Evolution and the critical role of the microbiota in the reduced mental and physical health associated with low socioeconomic status (SES).

The evolution of the gut-microbiota-brain axis in animals reveals that microbial inputs influence metabolism, the regulation of inflammation and the development of organs, including the brain. Inflammatory, neurodegenerative and psychiatric disorders are more prevalent in people of low socioeconomic status (SES). Many aspects of low SES reduce exposure to the microbial inputs on which we are in a state of evolved dependence, whereas the lifestyle of wealthy citizens maintains these exposures. This partially explains the health deficit of low SES, so focussing on our evolutionary history and on environmental and lifestyle factors that distort microbial exposures might help to mitigate that deficit. But the human microbiota is complex and we have poor understanding of its functions at the microbial and mechanistic levels, and in the brain. Perhaps its composition is more flexible than the microbiota of animals that have restricted habitats and less diverse diets? These uncertainties are discussed in relation to the encouraging but frustrating results of attempts to treat psychiatric disorders by modulating the microbiota.

The old friends hypothesis: evolution, immunoregulation and essential microbial inputs.

In wealthy urbanised societies there have been striking increases in chronic inflammatory disorders such as allergies, autoimmunity and inflammatory bowel diseases. There has also been an increase in the prevalence of individuals with systemically raised levels of inflammatory biomarkers correlating with increased risk of metabolic, cardiovascular and psychiatric problems. These changing disease patterns indicate a broad failure of the mechanisms that should stop the immune system from attacking harmless allergens, components of self or gut contents, and that should terminate inappropriate inflammation. The Old Friends Hypothesis postulates that this broad failure of immunoregulation is due to inadequate exposures to the microorganisms that drive development of the immune system, and drive the expansion of components such as regulatory T cells (Treg) that mediate immunoregulatory mechanisms. An evolutionary approach helps us to identify the organisms on which we are in a state of evolved dependence for this function (Old Friends). The bottom line is that most of the organisms that drive the regulatory arm of the immune system come from our mothers and family and from the natural environment (including animals) and many of these organisms are symbiotic components of a healthy microbiota. Lifestyle changes that are interrupting our exposure to these organisms can now be identified, and many are closely associated with low socioeconomic status (SES) in wealthy countries. These insights will facilitate the development of education, diets and urban planning that can correct the immunoregulatory deficit, while simultaneously reducing other contributory factors such as epithelial damage.

Evolution, the Immune System, and the Health Consequences of Socioeconomic Inequality.

Healthy development and function of essentially all physiological systems and organs, including the brain, require exposure to the microbiota of our mothers and of the natural environment, especially in early life. We also know that some infections, if we survive them, modulate the immune system in relevant ways. If we study the evolution of the immune and metabolic systems, we can understand how these requirements developed and the nature of the organisms that we need to encounter. We can then begin to identify the mechanisms of the beneficial effects of these exposures. Against this evolutionary background, we can analyze the ways in which the modern urban lifestyle, particularly for individuals experiencing low socioeconomic status (SES), results in deficient or distorted microbial exposures and microbiomes. Thus, an evolutionary approach facilitates the identification of practical solutions to the growing scandal of health disparities linked to inequality.

Microbial exposures that establish immunoregulation are compatible with targeted hygiene.

It is often suggested that hygiene is not compatible with the microbial exposures that are necessary for establishment of the immune system in early life. However, when we analyze the microbial exposures of modern humans in the context of human evolution and history, it becomes evident that whereas children need exposure to the microbiotas of their mothers, other family members, and the natural environment, exposure to the unnatural microbiota of the modern home is less relevant. In addition, any benefits of exposure to the infections of childhood within their household setting are at least partly replaced by the recently revealed nonspecific effects of vaccines. This article shows how targeting hygiene practices at key risk moments and sites can maximize protection against infection while minimizing any impact on essential microbial exposures. Moreover, this targeting must aim to reduce direct exposure of children to cleaning agents because those agents probably exert TH2-adjuvant effects that trigger allergic responses to normally innocuous antigens. Finally, we need to halt the flow of publications in the scientific literature and the media that blame hygiene for the increases in immunoregulatory disorders. Appropriately targeted hygiene behavior is compatible with a healthy lifestyle that promotes exposure to essential microorganisms.

Association of the Salivary Microbiome With Animal Contact During Early Life and Stress-Induced Immune Activation in Healthy Participants.

