been associated with metabolic diseases such as obesity and diabetes ^[1]. Some food contaminants such as the exogenous Advanced Glycation End-Products (AGEs), produced in thermally processed products, have been shown to contribute to the persistent inflammatory component of diabetes, aging, and heart failure ^[2]. The same AGEs can also be formed at 37 °C, being called endogenous AGES. The deleterious effects of higher circulating levels of AGEs on health may be mediated by their eponymous cell membrane receptor RAGE ^[3]. Nonetheless, the overconsumption of macronutrients also associated with “western”, processed foods, such as lipids and carbohydrates, plays a similar role in triggering the CLGI implicated in obesity and neuroinflammation, both at the local and systemic levels ^[4]. The consequences of high lipid intake, for instance, range from appetite dysregulation in the hypothalamic core to metabolic endotoxemia ^[5], with this last being mediated by toll-like receptor 4 (TLR4) ^[6].

The clinical differentiation of CLGI is still a matter of significant debate. The main biomarkers currently used in the diagnosis of inflammation, such as C-reactive protein (CRP), are not specific to CLGI. Hence, many studies in recent decades have attempted to define CLGI biomarkers in humans and rodent models, including the proposition that clusters of biomarkers may best define the state ^[7]. While Calder and colleagues ^[7][8][9] have published comprehensive reviews on CLGI in nutritional studies in humans, detailing the biomarkers used to assess inflammation, important discoveries have concomitantly been made in murine models: to our knowledge, however, no review has yet been published on this body of work. Our goal here is to summarize current discoveries on diet-induced CLGI and discuss the factors which influence CLGI biomarker expression in rodent models.

2. Inflammation and Chronic Low-Grade Inflammation (CLGI)

Inflammation is part of the body’s innate and adaptive immune defenses. It comprises a series of cellular and chemical signal barriers which aim to control and conquer endogenous and exogenous stimuli (essentially bacterial or viral infections) and trauma-related damage ^[10]. Inflammation can be either an acute or chronic process, but both have common aims: namely, to neutralize the source of injury, promote tissue repair, and drive a self-limited return to homeostatic conditions ^[11]. In contrast to acute inflammation (AI), chronic inflammation (CI) is a process linked to resolution failure and induces continuous recruitment of the cellular immune apparatus, promoting tissue damage ^[12].

Beyond the difference in duration between AI and CI, the degree of inflammatory response also determines whether inflammation becomes pathological. Under a non-overt inflammation scenario, a chronic, but low-grade inflammatory mechanism comprehends excessive metabolic stress correlated to the rise of circulating levels of inflammation signals ^[13]. Both CI and CLGI are borderline conditions sharing similar molecular mechanisms, but a distinct involvement of metabolic tissues characterizes CLGI progression. Indeed, CLGI is currently considered to be a possible factor in the pathological aggravation of obesity, type 2 diabetes mellitus (T2DM), atherosclerosis, or cancer ^[1][14].

Compared to the chronic but severe inflammation present in arthritis or Crohn’s disease ^[15], metabolic disorders (such as obesity) and some age-related conditions (such as frailty) have only a CLGI component ^[16]. This difference in the intensity of inflammation has orientated research seeking to characterize specific molecular patterns and biomarker clusters in order to develop predictive tools aimed at reducing the health and socioeconomic impacts of these pathologies.

Although the terms “Low-Grade Inflammation” or “Chronic Low-Grade Inflammation” are used interchangeably in the literature, we here take “chronic” to be a compulsory requirement for the low-grade inflammatory stimulus to promote some sort of pathological effect. Thus, based on the extensive literature currently published, CLGI can be formally defined as a pathological state lacking overt inflammation, but characterized by continuous and unresolved activation of inflammation mediators. It results in increased production of cytokines, reactive oxygen species, macrophage infiltration, adipocyte imbalance, or vascular damage; these effects are associated with metabolically active tissues such as adipose tissue, skeletal muscle, and the liver, implicating CLGI in metabolic diseases ^[1][17]. Age-related immunosenescence and the accumulation of cellular debris are also part of CLGI evolution in older people ^[18][19].

