The Microbiome Mediates Environmental Effects on Aging.

Humans' indigenous microbes strongly influence organ functions in an age- and diet-dependent manner, adding an important dimension to aging biology that remains poorly understood. Although age-related differences in the gut microbiota composition correlate with age-related loss of organ function and diseases, including inflammation and frailty, variation exists among the elderly, especially centenarians and people living in areas of extreme longevity. Studies using short-lived as well as nonsenescent model organisms provide surprising functional insights into factors affecting aging and implicate attenuating effects of microbes as well as a crucial role for certain transcription factors like forkhead box O. The unexpected beneficial effects of microbes on aged animals imply an even more complex interplay between the gut microbiome and the host. The microbiome constitutes the major interface between humans and the environment, is influenced by biosocial stressors and behaviors, and mediates effects on health and aging processes, while being moderated by sex and developmental stages.

The gut microbiome in human neurological disease: A review.

Almost half the cells and 1% of the unique genes found in our bodies are human, the rest are from microbes, predominantly bacteria, archaea, fungi, and viruses. These microorganisms collectively form the human microbiota, with most colonizing the gut. Recent technological advances, open access data libraries, and application of high-throughput sequencing have allowed these microbes to be identified and their contribution to neurological health to be examined. Emerging evidence links perturbations in the gut microbiota to neurological disease, including disease risk, activity, and progression. This review provides an overview of the recent advances in microbiome research in relation to neuro(auto)immune and neurodegenerative conditions affecting humans, such as multiple sclerosis, neuromyelitis optica spectrum disorders, Parkinson disease, Alzheimer disease, Huntington disease, and amyotrophic lateral sclerosis. Study design and terminology used in this rapidly evolving, highly multidisciplinary field are summarized to empower and engage the neurology community in this "newly discovered organ." Ann Neurol 2017;81:369-382.

Early life antibiotic-driven changes in microbiota enhance susceptibility to allergic asthma.

Allergic asthma rates have increased steadily in developed countries, arguing for an environmental aetiology. To assess the influence of gut microbiota on experimental murine allergic asthma, we treated neonatal mice with clinical doses of two widely used antibiotics--streptomycin and vancomycin--and evaluated resulting shifts in resident flora and subsequent susceptibility to allergic asthma. Streptomycin treatment had little effect on the microbiota and on disease, whereas vancomycin reduced microbial diversity, shifted the composition of the bacterial population and enhanced disease severity. Neither antibiotic had a significant effect when administered to adult mice. Consistent with the 'hygiene hypothesis', our data support a neonatal, microbiota-driven, specific increase in susceptibility to experimental murine allergic asthma.

Babies, bugs and brains: How the early microbiome associates with infant brain and behavior development.

Growing evidence is demonstrating the connection between the microbiota gut-brain axis and neurodevelopment. Microbiota colonization occurs before the maturation of many neural systems and is linked to brain health. Because of this it has been hypothesized that the early microbiome interactions along the gut-brain axis evolved to promote advanced cognitive functions and behaviors. Here, we performed a pilot study with a multidisciplinary approach to test if the microbiota composition of infants is associated with measures of early cognitive development, in particular neural rhythm tracking; language (forward speech) versus non-language (backwards speech) discrimination; and social joint attention. Fecal samples were collected from 56 infants between four and six months of age and sequenced by shotgun metagenomic sequencing. Of these, 44 performed the behavioral Point and Gaze test to measure joint attention. Infants were tested on either language discrimination using functional near-infrared spectroscopy (fNIRS; 25 infants had usable data) or neural rhythm tracking using electroencephalogram (EEG; 15 had usable data). Infants who succeeded at the Point and Gaze test tended to have increased Actinobacteria and reduced Firmicutes at the phylum level; and an increase in Bifidobacterium and Eggerthella along with a reduction in Hungatella and Streptococcus at the genus level. Measurements of neural rhythm tracking associated negatively to the abundance of Bifidobacterium and positively to the abundance of Clostridium and Enterococcus for the bacterial abundances, and associated positively to metabolic pathways that can influence neurodevelopment, including branched chain amino acid biosynthesis and pentose phosphate pathways. No associations were found for the fNIRS language discrimination measurements. Although the tests were underpowered due to the small pilot sample sizes, potential associations were identified between the microbiome and measurements of early cognitive development that are worth exploring further.

The impact of gut microbes in allergic diseases.

PURPOSE OF REVIEW: The prevalence of allergic diseases continues to rise globally in developed countries. Since the initial proposal of the hygiene hypothesis, there has been increasing evidence to suggest that the intestinal microbiota, particularly during early infancy, plays a critical role in regulating immune responses associated with the development of atopy. This review evaluates the key epidemiologic and mechanistic data published to date. RECENT FINDINGS: Epidemiological data have provided the framework for animal studies investigating the importance of gut commensals in allergy development. These studies provide new insights about the microbial regulation of mucosal immune responses inside and outside the gut, and how these effects may drive allergic inflammation in susceptible individuals. Specific immune cells have been identified as mediators of these microbiota-regulated allergic responses. SUMMARY: In the last year, technological advances have provided us with a better understanding of the gut microbiome in healthy and allergic individuals. Recent studies have identified the associations between particular gut microbes and different disease phenotypes, as well as identified immune cells and their mediators involved in allergy development. This research has provided a number of host and microbe targets that may be used to develop novel therapies suitable for the treatment or prevention of allergic diseases.