The prevalence of stress-associated somatic and psychiatric disorders is increased in environments offering a narrow relative to a wide range of microbial exposure. Moreover, different animal and human studies suggest that an overreactive immune system not only accompanies stress-associated disorders, but might even be causally involved in their pathogenesis. In support of this hypothesis, we recently showed that urban upbringing in the absence of daily contact with pets, compared to rural upbringing in the presence of daily contact with farm animals, is associated with a more pronounced immune activation following acute psychosocial stressor exposure induced by the Trier Social Stress Test (TSST). Here we employed 16S rRNA gene sequencing to test whether this difference in TSST-induced immune activation between urban upbringing in the absence of daily contact with pets (n = 20) compared with rural upbringing in the presence of daily contact with farm animals (n = 20) is associated with differences in the composition of the salivary microbiome. Although we did not detect any differences in alpha or beta diversity measures of the salivary microbiome between the two experimental groups, statistical analysis revealed that the salivary microbial beta diversity was significantly higher in participants with absolutely no animal contact (n = 5, urban participants) until the age of 15 compared to all other participants (n = 35) reporting either daily contact with farm animals (n = 20, rural participants) or occasional pet contact (n = 15, urban participants). Interestingly, when comparing these urban participants with absolutely no pet contact to the remaining urban participants with occasional pet contact, the former also displayed a significantly higher immune, but not hypothalamic-pituitary-adrenal (HPA) axis or sympathetic nervous system (SNS) activation, following TSST exposure. In summary, we conclude that only urban upbringing with absolutely no animal contact had long-lasting effects on the composition of the salivary microbiome and potentiates the negative consequences of urban upbringing on stress-induced immune activation.

Identification and characterization of a novel anti-inflammatory lipid isolated from Mycobacterium vaccae, a soil-derived bacterium with immunoregulatory and stress resilience properties.

RATIONALE: Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known. OBJECTIVES: Our objective was to identify and characterize novel anti-inflammatory molecules from M. vaccae NCTC 11659. METHODS: We have purified and identified a unique anti-inflammatory triglyceride, 1,2,3-tri [Z-10-hexadecenoyl] glycerol, from M. vaccae and evaluated its effects in freshly isolated murine peritoneal macrophages. RESULTS: The free fatty acid form of 1,2,3-tri [Z-10-hexadecenoyl] glycerol, 10(Z)-hexadecenoic acid, decreased lipopolysaccharide-stimulated secretion of the proinflammatory cytokine IL-6 ex vivo. Meanwhile, next-generation RNA sequencing revealed that pretreatment with 10(Z)-hexadecenoic acid upregulated genes associated with peroxisome proliferator-activated receptor alpha (PPARα) signaling in lipopolysaccharide-stimulated macrophages, in association with a broad transcriptional repression of inflammatory markers. We confirmed using luciferase-based transfection assays that 10(Z)-hexadecenoic acid activated PPARα signaling, but not PPARγ, PPARδ, or retinoic acid receptor (RAR) α signaling. The effects of 10(Z)-hexadecenoic acid on lipopolysaccharide-stimulated secretion of IL-6 were prevented by PPARα antagonists and absent in PPARα-deficient mice. CONCLUSION: Future studies should evaluate the effects of 10(Z)-hexadecenoic acid on stress-induced exaggeration of peripheral inflammatory signaling, central neuroinflammatory signaling, and anxiety- and fear-related defensive behavioral responses.

Less immune activation following social stress in rural vs. urban participants raised with regular or no animal contact, respectively.

Urbanization is on the rise, and environments offering a narrow range of microbial exposures are linked to an increased prevalence of both physical and mental disorders. Human and animal studies suggest that an overreactive immune system not only accompanies stress-associated disorders but might even be causally involved in their pathogenesis. Here, we show in young [mean age, years (SD): rural, 25.1 (0.78); urban, 24.5 (0.88)] healthy human volunteers that urban upbringing in the absence of pets (n = 20), relative to rural upbringing in the presence of farm animals (n = 20), was associated with a more pronounced increase in the number of peripheral blood mononuclear cells (PBMCs) and plasma interleukin 6 (IL-6) concentrations following acute psychosocial stress induced by the Trier social stress test (TSST). Moreover, ex vivo-cultured PBMCs from urban participants raised in the absence of animals secreted more IL-6 in response to the T cell-specific mitogen Con A. In turn, antiinflammatory IL-10 secretion was suppressed following TSST in urban participants raised in the absence of animals, suggesting immunoregulatory deficits, relative to rural participants raised in the presence of animals. Questionnaires, plasma cortisol, and salivary α-amylase, however, indicated the experimental protocol was more stressful and anxiogenic for rural participants raised in the presence of animals. Together, our findings support the hypothesis that urban vs. rural upbringing in the absence or presence of animals, respectively, increases vulnerability to stress-associated physical and mental disorders by compromising adequate resolution of systemic immune activation following social stress and, in turn, aggravating stress-associated systemic immune activation.

