Gut Microbiota, Host Gene Expression, and Aging

Abstract

Novel concepts of disease susceptibility and development suggest an important role of gastrointestinal microbiota and microbial pathogens. They can contribute to physiological systems and disease processes, even outside of the gastrointestinal tract. There is increasing evidence that genetics of the host influence and interact with gut microbiota. Moreover, aging-associated oxidative stress may cause morphologic alterations of bacterial cells, thus influencing the aggressive potential and virulence markers of an anaerobic bacterium and finally the type of interaction with the host. At the same time, microbiota may influence host gene expression and it is becoming apparent that it may occur through the regulation of microRNAs. They are short single-stranded noncoding RNAs that regulate posttranscriptional gene expression by affecting mRNA stability and/or translational repression of their target mRNAs. The introduction of -omics approaches (such as metagenomics, metaproteomics, and metatranscriptomics) in microbiota research will certainly advance our knowledge of this area. This will lead to greatly deepen our understanding of the molecular targets in the homeostatic interaction between the gut microbiota and the host and, thereby, promises to reveal new ways to treat diseases and maintain health.

AGING AND OXIDATIVE STRESS

Aging is defined as the process of becoming older, which is genetically determined and environmentally modulated. Recently, the identification of longevity-influencing genes, not only in lower organisms, but also in mammals, significantly strengthened the mechanistic connection between oxidative stress and aging.¹ The oxidative damage due to the excess of free radical species is thought to play an important role in the onset of mobility limitation, disability, and mortality. Thus, the evaluation of increased lipid peroxidation might represent a predictive marker of negative health-related events in elderly people.^2,3 Oxidative stress is also closely associated with platelet activation and inflammation, and an interaction among these 3 pathways might be the underpinnings of aging and age-related diseases.⁴ We have recently performed a study to address whether the levels of systemic biomarkers of lipid peroxidation, platelet activation, and inflammation might predict the incidence of major adverse health-related events in a large cohort of elderly people, participating in the Health Aging and Body Composition (Health ABC) study, a prospective cohort study investigating changes in body composition and weight-related health conditions in well-functioning older persons.⁵

In this study we assessed urinary levels of 8-iso-PGF_2α as a reliable, noninvasive index of lipid peroxidation in vivo.⁶ This is a member of the family of prostaglandin (PG)-like compounds, called F₂-isoprostanes, formed nonenzymatically through free radical catalyzed attack on esterified arachidonate followed by enzymatic release from cellular or lipoprotein phospholipids. Different from classic prostanoids, which result following an enzymatic action of the PGH-synthase (also known as cyclooxygenase, COX) from arachidonic acid, F₂-isoprostanes are formed from a free radical–catalyzed (nonenzymatic) mechanism.^7,8

Altered generation of F₂-isoprostanes has been reported in a variety of syndromes associated with oxidant stress, such as coronary ischemia-reperfusion syndromes, acute coronary syndromes, Alzheimer disease, chronic obstructive pulmonary disease, and cystic fibrosis. Their urinary levels have been reported to correlate with several cardiovascular risk factors, including hypercholesterolemia, hyperhomocysteinemia, diabetes mellitus, hypertension, and cigarette smoking.⁹ Elevated urinary 8-iso-PGF_2α levels have been reported in conjunction with platelet activation in women, with android obesity⁹ and in association with age and alcohol consumption.⁸

To detect the platelet function activation in vivo, it is appropriate to measure the urinary levels of 11-dehydro-TXB₂, which is the major enzymatic metabolite of TXA₂, that is, a potent proaggregatory and vasoconstrictor mediator which is generated from arachidonic acid by COX-1 activity in platelets.¹⁰

As a marker of systemic inflammation we assessed the serum levels of interleukin (IL-6). These biomarkers were assessed in a large number of participants in HABC study, and they were tested in Cox proportional hazard models as predictors of (severe) mobility disability and overall mortality.⁵ Interestingly, we found that the inflammatory marker IL-6 is a robust predictor for the onset of negative health-related events. Our results confirm the notion that the assessment of IL-6 levels represents a valuable and clinically meaningful biomarker for gerontology and geriatric medicine.⁵ In fact, IL-6 concentrations have been shown to predict subclinical (eg, sarcopenia, body composition modifications, insulin resistance)¹¹ as well as clinical (eg, physical impairment, disability, mortality)¹² conditions in older people. An interesting result of our study was that elderly participants with higher urinary levels of 8-iso-PGF_2α and 11-dehydro-TXB₂ presented a higher mortality risk.

