Epigenetics of Inflammation, Maternal Infection, and Nutrition^,,

Abstract

Studies have demonstrated that epigenetic changes such as DNA methylation, histone modification, and chromatin remodeling are linked to an increased inflammatory response as well as increased risk of chronic disease development. A few studies have begun to investigate whether dietary nutrients play a beneficial role by modifying or reversing epigenetically induced inflammation. Results of these studies show that nutrients modify epigenetic pathways. However, little is known about how nutrients modulate inflammation by regulating immune cell function and/or immune cell differentiation via epigenetic pathways. This overview will provide information about the current understanding of the role of nutrients in the epigenetic control mechanisms of immune function.

Modes of Epigenetic Modifications

Epigenetics is the study of heritable changes that affect gene expression without DNA base pair sequence changes (1). One of the best-known examples of epigenetic alterations involves DNA methylation of carbon number 5 of nucleotide base cytosines on 5′ in CpG islands (2) by methyl transferases. In the early 1990s, it was demonstrated that inherited genes function differently depending on whether they were from the mother or the father (3–5). During the developmental period, de novo methylation of CpG islands occurs in specific genes responsible for early embryonic development (6) and the levels of CpG island methylation can vary in tissue-specific methylation patterns (7). One of the best-known examples of an imprinted gene that is expressed in a parent-specific manner is insulin-like growth factor 2 (Igf2)⁶. Igf2 coding for fetal growth factor is expressed from the paternal allele and highly methylated in fetal liver but is undermethylated in fetal brain (7). The expression of Igf2 is regulated by maternal nutrient intake during fetal development (8–10), potentially resulting in disease phenotypes including obesity (10). Another well-known epigenetics modification involves histone modifications that include methylation, phosphorylation, ubiquitination, and acetylation (1). Together with CpG island methylation, histone modifications affect how accessible nucleosomes are to transcription machinery and transcription activation (11). Histone modifications may also change the binding capacity of other proteins to histones via alterations of local hydrophobicity (11) and by changing activation of RNA polymerase and other transcription coactivator binding (12).

Immune Dysfunction, Inflammation, and Epigenetics

Environmental stimuli, such as toxins or infectious agents, can alter immune cell activation through epigenetic modifications. For example, immune cell activation by pathogens such as bacteria was shown to induce changes in histone modifications, DNA methylation, and chromatin structure. These changes induce inflammatory gene transcription by programming the host immune cells (13). Although the role of epigenetic modifications in inflammatory diseases has been attracting increased attention recently, the exact epigenetic mechanisms underlying the different inflammatory diseases are poorly defined. Among the recent studies addressing the immune cell epigenetic modification leading to alterations in inflammatory status, many were conducted with the use of T lymphocytes. For example, activated lymphocytes have euchromatin, which is loosely coiled, transcriptionally active, and accessible, whereas naive and resting cells contain compact nuclei with inaccessible heterochromatin (14). Upon activation, T cells recruit the SWItch/sucrose nonfermentable (SWI/SNF) complex to the chromatin to control gene expression (14). SWI/SNF complexes are chromatin-remodeling complexes that use ATP hydrolysis to remodel nucleosomes and to modulate transcription (15). Epigenetic regulation, including histone modification and DNA methylation, is important for T cell differentiation, including T helper (Th) 1 and Th2 subsets, as well as for regulatory T cells and the Th17 lineage (16, 17). For example, histone modifications result in cluster of differentiation 4 (CD4)⁺ T cell differentiation into the Th2 phenotype (18). Native CD4⁺ T cells differentiate into specific Th cell phenotypes by chromatin remodeling, allowing increased binding to the regulator regions of interferon γ (IFN-γ) or IL-4 genes (19). Increased Th1 cells that secrete high amounts of IFN-γ are critical for host defense against intracellular pathogens by activating macrophages, whereas increased Th2 cells that produce high amounts of IL-4 are essential for a humoral immune response (2). Altering chromatin structure at the promoters for the Ifn-γ or Il-4 genes can tip the balance in the case of inflammatory lung disease; administration of a gram-negative bacterium to pregnant mice prevented an asthmalike phenotype in the progeny through epigenetic modifications (20). This protection was mediated via increased histone 4 (H4) acetylation in the Ifn-γ promoter and decreased H4 acetylation at the Il-4 promoter–dependent pathway, preventing a Th2 inflammatory response (20). DNA methylation changes also play a role in Th1/Th2-specific gene expression; for example, in a model of experimental asthma, CD4⁺ T cells experienced an increase in DNA methylation at the Ifn-γ promoter upon allergen challenge (21). Other factors that control T cell function such as IL-2, a T cell proliferative factor, are regulated via IL-6 (Il-6) promoter demethylation in T cells (22).