The Microbiota, Immunoregulation, and Mental Health: Implications for Public Health.

The hygiene or "Old Friends" hypothesis proposes that the epidemic of inflammatory disease in modern urban societies stems at least in part from reduced exposure to microbes that normally prime mammalian immunoregulatory circuits and suppress inappropriate inflammation. Such diseases include but are not limited to allergies and asthma; we and others have proposed that the markedly reduced exposure to these Old Friends in modern urban societies may also increase vulnerability to neurodevelopmental disorders and stress-related psychiatric disorders, such as anxiety and affective disorders, where data are emerging in support of inflammation as a risk factor. Here, we review recent advances in our understanding of the potential for Old Friends, including environmental microbial inputs, to modify risk for inflammatory disease, with a focus on neurodevelopmental and psychiatric conditions. We highlight potential mechanisms, involving bacterially derived metabolites, bacterial antigens, and helminthic antigens, through which these inputs promote immunoregulation. Though findings are encouraging, significant human subjects' research is required to evaluate the potential impact of Old Friends, including environmental microbial inputs, on biological signatures and clinically meaningful mental health prevention and intervention outcomes.

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice.

The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1-2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

Hygiene and other early childhood influences on the subsequent function of the immune system.

The immune system influences brain development and function. Hygiene and other early childhood influences impact the subsequent function of the immune system during adulthood, with consequences for vulnerability to neurodevelopmental and psychiatric disorders. Inflammatory events during pregnancy can act directly to cause developmental problems in the central nervous system (CNS) that have been implicated in schizophrenia and autism. The immune system also acts indirectly by "farming" the intestinal microbiota, which then influences brain development and function via the multiple pathways that constitute the gut-brain axis. The gut microbiota also regulates the immune system. Regulation of the immune system is crucial because inflammatory states in pregnancy need to be limited, and throughout life inflammation needs to be terminated completely when not required; for example, persistently raised levels of background inflammation during adulthood (in the presence or absence of a clinically apparent inflammatory stimulus) correlate with an increased risk of depression. A number of factors in the perinatal period, notably immigration from rural low-income to rich developed settings, caesarean delivery, breastfeeding and antibiotic abuse have profound effects on the microbiota and on immunoregulation during early life that persist into adulthood. Many aspects of the modern western environment deprive the infant of the immunoregulatory organisms with which humans co-evolved, while encouraging exposure to non-immunoregulatory organisms, associated with more recently evolved "crowd" infections. Finally, there are complex interactions between perinatal psychosocial stressors, the microbiota, and the immune system that have significant additional effects on both physical and psychiatric wellbeing in subsequent adulthood. This article is part of a Special Issue entitled Neuroimmunology in Health And Disease.

Microbiota, immunoregulatory old friends and psychiatric disorders.

Regulation of the immune system is an important function of the gut microbiota. Increasing evidence suggests that modern living conditions cause the gut microbiota to deviate from the form it took during human evolution. Contributing factors include loss of helminth infections, encountering less microbial biodiversity, and modulation of the microbiota composition by diet and antibiotic use. Thus the gut microbiota is a major mediator of the hygiene hypothesis (or as we prefer, "Old Friends" mechanism), which describes the role of organisms with which we co-evolved, and that needed to be tolerated, as crucial inducers of immunoregulation. At least partly as a consequence of reduced exposure to immunoregulatory Old Friends, many but not all of which resided in the gut, high-income countries are undergoing large increases in a wide range of chronic inflammatory disorders including allergies, autoimmunity and inflammatory bowel diseases. Depression, anxiety and reduced stress resilience are comorbid with these conditions, or can occur in individuals with persistently raised circulating levels of biomarkers of inflammation in the absence of clinically apparent peripheral inflammatory disease. Moreover poorly regulated inflammation during pregnancy might contribute to brain developmental abnormalities that underlie some cases of autism spectrum disorders and schizophrenia. In this chapter we explain how the gut microbiota drives immunoregulation, how faulty immunoregulation and inflammation predispose to psychiatric disease, and how psychological stress drives further inflammation via pathways that involve the gut and microbiota. We also outline how this two-way relationship between the brain and inflammation implicates the microbiota, Old Friends and immunoregulation in the control of stress resilience.