Using a similar biomarker approach, it has been possible to identify novel mechanisms of cardiovascular disease development involving gastrointestinal bacterial infection–induced oxidative stress.¹³ In fact, in dyspeptic individuals with H. pylori infection, it was found that enhanced in vivo lipid peroxidation was associated with platelet activation. Reversibility of the hemostatic abnormality after successful eradication of H. pylori suggests the role of bacterial infection in enhanced platelet activation, in this setting. This finding may have clinical implications for cardiovascular risk management.

Altogether, the scientific findings reported here provide evidences of the important role of oxidative stress in aging-associated diseases. Moreover, they enlighten the role of gastrointestinal microbiota and microbial pathogens in disease susceptibility, even outside of the gastrointestinal tract, by modulating the extent of oxidative stress.

HOST-BACTERIAL RELATIONSHIPS IN HEALTH AND DISEASE

The mammalian gut represents a complex ecosystem colonized by a vast community of organisms that collectively comprise the microbiota, including up to 100 trillion (10¹⁴) microbes. The intestinal microbiota is not homogenous. The number of bacterial cells present in the mammalian gut shows a continuum that goes from 10¹ to 10³ bacteria per gram of contents in the stomach and duodenum, progressing to 10⁴ to 10⁷ bacteria per gram in the jejunum and ileum and culminating in 10¹¹ to 10¹² cells per gram in the colon.¹⁴ In addition, different bacterial groups are enriched at different sites when comparing biopsy samples of the small intestine and colon from healthy individuals and also a great latitudinal variation in the microbiota composition, that is, between intestinal lumen, mucus layer, and in proximity of the epithelium, has been reported.¹⁴ Microbial community is host specific and it is established early in life and influenced soon after birth by maternal environment. Diet, exposure to antibiotics, pathogens, and parasites can also influence compositional features of the microbiota.¹⁴

Interactions between the mammalian host and the intestinal microbiota require a delicate balance that must be actively maintained by both host and microbe to achieve a healthy steady state¹⁵; the disruption of this equilibrium may induce highly unwanted processes in the host (Fig. 1).

FIGURE 1: Gut microbiota-host balance. Schematic representation of the gut microbiota-host balance as result of their coevolution. Failure to achieve or maintain this complex equilibrium may perturbate the healthy steady state, causing intestinal or intestinal-related diseases and systemic complications.

The mechanisms through which microbiota exert their beneficial or detrimental influences remain largely undefined. Commensal bacterial colonization of the host digestive tract has been shown to modulate the expression of a number of host genes.¹⁵ In germ-free mice colonized with Bacteroides thetaiotaomicron (a prominent member of the adult mouse and human gut microflora), global intestinal transcriptional responses to colonization were observed with DNA microarrays. The results of these studies showed that commensals are able to modulate expression of host genes involved in several important intestinal functions, including nutrient absorption, mucosal barrier fortification, xenobiotic metabolism, angiogenesis, and postnatal intestinal maturation.¹⁵ Despite the mounting evidence of microbiota capability to modulate host gene expression, underlying molecular mechanisms remain elusive. Recently, a role of microRNAs (miRNAs) in microbiota-mediated regulation of host gene expression has been proposed.¹⁶ miRNAs are small noncoding RNAs that posttranscriptionally regulate gene expression by binding to the 3′-untranslated regions (3′-UTRs) of target mRNAs. Such binding is not homologous, allowing a single miRNA to potentially regulate hundreds of genes.¹⁷ Interestingly, germ-free mice colonization with the microbiota from pathogen-free mice revealed that mmu-miR-665, one of the dysregulated miRNAs during colonization of germ-free mice, was able to downregulate Abcc3 expression, in murine macrophage RAW 264.7 cell line. Abcc3 belongs to the multidrug resistance–associated protein family, which mediates the metabolism of xenobiotics and endogenous toxins, an intestinal function dysregulated in response to colonization of mice.¹⁶

