The Microbiome of the Built Environment and Human Behavior: Implications for Emotional Health and Well-Being in Postmodern Western Societies.

It is increasingly evident that inflammation is an important determinant of cognitive function and emotional behaviors that are dysregulated in stress-related psychiatric disorders, such as anxiety and affective disorders. Inflammatory responses to physical or psychological stressors are dependent on immunoregulation, which is indicated by a balanced expansion of effector T-cell populations and regulatory T cells. This balance is in part driven by microbial signals. The hygiene or "old friends" hypothesis posits that exposure to immunoregulation-inducing microorganisms is reduced in modern urban societies, leading to an epidemic of inflammatory disease and increased vulnerability to stress-related psychiatric disorders. With the global trend toward urbanization, humans are progressively spending more time in built environments, thereby, experiencing limited exposures to these immunoregulatory "old friends." Here, we evaluate the implications of the global trend toward urbanization, and how this transition may affect human microbial exposures and human behavior.

Microbial 'old friends', immunoregulation and socioeconomic status.

The immune system evolved to require input from at least three sources that we collectively term the 'old friends': (i) the commensal microbiotas transmitted by mothers and other family members; (ii) organisms from the natural environment that modulate and diversify the commensal microbiotas; and (iii) the 'old' infections that could persist in small isolated hunter-gatherer groups as relatively harmless subclinical infections or carrier states. These categories of organism had to be tolerated and co-evolved roles in the development and regulation of the immune system. By contrast, the 'crowd infections' (such as childhood virus infections) evolved later, when urbanization led to large communities. They did not evolve immunoregulatory roles because they either killed the host or induced solid immunity, and could not persist in hunter-gatherer groups. Because the western lifestyle and medical practice deplete the 'old' infections (for example helminths), immunoregulatory disorders have increased, and the immune system has become more dependent upon microbiotas and the natural environment. However, urbanization maintains exposure to the crowd infections that lack immunoregulatory roles, while accelerating loss of exposure to the natural environment. This effect is most pronounced in individuals of low socioeconomic status (SES) who lack rural second homes and rural holidays. Interestingly, large epidemiological studies indicate that the health benefits of living close to green spaces are most pronounced for individuals of low SES. Here we discuss the immunoregulatory role of the natural environment, and how this may interact with, and modulate, the proinflammatory effects of psychosocial stressors in low SES individuals.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: darwinian medicine and the 'hygiene' or 'old friends' hypothesis.

The current synthesis of the 'hygiene hypothesis' suggests that the recent increase in chronic inflammatory disorders is at least partly attributable to immunodysregulation resulting from lack of exposure to microorganisms that have evolved an essential role in the establishment of the immune system. This document provides a background for discussion of the following propositions. 1. The essential role of these organisms is an example of 'evolved dependence'. 2. The most relevant organisms are those that co-evolved with mammals, and already accompanied early hominids in the Paleolithic. 3. More recently evolved 'childhood infections' are not likely to have evolved this role, and recent epidemiology supports this contention. 4. This mechanism is interacting with other modern environmental changes that also lead to enhanced inflammatory responses [inappropriate diet, obesity, psychological stress, vitamin D deficiency, pollution (dioxins), etc.]. 5. The range of chronic inflammatory disorders that is affected is potentially larger than usually assumed [allergies, autoimmunity, inflammatory bowel disease, but also vascular disease, some cancers, depression/anxiety (when accompanied by raised inflammatory cytokines), and perhaps neurodegenerative disorders and type 2 diabetes].

FOXP3 gene expression in a tuberculosis case contact study.

Regulatory T lymphocytes (T(regs)) that express FOXP3 are involved in the beneficial attenuation of immunopathology, but are also implicated in down-regulation of protective responses to infection. Their role in tuberculosis (TB) is unknown. We classified 1272 healthy TB contacts according to their tuberculin skin test (TST) and interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) results and 128 TB cases, and studied the expression of FOXP3 and interleukin (IL)-10 in blood samples. Compared to the uninfected contact group (TST(-), ELISPOT(-)), we observed higher levels of FOXP3 mRNA in blood from TB patients (< 0.001), but IL-10 expression was slightly lower (P = 0.04). In contrast, FOXP3 expression levels were significantly lower (P = 0.001) in the recently infected contacts (TST(+), ELISPOT(+)) but there was no difference for IL-10 (P = 0.74). We hypothesize that during early/subclinical TB, most of which will become latent, FOXP3(+) T(regs) may be sequestered in the lungs, but when TB becomes progressive, FOXP3 reappears at increased levels in the periphery. While these findings do not reveal the role, beneficial or harmful, of T(regs) in TB, they emphasize the probable importance of these cells.

Interpretation of Mycobacterium tuberculosis antigen-specific IFN-gamma release assays (T-SPOT.TB) and factors that may modulate test results.