Regulation of the immune system by biodiversity from the natural environment: an ecosystem service essential to health.

Epidemiological studies suggest that living close to the natural environment is associated with long-term health benefits including reduced death rates, reduced cardiovascular disease, and reduced psychiatric problems. This is often attributed to psychological mechanisms, boosted by exercise, social interactions, and sunlight. Compared with urban environments, exposure to green spaces does indeed trigger rapid psychological, physiological, and endocrinological effects. However, there is little evidence that these rapid transient effects cause long-term health benefits or even that they are a specific property of natural environments. Meanwhile, the illnesses that are increasing in high-income countries are associated with failing immunoregulation and poorly regulated inflammatory responses, manifested as chronically raised C-reactive protein and proinflammatory cytokines. This failure of immunoregulation is partly attributable to a lack of exposure to organisms ("Old Friends") from mankind's evolutionary past that needed to be tolerated and therefore evolved roles in driving immunoregulatory mechanisms. Some Old Friends (such as helminths and infections picked up at birth that established carrier states) are almost eliminated from the urban environment. This increases our dependence on Old Friends derived from our mothers, other people, animals, and the environment. It is suggested that the requirement for microbial input from the environment to drive immunoregulation is a major component of the beneficial effect of green space, and a neglected ecosystem service that is essential for our well-being. This insight will allow green spaces to be designed to optimize health benefits and will provide impetus from health systems for the preservation of ecosystem biodiversity.

Microbial 'Old Friends', immunoregulation and stress resilience.

Chronic inflammatory diseases (autoimmunity, allergy and inflammatory bowel diseases) are increasing in prevalence in urban communities in high-income countries. One important factor is reduced exposure to immunoregulation-inducing macro- and microorganisms and microbiota that accompanied mammalian evolution (the hygiene hypothesis or 'Old Friends' mechanism). Reduced exposure to these organisms predisposes to poor regulation of inflammation. But inflammation is equally relevant to psychiatric disorders. Inflammatory mediators modulate brain development, cognition and mood, and accompany low socioeconomic status and some cases of depression in developed countries. The risk of all these conditions (chronic inflammatory and psychiatric) is increased in urban versus rural communities, and increased in immigrants, particularly if they move from a low- to a high-income country during infancy, and often the prevalence increases further in second generation immigrants, suggesting that critical exposures modulating disease risk occur during pregnancy and infancy. Diminished exposure to immunoregulation-inducing Old Friends in the perinatal period may enhance the consequences of psychosocial stressors, which induce increased levels of inflammatory mediators, modulate the microbiota and increase the risk for developing all known psychiatric conditions. In later life, the detrimental effects of psychosocial stressors may be exaggerated when the stress occurs against a background of reduced immunoregulation, so that more inflammation (and therefore more psychiatric symptoms) result from any given level of psychosocial stress. This interaction between immunoregulatory deficits and psychosocial stressors may lead to reduced stress resilience in modern urban communities. This concept suggests novel interpretations of recent epidemiology, and novel approaches to the increasing burden of psychiatric disease.

Pathways underlying afferent signaling of bronchopulmonary immune activation to the central nervous system.

Bronchopulmonary inflammation, such as that associated with asthma, activates afferent neural pathways. We recently demonstrated that localized inflammation in the lungs, induced by intratracheal administration of ovalbumin in ovalbumin-preimmunized mice (an animal model of asthma) results in activation of the dorsolateral part of the nucleus of the solitary tract, a major target of vagal afferent fibers innervating the lungs and airways. Activation of the nucleus of the solitary tract was evident in the absence of activation of the area postrema, a circumventricular organ, consistent with the hypothesis that localized inflammation in the bronchopulmonary system can signal to the central nervous system via specific neural pathways, in the absence of circulating proinflammatory mediators. The pattern of brain activation in ovalbumin-challenged mice differs from the pattern of activation in mice challenged with heat-killed Mycobacterium vaccae, suggesting that qualitative aspects of bronchopulmonary inflammation determine the overall pattern of brain activation. The mechanisms through which localized bronchopulmonary inflammation signals to the central nervous system is poorly understood, but appears to involve both vagal and spinal afferent pathways. In this chapter, we review our current understanding of the anatomical pathways through which localized inflammation in the bronchopulmonary system influences central nervous system function.