Another major immune cell type that has been studied widely is the macrophage. Macrophages are key regulatory cell types in the immune response and can be polarized to relatively stable macrophage type 1 or 2 (M1 or M2) phenotypes in vitro and in vivo. Stimulation with cytokines such as IFN-γ and/or by bacterial and viral products leads to M1 polarization. This polarization results in epigenetic changes including DNA methylation and acetylation and methylation of histones of genes involved in the inflammatory process. M1 polarized macrophages are involved in the immune response during inflammation, extracellular matrix destruction, immune response to tumors, and apoptosis and mediate the proinflammatory effects attributed to adipose tissue macrophages. This polarization is dependent on many factors. Stimulation with cytokines such as IL-4, IL-13, or IL-10 and/or certain parasite-derived antigens or allergens leads to M2 polarization. This polarization is dependent on induction of demethylases that act on histones that bind to the promoter of genes associated with the M2 phenotype (23). M2 polarized macrophages are involved in the immune response during parasitic and allergic diseases as well as insulin resistance, atherosclerosis, extracellular matrix remodeling, cell proliferation, and angiogenesis. The histone lysine demethylase Jumonji domain-containing protein (JMJD) gene is expressed in macrophages (24), and decreased activity of the JMJD protein results in increased histone H3 lysine 9 (H3K9) methylation and subsequent decreases in macrophage inflammatory chemokine production (25). Interestingly, nutrient status, particularly nutrient starvation, was shown to increase expression of JMJD (26).

Maternal Infection and Epigenetics

The prenatal environment influences the risk of inflammatory disease in adulthood. For example, maternal infection or inflammation can lead to preterm birth, which is itself linked to a number of chronic diseases. Many of these adverse long-term outcomes, including cardiovascular disease, asthma, and obesity, have an inflammatory component. The mechanisms remain unclear, but inflammation may alter the appropriate maintenance of epigenetic profiles during pregnancy. Neonatal T cells, for instance, produce very little IFN-γ due to a number of factors, including low-level expression of the transcription factor T-box gene that encodes transcription factor 2 (Tbet2). However, maintaining the fine control over this Th1 cytokine in utero probably also involves epigenetic factors, including hypermethylation of the Ifn-γ promoter (27). This normal shift away from potentially dangerous Th1 responses during the neonatal period could be altered by the maternal cytokine environment. Indeed, a recent study demonstrated that the maternal cytokine response determined the corresponding cytokine responses in infants, irrespective of the mother’s atopic status or environmental factors (28). These studies offer the intriguing possibility that cytokine imprinting may be linked to epigenetic modifications. Animal studies demonstrated this supposition experimentally. Increasing histone acetylation at Ifn-γ and decreasing histone acetylation at the Il-4 promoters, for instance, were shown to protect the progeny of pregnant mice from asthmalike symptoms (discussed previously; 20). Such epigenetic modifications have been implicated in early childhood diseases ranging from autism to allergy (29–31). Maternal exposures to farm environments can increase the number of T regulatory (Treg) cells in the cord blood of their infants, which is associated with decreased Th2 cytokines and may be linked to demethylation at the forkhead box P3 (FOXP3) promoter (32). The exposure of the mother to allergens in the environment may protect her infant from subsequent atopy and asthma.

Abnormal immune responses to common childhood infections, such as parvovirus B19, are linked to epigenetic alterations that may influence cancer (33). However, a clear link between maternal infections and subsequent epigenetic alterations in the fetus, and the mechanisms involved, remains lacking. Recent studies offer some clues. For example, recurrent urinary tract infections are common in pregnant women and can cause preterm birth and low birth weight (34). Uropathogenic Escherichia coli infection of bladder epithelial cells results in hypermethylation of the tumor suppressor gene cyclin-dependent kinase 2a (CDKN2A) (35), which is also methylated in human papilloma virus 16 (HPV-16)–induced epithelial dysplasia (36).

Whether maternal infection with a urinary tract pathogen directly induces epigenetic changes in the fetus is unclear. However, one could envision a scenario in which neonatal exposures to common pathogens leads to alterations in the epigenetic landscape of the fetus, increasing later susceptibility to chronic disease or cancer. Indeed, viruses and bacteria can act as epimutagens or “biological stressors” that induce epigenetic alterations (37, 38) either directly or indirectly through the induction of inflammation.