BACKGROUND: Data about T cell antigen-specific (ESAT-6 and CFP-10) IFN-gamma release assays (IGRAs) during and after completion of anti-tuberculous (TB) treatment are limited and highly discordant. Thus, the utility of IGRAs as a surrogate marker of mycobacterial burden remain unclear. METHODS: To investigate factors that modulate IGRA responses during anti-TB treatment we used a standardised assay (T-SPOT.TB) in 33 patients with culture positive tuberculosis. RESULTS: Significantly more patients in the early (< or = 4 months of anti-TB treatment) rather than the late phase (> 4 months or completed anti-TB treatment) had positive IGRA responses [10/12 (83%) vs 4/21 (19%); p < or = 0.01]. Thus, 17/21 (81%) in the late phase or who had completed treatment (mean duration of treatment = 8.7 months) were IGRA negative, despite having robust antigen-specific recall proliferative responses. In these 17 patients prolonged incubation (5 days vs overnight), use of different antigen preparations (protein vs peptide) and addition of endotoxin, failed to elicit positive responses. CONCLUSIONS: In treated TB patients the discordant IGRA data remain unexplained by variation in laboratory protocols and are more likely due to host or environmental factors. In a low burden setting IGRAs may be a promising surrogate marker of mycobacterial disease burden.

Identification of an immune-responsive mesolimbocortical serotonergic system: potential role in regulation of emotional behavior.

Peripheral immune activation can have profound physiological and behavioral effects including induction of fever and sickness behavior. One mechanism through which immune activation or immunomodulation may affect physiology and behavior is via actions on brainstem neuromodulatory systems, such as serotonergic systems. We have found that peripheral immune activation with antigens derived from the nonpathogenic, saprophytic bacterium, Mycobacterium vaccae, activated a specific subset of serotonergic neurons in the interfascicular part of the dorsal raphe nucleus (DRI) of mice, as measured by quantification of c-Fos expression following intratracheal (12 h) or s.c. (6 h) administration of heat-killed, ultrasonically disrupted M. vaccae, or heat-killed, intact M. vaccae, respectively. These effects were apparent after immune activation by M. vaccae or its components but not by ovalbumin, which induces a qualitatively different immune response. The effects of immune activation were associated with increases in serotonin metabolism within the ventromedial prefrontal cortex, consistent with an effect of immune activation on mesolimbocortical serotonergic systems. The effects of M. vaccae administration on serotonergic systems were temporally associated with reductions in immobility in the forced swim test, consistent with the hypothesis that the stimulation of mesolimbocortical serotonergic systems by peripheral immune activation alters stress-related emotional behavior. These findings suggest that the immune-responsive subpopulation of serotonergic neurons in the DRI is likely to play an important role in the neural mechanisms underlying regulation of the physiological and pathophysiological responses to both acute and chronic immune activation, including regulation of mood during health and disease states. Together with previous studies, these findings also raise the possibility that immune stimulation activates a functionally and anatomically distinct subset of serotonergic neurons, different from the subset of serotonergic neurons activated by anxiogenic stimuli or uncontrollable stressors. Consequently, selective activation of specific subsets of serotonergic neurons may have distinct behavioral outcomes.

Killed Mycobacterium vaccae suspension in children with moderate-to-severe atopic dermatitis: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: The hygiene hypothesis is often proposed to explain the high prevalence of atopy in the western world. Dysregulation of the immune system may result from inadequate exposure to micro-organisms such as mycobacteria. A small trial suggested that a killed extract of Mycobacterium vaccae ameliorates atopic dermatitis (AD). OBJECTIVES: To confirm in a large clinical trial whether killed M. vaccae ameliorates AD in 5-16-year-old children. METHODS: This was a randomized, placebo-controlled, double-blind, multi-centre study of the effect of intradermal injection of killed M. vaccae (0.1 or 1 mg) on patients, aged 5-16, with moderate-to-severe AD. Patients were followed up for 24 weeks. The primary end point was the change in severity of AD at 12 weeks, assessed using the six area, six-sign, atopic dermatitis (SASSAD) score. Secondary end points included changes in disease extent, patient's global assessment and children's dermatology life quality index. RESULTS: There were 166 patients randomized. The mean SASSAD score fell to a similar degree at week 12 in all treatment arms: from 33 to 24, (26%) in the high-dose group, from 30 to 23 (25%) in the low-dose group and from 36 to 27 (24%) in the placebo group (P>0.05). Secondary end points followed the same trend. Adverse events were generally those expected to occur in this population. Injection site reactions occurred in 32 patients at week 4. CONCLUSIONS: M. vaccae was no more effective than the placebo in ameliorating the severity of AD.

Smoking and tuberculosis: the epidemiological association and immunopathogenesis.