In the complex web of relationships between the host and gut microbiota, host not only tolerates, but has evolved to require colonization for normal host physiology, such as immune development and function, nutrient processing, and behavior and stress responses.¹⁵ Germ-free animals that have been raised in the complete absence of microbial exposure present undeveloped tissue architecture, deficiency in nutrient and vitamin absorption, and significant susceptibility to gastrointestinal infection.¹⁵ Moreover, the importance of the gut microbiota in the development of both the intestinal mucosal and systemic immune systems can be readily appreciated from studies of germ-free animals. Although the systemic immune system is designed to react in almost an automatic manner to any microbial agent it detects, the mucosal immune system must be more tentative in its response so as to preserve the critical partnership with the gut bacteria. Therefore, the challenge to the mucosal immune system is to selectively and actively tolerate the gut microbiota during steady-state conditions, while being able to mount an appropriate inflammatory response during an incidence of disease or infection.¹⁵

The mammalian gut has evolved numerous physical and molecular mechanisms for maintaining this delicate balance with commensal organisms. It is well known that interleukin 10 (IL-10) and regulatory T cells (Tregs) are involved in maintaining tolerance to intestinal microorganisms.^15,18 However, recently, suppressor of cytokine signaling 1 (SOCS1) has been identified as an essential mediator of immune tolerance in the intestine and an essential role for prostaglandin E2 (PGE₂) in the maintenance of tolerance within the intestine in the absence of Tregs has been described, thus suggesting that SOCS1 and PGE₂, potentially interacting together, may act as an alternative intestinal tolerance mechanism distinct from IL-10 and Tregs.¹⁸

Dysregulation of the intestinal mucosa homeostasis leads to a multitude of pathologic conditions, from the obvious case of inflammatory bowel diseases (IBD) to the more unexpected activation of chronic human immunodeficiency virus infection¹⁴ (Fig. 1).

Colonization of the intestinal mucosa by bacterial enteric pathogens results in the induction of a strong inflammatory response aimed at controlling the offending pathogen. However, this inflammatory response has also been shown to decrease the viability of the gut microbiota, allowing the pathogen to occupy the vacated niches.¹⁴ Commensal bacteria of the microbiota have been strongly implemented in the initiation and progression of IBDs, which include Crohn’s disease and ulcerative colitis. The disease is, generally, thought to be mediated by an overt T-cell inflammatory response that is perpetuated by stimulation from microbial antigens. IBD may be caused in part by overall changes in the development or composition of the intestinal microbiota, known as dysbiosis.¹⁵ Currently, it remains unknown whether dysbiosis of the gut microbiota is a result of IBD or the cause of inflammation. These changes in the microbial community may result in a reduction of protective commensal organisms.

Disproportionate proinflammatory signaling at the gastrointestinal mucosa (such as that induced by the presence of an excessively colitogenic microbiota) may result in increased sloughing and repair of the intestinal epithelium. This process can ultimately result in the formation of neoplasia and malignancy¹⁴ (Fig. 1). Probably, the best-known and most-studied example of a microbiota-induced gastrointestinal malignancy is the H. pylori-mediated gastric carcinoma.¹⁹ Because of its wide diffusion, this bacterium may be considered as part of the gastric microbiota. Its presence induces a persistent immune response in the host, resulting in a state of inflammation at the gastric mucosa that ultimately leads to malignant transformations at the gastric epithelium.¹⁴

In some circumstances, members of the gut microbiota migrate beyond their tightly regulated borders, and this disruption can cause systemic complications, promoting a new repertoire of diseases, that is, type 2 diabetes, atherosclerosis, systemic inflammatory response syndrome, and burn injury.¹⁴ The systemic presence of the intestinal microbiota and resulting associated complications may be due to outside stresses on the host (such as injury, infection, or unhealthy diet), which can promote a dysregulation of intestinal mucosal homeostasis, resulting in a “leaky gut” syndrome and translocation of gut bacteria or bacterial products to systemic sites.

TECHNIQUES TO STUDY MICROBIOTA DIVERSITY

A number of techniques have been applied to analyze the composition, abundance, and function of the gastrointestinal microbiota over the last several decades. The analysis method of choice depends on the question being asked as well as the time and cost restrictions associated with the task.