Regulation of inflammation by interleukin-4: a review of "alternatives".

Studies of IL-4 have revealed a wealth of information on the diverse roles of this cytokine in homeostatic regulation and disease pathogenesis. Recent data suggest that instead of simple linear regulatory pathways, IL-4 drives regulation that is full of alternatives. In addition to the well-known dichotomous regulation of Th cell differentiation by IL-4, this cytokine is engaged in several other alternative pathways. Its own production involves alternative mRNA splicing, yielding at least two functional isoforms: full-length IL-4, encoded by the IL-4 gene exons 1-4, and IL-4δ2, encoded by exons 1, 3, and 4. The functional effects of these two isoforms are in some ways similar but in other ways quite distinct. When binding to the surface of target cells, IL-4 may differentially engage two different types of receptors. By acting on macrophages, a cell type critically involved in inflammation, IL-4 induces the so-called alternative macrophage activation. In this review, recent advances in understanding these three IL-4-related branch points--alternative splicing of IL-4, differential receptor engagement by IL-4, and differential regulation of macrophage activation by IL-4--are summarized in light of their contributions to inflammation.

Can we vaccinate against depression?

Major depression is common in the context of autoimmune and inflammatory diseases and is frequently associated with persistently raised levels of proinflammatory cytokines and other markers of inflammation, even in the absence of another diagnosable immune pathology to account for these findings. Therefore immunoregulation-inducing vaccines or manipulations of the gut microbiota might prevent or treat depression. These strategies are already undergoing clinical trials for chronic inflammatory disorders, such as allergies, autoimmunity and inflammatory bowel disease. In this article, we summarize data suggesting that this approach might be effective in depression and encourage the initiation of clinical vaccination trials in this disorder.

Hygiene hypothesis and autoimmune diseases.

Throughout the twentieth century, there were striking increases in the incidences of many chronic inflammatory disorders in the rich developed countries. These included autoimmune disorders such as Type 1 diabetes and multiple sclerosis. Although genetics and specific triggering mechanisms such as molecular mimicry and viruses are likely to be involved, the increases have been so rapid that any explanation that omits environmental change is incomplete. This chapter suggests that a series of environmental factors, most of them microbial, have led to a decrease in the efficiency of our immunoregulatory mechanisms because we are in a state of evolved dependence on organisms with which we co-evolved (and that had to be tolerated) as inducers of immunoregulatory circuits. These organisms ("Old Friends") are depleted from the modern urban environment. Rather than considering fetal programming by maternal microbial exposures, neonatal programming, the hygiene hypothesis, gut microbiota, and diet as separate and competing hypotheses, I attempt here to integrate these ideas under a single umbrella concept that can provide the missing immunoregulatory environmental factor that is needed to explain the recent increases in autoimmune disease.

Hygiene and other early childhood influences on the subsequent function of the immune system.

The current 'Darwinian' synthesis of the hygiene (or 'Old Friends') hypothesis suggests that the increase in chronic inflammatory disorders that started in Europe in the mid-19th century and progressed until the late 20th century is at least partly attributable to immunodysregulation resulting from lack of exposure to microorganisms that were tasked by co-evolutionary processes with establishing the 'normal' background levels of immunoregulation, a role that they perform in concert with the normal microbiota. This is an example of 'evolved dependence'. The relevant organisms co-evolved with mammals, already accompanied early hominids in the Paleolithic era and are associated with animals, mud and faeces. These organisms often establish stable carrier states, or are encountered continuously in primitive environments as 'pseudocommensals' from mud and water. These organisms were not lost during the first epidemiological transition, which might even have resulted in increased exposure to them. However, the crucial organisms are lost progressively as populations undergo the second epidemiological transition (modern urban environment). Recently evolved sporadic 'childhood infections' are not likely to have evolved immunoregulatory roles, and epidemiology supports this contention. The consequences of the loss of the Old Friends and distortion of the microbiota are aggravated by other modern environmental changes that also lead to enhanced inflammatory responses (obesity, vitamin D deficiency, pollution (dioxins), etc.). The range of chronic inflammatory disorders affected may be larger than had been assumed (allergies, autoimmunity, inflammatory bowel disease, but also coeliac disease, food allergy, vascular disease, some cancers, and depression/anxiety when accompanied by raised inflammatory cytokines).