There is increasing evidence of a link between tuberculosis and smoking. This paper reviews the epidemiological evidence from the UK, China, India and the USA, summarizing some of the main papers which indicate an association. Where an association has been found there seems to be an increase in tuberculosis case rates of between two- and four-fold for those smoking in excess of 20 cigarettes a day, but it may be difficult to control for other factors, particularly alcohol consumption. The final part of the paper reviews possible mechanisms. A likely possibility is that nicotine turns off the production of TNF-alpha by the macrophages in the lungs, rendering the patient more susceptible to the development of progressive disease from latent Mycobacterium tuberculosis infection.

Intragastric administration of Mycobacterium vaccae inhibits severe pulmonary allergic inflammation in a mouse model.

BACKGROUND: Coexistence with harmless microorganisms such as lactobacilli, saprophytic mycobacteria and some helminths, throughout evolution, may have shaped the host immune system. Exposure to such organisms may have therapeutic benefits by triggering immunoregulatory mechanisms that control inappropriate immune responses to self, gut contents or allergens. OBJECTIVE: We determined whether treatment with Mycobacterium vaccae by gavage influences the host immune response both locally and systemically. We also investigated whether delivery by this route prevents severe symptoms of disease in a murine model of pulmonary allergic inflammation. RESULTS: A single intragastric administration of M. vaccae induced a transient increase in the production of IL-10 and IFN-gamma by mesenteric lymph nodes cells and splenocytes. In addition, in a mouse model of pulmonary allergic inflammation, a single treatment with M. vaccae by gavage not only diminished the total cellular infiltrate and the eosinophilic component induced by subsequent intratracheal allergen challenge, but also biased local and systemic cytokine production towards IL-10. Delivery of M. vaccae by gavage was as effective as subcutaneous treatment. CONCLUSION: This is the first report to suggest that heat-killed mycobacteria can down-regulate symptoms of allergic inflammation by the intragastric route. These data suggest an alternative route of treatment with M. vaccae for patients with allergic conditions.

Microbes, immunoregulation, and the gut.

Two distinct, but rapidly converging, areas of research (the hygiene hypothesis and the study of probiotic/prebiotic effects) have emphasised the need to understand, and ultimately to manipulate, our physiological interactions with commensal flora, and with other transient but harmless organisms from the environment that affect immunoregulatory circuits. The story began with allergic disorders but now inflammatory bowel disease is increasingly involved.

Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders.

In the rich, developed parts of the world there has been a steady and simultaneous increase in at least three groups of disease: (1) allergies, (2) inflammatory bowel diseases (IBD; e.g. Crohn's disease and ulcerative colitis) and (3) autoimmunity (e.g. type 1 diabetes and multiple sclerosis). Because the medical world is so compartmentalised it was some time before the connection between these increases was noticed and understood. There is now evidence that the simultaneous increase in these diseases of immunodysregulation is at least partly attributable to malfunction of regulatory T cells (Treg). This paper provides an overview of relevant work in each of these fields of medicine (though with emphasis on the allergic disorders), and concludes that the increasing failure of Treg is a consequence of diminished exposure to certain micro-organisms that are "old friends", because of their continuous presence throughout mammalian evolution. These organisms, which include saprophytic mycobacteria, helminths and lactobacilli, are recognised by the innate immune system as harmless, and as adjuvants for Treg induction. Polymorphisms of components of the innate immune system such as TLR2 and NOD2 appear to define subsets of the population that will develop immunoregulatory disorders when living in the modern environment. A further role of the "old friends" and of the Treg that they induce might be to maintain the levels of regulatory IL-10 secreting macrophages and antigen-presenting cells, which are depleted in asthma and Crohn's disease. These concepts are leading to novel therapies based on harmless organisms or their components. Phase I/II clinical trials have yielded some statistically significant results, and phase II trials are in progress.

The effects of Mycobacterium vaccae on allergen-induced airway responses in atopic asthma.

T-helper (Th)2 cytokines play a central role in asthma. Therefore, a double-blind randomised study was conducted to investigate whether heat-killed Mycobacterium vaccae (SRL172), a potent downregulator of Th2 cytokines, can reduce allergen-induced airway responses in patients with atopic asthma. A total 24 male asthmatics participated in this study. A bronchial allergen challenge was performed along with early (EAR) and late asthmatic responses (LAR) 2 weeks before and 3 weeks after a single intradermal injection of SRL172 or placebo. Before and after treatment, serum immunoglobulin (Ig)E levels and in vitro production of interleukin (IL)-5 by peripheral blood lymphocytes were studied. Neither treatment affected the EAR. SRL172 caused a mean 34% reduction of the area under the curve of the forced expiratory volume in one second (FEV1) changes during the LAR, which failed to reach conventional statistical significance when compared with placebo. SRL172 also caused a mean 25% decrease in the maximum fall in FEV1 during LAR, but this was not significantly different from placebo. SRL172 caused a reduction in serum IgE and IL-5 synthesis in vitro 3 weeks post-treatment (p = 0.07). This study shows a trend toward significance for the effects of heat-killed Mycobacterium vaccae (SRL172) on allergen-induced airway responses. Further clinical trials, involving multiple dosing, are needed.