Up to the 1990s, knowledge of the gut microbiota was limited because bacteriological culture was the only technique available to characterize its composition and most gastrointestinal organisms cannot be cultured in the available defined media. Our knowledge about bacterial diversity in the human gastrointestinal tract has increased concomitantly with the development of different molecular approaches such as fingerprinting techniques of 16S rDNA amplicons, sequencing of 16S rDNA clones, fluorescent in situ hybridization, DNA microarrays and, more recently, high-throughput sequencing^20,21 (Fig. 2). The use of these techniques revealed that the composition of the intestinal microbiota varies between individuals due to host genotype, age, health status, and diet (Fig. 1), although the predominant population is fairly stable under normal conditions. Although the techniques elucidating the composition and number of the gut bacterial communities promote our knowledge of the identities of our microbial inhabitants, it does little to tell us about their function.

FIGURE 2: Techniques used to study the gut microbiota. Schematic overview of the most used approaches to characterize the gut microbiota, including fingerprinting techniques of 16 S rDNA amplicons, sequencing of 16 S rDNA clones, fluorescent in situ hybridization, DNA microarrays or, more recently, high-throughput sequencing and the more innovative metagenomic, metaproteomic, and metatranscriptomic approaches. Modified from Fraher MH, et al.²¹

Metagenomics is one of the newest additions to the repertoire of microbial community analysis tools. It refers to culture-independent and sequencing-based studies of the collective set of genomes of mixed microbial communities (metagenomes) and it aims to explore their compositional and functional characteristics.²² This method is unique because it is not dependent on the cloning and sequencing of specific genes. Instead, it provides a detailed survey of all the genes that exist within a particular community, providing insight to both structure (composition) and function in a single experiment.¹⁴ Even if metagenomic approach provides extensive information about microbiota diversity, gene content, and their potential functions, it leaves many unanswered questions such as: (i) are the predicted genes expressed? (ii) what are the conditions for the expression of these genes? and (iii) what is the extent of their expression?

To clarify these points is possible by using metaproteomics and metatranscriptomics. Metatranscriptomics retrieves and sequences environmental mRNAs from a microbial ecosystem (as opposed to the DNA content which is analyzed in metagenomics approaches) to assess what genes may be expressed in that community.²⁰ It generates structural information (ie, community membership) while simultaneously obtaining functional insights about a microbial community.¹⁴

Proteomics of isolated microbes is an established and powerful method to determine global expression profiles. However, metaproteomics, that is, studying the proteome of a complex environmental system like the human intestinal microbiota, is an emerging field within the area of proteomics that is characterized by a high level of complexity.²³ Metaproteomics utilizes nontargeted shotgun mass spectrometry to assess the diversity and abundance of proteins contained within the gut metaproteome.¹⁴ Several comparisons have been made between the results of metaproteomic and metagenomic analysis of 2 separate sets of human fecal samples. The proteins identified in human fecal samples differed significantly from the distribution of proteins predicted using metagenomics, suggesting that functional gene analysis does not necessarily correlate well with gene expression levels.¹⁴ To confirm the accuracy of these observations, however, comparisons need to be made between matched datasets.

CONCLUSIONS AND PERSPECTIVES

In the last century, several studies have defined new milestones for understanding the microbial ecology of the gastrointestinal ecosystem and assessing how the “microbial world within us” impacts our everyday life. The current genomic revolution offers an unprecedented opportunity to identify the molecular foundations of complex interactions between gut microbiota and host both in healthy and diseased conditions. In fact, the introduction of -omics approaches strongly improved the tools available for microbiota research, and especially the shift from “who’s there?” to “what do they do?” type of approaches will certainly advance our knowledge of this area. However, population complexity and diversity between individuals make this challenging. Although “meta” family of function-focused analyses encounters several practical problems, some of which are still technical, it may provide fruitful evidence in detecting associations between molecular markers (genes, proteins, and metabolites) and disease, thus providing new comprehensive tools for the diagnosis. On the basis of wide microbiota descriptive and functional charts, they may be used in view of personalized physiology and nutrition understanding, thus providing patient-tailored therapies.