3. High-AGE Diets and CLGI Initiation in Murine Models

Advanced Glycation End-Products (AGEs) are the result of non-enzymatic, post-translational reactions between reducing carbonyls and protein-amino groups, nucleic acids, or aminophospholipids ^[20]. The formation of AGEs by the Maillard reaction gained prominence in human health research during the 1950s, after the discovery of glycated hemoglobin under physiological conditions and its reported correlation with glycemic levels in diabetic patients ^[21]. The glycation of cellular proteins has a negative effect on cell and tissue function, molecular aging, and chronic disease development ^[22][23]. In addition to their endogenous occurrence, AGEs are also formed during the thermal processing of foods, significantly increasing humans’ exposure to dietary AGEs (dAGEs). Food-borne AGEs are part of a heterogeneous group of chemically stable molecules resulting from the Maillard reaction, some of which are implicated in benignly improving flavors, aromas, and browning, while others are thought to be involved in adverse health effects (e.g., chronic inflammation, degenerative diseases, aging, insulin resistance) ^[22][23].

AGEs have a close relationship with inflammation and oxidative stress which is mediated by RAGE, their eponymous receptor which is part of the immunoglobulin superfamily and participates in immune surveillance in the lungs, liver, vascular endothelium, monocytes, dendritic cells, and neurons, to name only the major locations identified so far ^[24]. RAGE is a promiscuous receptor for which multiple ligands have been identified (e.g., HMGB1, S100, multiple AGEs), making it an important pattern recognition receptor (PRR) and inflammation trigger ^[3]. Activation of the RAGE-AGE axis has been described as a key mechanism leading to the production of pro-inflammatory cytokines, which leads to the maladaptive tissue remodeling caused by the modulation of genes and proteins implicated in extracellular matrix composition, cellular connectivity, elasticity, and tissue flexibility ^[25]. In this way, the long-term consumption of dAGEs would expose the immune system to CLGI activation ^[26].

Protein-bound CML is a high-affinity RAGE ligand ^[27][28]. The pro-inflammatory effect of dCML has been demonstrated in a comparison between wild-type and RAGE knockout animals receiving a CML-enriched diet (50, 100, and 200 µg CML/g food ). In wild-type animals receiving dCML, a dose-dependent increase in expression of VCAM-1 was observed, both histologically and in mRNA expression, while RAGE expression was increased significantly only at the protein level. The RAGE knockout animals were apparently protected from an increase in these inflammation triggers, and no significant VCAM-1 expression increase with dCML dose was reported in this genotype ^[29]. Such a protective effect over RAGE knockout animals was previously demonstrated in obese male mice that received both fat and AGE enriched diet. Harcourt et al. ^[30] reported that MCP-1 levels, both in plasma and kidneys of RAGE knockout mice, were reduced followed by improved MIP (macrophage migration inhibitory factor) level in the same samples. Further, the influence of the RAGE-AGE axis upon the promotion of inflammation was highlighted by the application of alagebrium, an AGE cross-link breaker currently investigated as an anti-AGE compound ^[31]. In animals receiving alagebrium, MCP-1 and MIP levels behaved in the same way as in RAGE knockout in addition to improved glycemic control ^[30]. The potential deleterious effect of dCML can also be inferred from the endocrine perspective, as reported in experiments on mice with ovarian hormone dysfunction. Thornton et al. ^[32] compared the effect of a CML-enriched and low-AGE (L-AGE) diet on ovary dysfunctions in C57BL/6 mice. The results, after 13 weeks, from mice receiving the high-AGE (H-AGE) diet showed a dysregulation of the estrous cycle and superovulation followed by an upregulation of macrophage marker F4/80 mRNA expression. The local expression of macrophage biomarkers was significantly lower in animals in receipt of a low-AGE (L-AGE) diet. Other CLGI biomarkers were examined in the ovarian tissues, with the expression of pro-inflammatory cluster of differentiation 11 (CD11) increasing and anti-inflammatory CD206 decreasing among the H-AGE mice. This may be explained by the different RAGE expression among the follicular cell types in response to gonadotrophins, since dAGEs can interfere in the gonadal cycle ^[33]. Chatzigeorgiou et al. ^[34] reported that peripheral blood mononuclear cells (PBMCs) isolated from female mice receiving an H-AGE diet had both RAGE and scavenger receptor type A (SR-A) downregulation which could be involved in dAGEs accumulation in endocrine tissues as the ovaries.