Immunological and pathological comparative analysis between experimental latent tuberculous infection and progressive pulmonary tuberculosis.

Mycobacterium tuberculosis produces latent infection or progressive disease. Indeed, latent infection is more common since it occurs in one-third of the world's population. We showed previously, using human material with latent tuberculosis, that mycobacterial DNA can be detected by in situ PCR in a variety of cell types in histologically-normal lung. We therefore sought to establish an experimental model in which this phenomenon could be studied in detail. We report here the establishment of such a model in C57Bl/6 x DBA/2 F1 hybrid mice by the intratracheal injection of low numbers of virulent mycobacteria (4000). Latent infection was characterized by low and stable bacillary counts without death of animals. Histological and immunological study showed granulomas and small patches of alveolitis, with high expression of tumour necrosis factor alpha (TNFalpha), inducible nitiric oxide synthase (iNOS), interleukin 2 (IL-2) and interferon gamma (IFNgamma). In contrast, the intratracheal instillation of high numbers of bacteria (1 x 106) produced progressive disease. These animals started to die after 2 months of infection, with very high bacillary loads, massive pneumonia, falling expression of TNF-alpha and iNOS, and a mixed Th1/Th2 cytokine pattern. In situ PCR to detect mycobacterial DNA revealed that the most common positive cells in latently-infected mice were alveolar and interstitial macrophages located in tuberculous lesions, but, as in latently-infected human lung, positive signals were also seen in bronchial epithelium, endothelial cells and fibroblasts from histologically-normal areas. Our results suggest that latent tuberculosis is induced and maintained by a type 1 cytokine pattern plus TNFalpha, and that mycobacteria persist intracellularly in lung tissue with and without histological evidence of a local immune response.

Prevention of experimental myointimal hyperplasia by immunomodulation.

INTRODUCTION: we have tested the hypothesis that treatment with a mycobacterial preparation that modulates the antibody response, would diminish restenosis in a rat angioplasty model. MATERIALS/METHODS: male Sprague-Dawley rats were used. All immunisations were given subcutaneously. Group A (control) received normal saline on days 0, 21, and 42. Group B received SRL172 on days 0, 21, and 42. Group C received SRL172 on days 0, 21, and 42, and hsp65/Incomplete Freund's on days 21 and 42. Group D received hsp65/Freund's on days 21 and 42. Right common carotid arteries were balloon-injured on day 63 using a standard technique known to produce MIH and animals were sacrificed on day 77. For each carotid 6 microm cross sections were cut from paraffin blocks. Cross-sectional areas were measured by computerised planimetry. RESULTS: balloon injury resulted in MIH in all animals. Data represents mean+/-SEM for the percentage of area enclosed within the internal elastic lamina occupied by MIH (% MIH); which for groups A, B, C, and D was 85+/-11, 24+/-3, 27+/-7, and 17+/-3 respectively. All the treatment groups had significantly less MIH when compared to the control group but no statistically significant difference was found between any of the treatment groups. CONCLUSIONS: this is the first report that immunomodulation with mycobacterial material suitable for use in man, can reduce MIH. Since such modulation has low risk, this raises the prospect of an important new therapeutic modality to combat restenosis.

Interleukin-4 and its alternatively spliced variant (IL-4delta2) in patients with atopic asthma.

The interleukin-4 (IL-4) splice variant (IL-4delta2) is known to antagonize many biological activities of IL-4, and this challenges our understanding of the role of IL-4 in asthma. Studies that have used nonspecific antibodies, probes, and/or primers to quantify IL-4 in clinical samples would not have distinguished the expression of IL-4 from IL-4delta2. This is the first study to examine patients with chronic asthma and atopy for IL-4delta2 mRNA in their peripheral blood mononuclear cells without antigen stimulation, using a quantitative nested reverse-transcription polymerase chain reaction (RT-PCR) protocol. The median IL-4 mRNA copy number in cells from the patients with asthma was 2.8 logs higher than in a comparator group of patients with tuberculosis (p = 0.0005) and 4.5 logs higher (p = 0.0004) than in healthy control subjects. In contrast, IL-4delta2 expression in cells from patients with asthma was similar to that seen in cells from patients with tuberculosis. Hence, the median ratio of IL-4 to IL-4delta2 was 500-fold higher in the patients with asthma when compared with either patients with tuberculosis or healthy control subjects. The relative expression of IL-4 and IL-4delta2 may be a reason for the functional diversity of Th2 cells in different clinical conditions, and a hitherto unexplored mechanism for the pulmonary pathology in patients with atopic asthma.