Another example of epigenetic alterations induced by a bacterial infection comes from the bacterium Listeria monocytogenes, a food-borne pathogen that is especially dangerous for pregnant women and the fetus. L. monocytogenes activates MAPK signaling pathways, leading to specific histone modifications and transcriptional activation of MAPK-induced inflammatory genes (39). L. monocytogenes also causes a decrease in the modification of histones on another set of immunity-related genes such as chemokine (C-X-C motif) ligand 2 (Cxcl2) (40). The ability of the bacterium to modulate immune gene expression through chromatin modification offers a glimpse into how early-life infection could influence immune response and modulation in later life. Indeed, a recent study of DNA methylation patterns in preterm birth caused by infection demonstrated a significant increase in methylation at the regulatory regions for pleomorphic adenoma gene 1 (PLAG1) (41). PLAG1 is a transcription factor that stands at the nexus of growth and development, and dysregulation of PLAG1 expression has been associated with cancer (42). Clearly, further studies are needed to uncover the complex interactions between epigenetic changes induced by maternal infection and the long-term consequences for both mother and child.

Nutritional and Bioactive Factors That Regulate Inflammation via Epigenetic Mechanisms

Progress has been made in identifying the cellular and molecular signaling mechanisms underlying anti-inflammatory effects of numerous nutrients. Macronutrients (e.g., n–3 PUFAs), micronutrients (e.g., vitamins A, D, C, and E; folic acid; selenium) and other bioactive compounds (e.g., flavonoids) have all been targets of investigation. However, only a few studies addressed the regulatory role that nutrients play to modulate inflammation via epigenetic pathways. Maternal overnutrition and obesity resulting in dyslipidemia and increased systemic inflammation were shown to have profound effects on the developing embryo and the fetus in utero (43). A maternal high-saturated-fat diet was shown to induce inflammation pathways in the offspring in animal (44–46) and in human (47, 48) studies. Among the micronutrients, folic acid is a well-known one-carbon donor for methylation and synthesis of DNA. These functions of folic acid are especially critical during early postnatal development because rapid cell growth and proliferation are taking place during this period. Higher inflammation during this developmental period poses a higher risk of disease development (49) due to immune system overactivation. Anti-inflammatory nutrients such as folic acid can be beneficial in preventing inflammatory responses (50, 51). Recent in vitro studies demonstrated that folic acid–deficient conditions increased the expression of inflammatory mediators such as Il-β, Il-6, Tnf-α, and monocyte chemoattractant protein-1 (Mcp-1) in the mouse monocyte cell line RAW264.7 (52). Increased methylation of the promoter regions of tumor suppressor genes is associated with carcinogenesis, whereas decreased methylation of tumor suppressor genes is associated with a reduction in tumor development. In vivo, increased consumption of folate and folate-rich foods is associated with decreased promoter methylation of genes associated with tumor suppression (53). Promoter CpG island methylation levels in colorectal mucosa were related to RBC folate concentrations in human studies (54). In addition, high intake of maternal folic acid reduced cancer risk in offspring in a rat model by altering histone deacetylation (55). Another micronutrient that alters epigenetic pathways is vitamin D. Deficiency or low concentrations of vitamin D are associated with increased inflammation, and the anti-inflammatory actions of vitamin D may be due to changes in DNA methylation and histone modifications. Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the brain and spinal cord, and increased severity of MS is associated with decreased vitamin D status (56). Vitamin D suppresses inflammatory responses in adult humans with MS by decreasing IL-17 gene expression by blocking of nuclear factor necessary for activating T cells (NFAT) transcription factors and by recruitment of histone deacetylase (HDAC) (57). IL-12, a proinflammatory cytokine, is involved in activation and differentiation of CD4⁺ T cells into Th1 cells. Vitamin D suppresses Il-12 promoter activation by modulating histone modification (58). Moreover, severe vitamin D deficiency is associated with methylation changes in peripheral blood leukocyte DNA in humans (59). Among the bioactive nutrients, curcumin and curcumin-derived synthetic analogs control inflammation responses and body weight by altering DNA methylation (60) and via micro-RNA (61). In addition, curcumin inhibits high-glucose-induced NF-κB activation, proinflammatory cytokine production, and histone acetylase activity in human monocytic THP-1 cells (62). Other plant polyphenolic bioactive compounds, such as epigallocatechin gallate, inhibit inflammation via modulation of histone acetyltransferase activity (63). Few studies have addressed how phytochemical nutrients suppress the transcription activation processes via epigenetic modification (64, 65). Inflammatory cytokine gene expression may also be modulated by histone deacetylase inhibitors (HDACi) through destabilization of the DNA methylation enzyme mRNA, which then leads to decreased inflammatory gene expression (66, 67). In line with this observation, HDACi suppressed IL-1β–, TNF-α–, and Toll-like receptor ligand–induced rheumatoid arthritis synovial macrophage production of IL-6 cytokines (66). Determination of the possible nutrient control of HDACi would have important implications, because HDACi-regulated reduction in cytokine production might suppress inflammatory cytokine production in rheumatoid arthritis. Currently, however, relatively little is known regarding the potential effects of anti-inflammatory nutrients on HDACi regulation of cytokine gene expression. Interestingly, ginger, a commonly used dietary herbal product, was shown to have broad anti-inflammatory properties (68). For example, a study by Shim et al. (69) showed that bioactive compounds found in ginger extracts, such as 6-shogaol, have anti-inflammatory effects on primary rat astrocytes production of inflammatory cytokines by HDAC inhibition when these cells were stimulated with LPS. Furthermore, another recent study showed that injection of Alzheimer disease mice with 6-shogaol had beneficial effects such as improved memory by increased glial cell activation and brain neuronal survival (70). Although reduction in neuronal inflammation results in beneficial effects, whether improved memory in animals with Alzheimer disease is directly mediated via modulation of HDACi remains to be determined. Other dietary bioactive compounds such as allyl sulfur compounds found in garlic were shown to inhibit inflammation (71) and cancer cell growth (mouse erythroleukemia) via modulating acetylation of histone (72). Plant flavonoids, including apigenin (73), which is found in high concentrations in parsley (74), and chrysin (75), which is found in medicinal herbs (76), were shown to inhibit HDAC. In addition, although a better known function is its role as metabolic FA fuel for gut bacteria, the dietary fiber component butyrate was shown to induce cell growth arrest, inhibit DNA synthesis, induce cell differentiation, and regulate gene expression by inhibiting HDAC (77, 78).