An analytical approach employing CML isotopes has demonstrated that dCML accumulates primarily in the kidneys, but also in the ileum, colon, and lungs in a RAGE-independent manner ^[35]. RAGE is important in triggering respiratory allergies, being involved in complications of lung cancer, asthma, and bronchoalveolar inflammation ^[36]. To investigate the involvement of the AGE-RAGE axis in respiratory inflammation, mice were exposed to an H-AGE diet over 4 weeks and the bronchoalveolar lavage was analyzed. This presented higher polymorphonuclear (PMN) cells, cytokines (IL1B, IL-6, and MM1, MMP-2), and TNFsRII, all of which may contribute to aggravation of lung injury, and which were associated with the triggering of inflammation by circulating dAGEs. Here, then, the responsiveness of the lungs to dAGEs is likely to be associated with pulmonary RAGE expression ^[37].

4. Perspectives and Conclusions

Based on the data presented in this review, the involvement of diet in the induction and progression of CLGI has been clearly demonstrated in murine models. Since multiple factors which contribute to CLGI initiation probably occur simultaneously (e.g., higher AGE concentration in the circulation, endotoxemia), a better understanding of the interaction of inflammation biomarkers in different organs and tissues would help to elucidate key biomarkers of CLGI, as well as identify the most susceptible among them, pinpointing the potential physiological implications. The murine models described in this review have shed significant light on CLGI, particularly the importance of local (tissue level) measurements of CLGI biomarkers. The use of murine models is also helping to illuminate CLGI crosstalk among different organs due to the ease of sample collection, preparation, and analysis compared with other animal models or clinical studies. Indeed, significant advances have been made in the attribution of the physiological effects of CLGI, and the identification of potential biomarkers induced by target compounds such as dAGEs and dietary lipids in these models. They have enabled the study of direct effects of key dietary influences on animal physiology, but some limitations on the translational use of future biomarkers in clinical applications between murine models and humans may arise (e.g., CRP). In the future, the current use of qualitative or semiquantitative techniques for CLGI biomarker analysis should give way to more extensive and specific analytical techniques, such as proteomics, transcriptomics and metabolomics, that could drive research on both screening and quantification of CLGI biomarkers in the whole organism.

From the nutritional perspective, strategies for the prevention of CLGI are required to reduce the impacts of several metabolic diseases, as well as to promote healthy aging. A balanced diet is often recommended to maintain good health ^[38], and evidence is also emerging from murine models that CLGI may be attenuated through the use of natural products, hinting at the possibility that dietary intervention may have the potential to limit initiation and/or progression of CLGI. Indeed, the use of probiotics or extracts of certain berries has been reported to ameliorate diet-induced CLGI in murine models under high-fat diet (HFD) ^[39][40][41], though these studies require confirmation in clinical studies in humans.

Lastly, current research on diet-induced CLGI has highlighted the utility of simultaneously examining several different inflammation biomarkers. Ongoing research has confirmed the involvement of several cytokines and other inflammation biomarkers in both the initiation and progression of CLGI, but their use for the prediction, and/or determination of CLGI still lacks consensus. We remain some way from defining a clinical diagnostic test for CLGI, but future CLGI treatment may target important receptors such as TLR4 and RAGE for the prevention of CLGI initiation, and diagnostics may rely upon the recent evolution of non-invasive biomarkers (e.g., lipocalin-2). With respect to the prospection of CLGI biomarkers, candidates should be systematically investigated to fill gaps in the whole-organism picture of inflammatory responses in different organs and tissues. From the diagnostics perspective, biomarker research should integrate current knowledge on the use of multiple biomarkers able to predict CLGI more robustly.

The overconsumption of AGEs (especially dCML) and obesogenic foods leads to several physiological effects which contribute to the onset of CLGI, and their study has contributed greatly to our understanding. Several different classes of biomarkers have been reported as important, but the levels of cytokines such as TNF-α and IL-6, as well as chemokines such as MCP-1 and adhesion molecules (e.g., VCAM1), are repeatedly cited as potential biomarkers in CLGI research in murine models. However, progress in this field can be only made with more robust analytical protocols. Particular attention should be paid to the quality and homogeneity of the dietary regimes employed, the simultaneous analysis of multiple organs under the same dietary protocol, and establishing both normal and pathological levels of key biomarkers already identified. Metabolomics studies that include multiple CLGI biomarkers have potential in this regard and offer the possibility of defining clusters or panels of biomarkers capable of specifically targeting this subtle, multi-layered and complex inflammatory process.