Maternal Nutrition Influence on Inflammation, Immune Response to Infection, and Epigenetics

Overnutrition or nutrient deficiency during pregnancy can have devastating effects on the health of offspring at early or late stages of their life. It is becoming clear that poor maternal nutrition has a negative impact on fetal gene expression through epigenetic programming (75). These changes likely involve covalent modifications of DNA and chromatin remodeling. Inadequate fetal and early-age poor nutrition is associated with an increased risk of metabolic syndrome later in life. The Dutch Winter Hunger Study, for example, showed that nutrient deficiency during mid- and late gestation was associated with hyperglycemia in middle age (75). Furthermore, growth in utero as well as in early childhood is greatly influenced by maternal macronutrient intake, suggesting the importance of a maternal diet that contains sufficient macronutrients for adequate fetal and child development. Indeed, low circulating concentrations of vitamin B-12 and high circulating concentrations of total homocysteine predicted intrauterine growth retardation (75). Micronutrients including zinc are also essential to the epigenome. Zinc is required for methylation, and its deficiency is suggested to contribute to atherosclerosis, inflammation, and diabetes. Several studies suggest that zinc deficiency at the stages of intrauterine and infant life contributes to the pathogenesis of inflammation and metabolic diseases in adulthood (75). Furthermore, maternal zinc deficiency was shown to impair immunity in the offspring of mice (75). Methylation can be a double-edged sword, however: in utero supplementation with cofactors and methyl donors required for methyl metabolism aggravated allergic airway diseases; the transcription factor runt-related transcription factor 3 (Runx3), a negative regulator of asthma-like disease in mice, was excessively methylated in this model system (79). The importance of adequate maternal nutrition for the progeny’s health should be highlighted, because the effects of maternal zinc deficiency in offspring persist during developmental stages despite intake of appropriate amounts of zinc. Thus, macro- and micronutrient deficiencies, either in intrauterine or during infant stages, can contribute to serious human health problems throughout life.

Another example of maternal nutrition deficiency that can affect the progeny’s health is the intake of a diet restricted in proteins during conception through pregnancy and lactation. Several reports showed that a maternal diet low in protein caused metabolic abnormalities, impaired immunity, increased sensitivity to oxidative stress, and glucose dyshomeostasis (75). Further studies from our laboratory showed that a low-protein prenatal and high-fat postnatal diet in Sprague-Dawley rats increased the number of small adipocytes in adipose tissue and decreased insulin sensitivity (75). Such data are suggestive of alterations in the DNA methylation within adipocytes and a subsequent increased risk of developing type 2 diabetes.

FAs are essential components of the human diet because adequate amounts of these fats are required for health and normal growth. In general, high intake of diets rich in n–3 FAs such as EPA, DHA, and α-linolenic acid (18:3n−3) confer protective effects. A recent study examined whether dietary n–3 PUFAs influence leptin and leptin receptor promoter DNA methylation. Results showed no effects of n–3 PUFAs on promoter DNA methylation in leptin and leptin receptor genes (75). Another study examined effects of fish oil supplementation on global DNA methylation. Although the exact underlying mechanisms involved in the modulation of insulin sensitivity by DNA methylation remain to be investigated, fish oil supplementation for 2 generations increased insulin-stimulated glucose uptake and insulin sensitivity, lowered blood lipid concentrations, and decreased global DNA methylation in the liver (75). Interestingly, the anticarcinogenic effects of n–3 PUFAs were tested in U937 leukemia cells in vitro. n–3 PUFA treatment induced the myeloid lineage-specific transcription factors CCAAT/enhancer-binding protein binding to the macrophage colony-stimulating factor (M-CSF) receptor promoter, resulting in enhanced M-CSF receptor gene expression via methylation alterations of the CpG islands in the M-CSF gene promoter region (75). These data suggest that maternal dietary FAs, especially n–3 PUFAs, modulate inflammatory functions in various immune cells by epigenetic mechanisms. High-fat diets alone (75) or when combined with high fructose intake by female rats during prenatal and throughout postnatal life were shown to increase the risk of renal and metabolic diseases later in life (75). Maternal obesity and/or high-fat diet intake predispose offspring to various vascular diseases involving hormonal dysregulation and inflammatory cytokine release. Phytochemical bioactive compounds called indoles are found in commonly consumed cruciferous vegetables such as broccoli, cabbage, and Brussels sprouts (80). It was shown that maternal indole supplementation reduces bisphenol A–induced prostatic tissue lesions and inflammation in a rat model study (81). Flavonoid quercetins are found in red onions, citrus fruits, and red wine (82). Although quercetins are known to have beneficial effects such as lipid (83) and blood pressure (84)–lowering effects, whether maternal diets supplemented with quercetins have anti-inflammatory effects is not clear. For example, although maternal quercetin intake reduces endoplasmic reticulum stress–associated stress and inflammation (85), maternal flavonoid supplementation also was shown to increase hepatic inflammatory cytokine expression in the liver (86) and increase the risk of carcinogenesis (87). Interestingly, intake of specific supplements such as green tea was shown to mitigate high-fat–induced metabolic abnormalities such as insulin resistance in rat offspring (75). The addition of green tea extract, which contains high amounts of flavonoid catechins compared with maternal high-fat diet alone, also resulted in increased serum adiponectin concentrations (88).

Summary and Conclusions

In summary, maternal nutrition has a considerable impact on fetal metabolic programming. Specifically, high-fat diets, micro- and macronutrient deficiencies, and low-protein diet intake during conception through lactation are all deleterious to fetal and infant development. The deleterious effects involve alterations in gene expression and epigenetic programming that converge to induce inflammation, impair the immune system, and cause a series of pathologic conditions at both early and late stages of life. Maternal nutrient intake, maternal infection, obesity, environmental pollution and toxicants, and psychological stress may all influence immune function and inflammation in utero (Figure 1). Of these, maternal nutrition may be a key factor. Both micro- and macronutrients modulate immune cell proinflammatory cytokine and chemokine secretory functions, immune cell differentiation, and immune cell proliferation by altering promoter region DNA methylation and histone modification. Because increased chronic inflammation causes metabolic complications and further contributes to the development of diabetes, cancer, and cardiovascular disease, increased anti-inflammatory nutrient and bioactive compounds may help reduce in utero programming of immune cells that control inflammatory response in the offspring. Future controlled human supplementation studies are needed to make nutrient supplementation recommendations. Both nutritional influences and infections likely induce global epigenetic changes, making it difficult to link specific changes in gene expression to any particular epigenetic modification. The rapidly expanding literature on maternal influences on fetal development and later disease states must therefore be interpreted carefully. However, new advances in next-generation sequencing and other technologies will enable researchers to unravel these complex networks and link specific gene expression changes to specific epigenetic modifications induced by infections and nutritional influences.

FIGURE 1.

Epigenetic regulators of immune and inflammatory functions in offspring. Maternal nutrient intake, infection, obesity, environmental pollution, and stress contribute to epigenetic changes in the fetus by DNA methylation and histone modifications. These epigenetic changes, in turn, alter immune function and inflammatory responses by activating inflammatory cytokines and chemokines, resulting in increased risk of acute and chronic diseases in offspring.