The human transcriptome during nontyphoid Salmonella and HIV coinfection reveals attenuated NFkappaB-mediated inflammation and persistent cell cycle disruption.

BACKGROUND: Invasive nontyphoid Salmonella (iNTS) disease is common and severe in adults with human immunodeficiency virus (HIV) infection in Africa. We previously observed that ex vivo macrophages from HIV-infected subjects challenged with Salmonella Typhimurium exhibit dysregulated proinflammatory cytokine responses. METHODS: We studied the transcriptional response in whole blood from HIV-positive patients during acute and convalescent iNTS disease compared to other invasive bacterial diseases, and to HIV-positive and -negative controls. RESULTS: During iNTS disease, there was a remarkable lack of a coordinated inflammatory or innate immune signaling response. Few interferon γ (IFNγ)-induced genes or Toll-like receptor/transcription factor nuclear factor κB (TLR/NFκB) gene pathways were upregulated in expression. Ex vivo lipopolysacharide (LPS) or flagellin stimulation of whole blood, however, showed that convalescent iNTS subjects and controls were competent to mount prominent TLR/NFκB-associated patterns of mRNA expression. In contrast, HIV-positive patients with other invasive bacterial infections (Escherichia coli and Streptococcus pneumoniae) displayed a pronounced proinflammatory innate immune transcriptional response. There was also upregulated mRNA expression in cell cycle, DNA replication, translation and repair, and viral replication pathways during iNTS. These patterns persisted for up to 2 months into convalescence. CONCLUSIONS: Attenuation of NFκB-mediated inflammation and dysregulation of cell cycle and DNA-function gene pathway expression are key features of the interplay between iNTS and HIV.

The Gut Mycobiome in Parkinson's Disease.

The gut microbiome has been increasingly implicated in Parkinson's disease (PD); however, most existing studies employ bacterial-specific sequencing, and have not investigated non-bacterial microbiome constituents. Here, we use fungal-specific internal transcribed spacer (ITS)-2 amplicon sequencing in a cross-sectional PD cohort to investigate associations between the fungal gut microbiome and PD. Fungal load among participants was extremely low, and genera identified were almost exclusively of proposed dietary or environmental origin. We observed significantly lower fungal DNA relative to bacterial DNA among PD patients. No fungi differed in abundance between patients and controls, nor were any associated with motor, cognitive, or gastrointestinal features among patients.

Biogeography of the Relationship between the Child Gut Microbiome and Innate Immune System.

The gut microbiome is a well-recognized modulator of host immunity, and its compositions differ between geographically separated human populations. Systemic innate immune responses to microbial derivatives also differ between geographically distinct human populations. However, the potential role of the microbiome in mediating geographically varied immune responses is unexplored. We here applied 16S amplicon sequencing to profile the stool microbiome and, in parallel, measured whole-blood innate immune cytokine responses to several pattern recognition receptor (PRR) agonists among 2-year-old children across biogeographically diverse settings. Microbiomes differed mainly between high- and low-resource environments and were not strongly associated with other demographic factors. We found strong correlations between responses to Toll-like receptor 2 (TLR2) and relative abundances of Bacteroides and Prevotella populations, shared among Canadian and Ecuadorean children. Additional correlations between responses to TLR2 and bacterial populations were specific to individual geographic cohorts. As a proof of concept, we gavaged germfree mice with human donor stools and found murine splenocyte responses to TLR stimulation were consistent with responses of the corresponding human donor populations. This study identified differences in immune responses correlating to gut microbiomes across biogeographically diverse settings and evaluated biological plausibility using a mouse model. This insight paves the way to guide optimization of population-specific interventions aimed to improve child health outcomes.IMPORTANCE Both the gut microbiome and innate immunity are known to differ across biogeographically diverse human populations. The gut microbiome has been shown to directly influence systemic immunity in animal models. With this, modulation of the gut microbiome represents an attractive avenue to improve child health outcomes associated with altered immunity using population-specific approaches. However, there are very scarce data available to determine which members of the gut microbiome are associated with specific immune responses and how these differ around the world, creating a substantial barrier to rationally designing such interventions. This study addressed this knowledge gap by identifying relationships between distinct bacterial taxa and cytokine responses to specific microbial agonists across highly diverse settings. Furthermore, we provide evidence that immunomodulatory effects of region-specific stool microbiomes can be partially recapitulated in germfree mice. This is an important contribution toward improving global child health by targeting the gut microbiome.

Robust TLR4-induced gene expression patterns are not an accurate indicator of human immunity.

BACKGROUND: Activation of Toll-like receptors (TLRs) is widely accepted as an essential event for defence against infection. Many TLRs utilize a common signalling pathway that relies on activation of the kinase IRAK4 and the transcription factor NFkappaB for the rapid expression of immunity genes. METHODS: 21 K DNA microarray technology was used to evaluate LPS-induced (TLR4) gene responses in blood monocytes from a child with an IRAK4-deficiency. In vitro responsiveness to LPS was confirmed by real-time PCR and ELISA and compared to the clinical predisposition of the child and IRAK4-deficient mice to Gram negative infection. RESULTS: We demonstrated that the vast majority of LPS-responsive genes in IRAK4-deficient monocytes were greatly suppressed, an observation that is consistent with the described role for IRAK4 as an essential component of TLR4 signalling. The severely impaired response to LPS, however, is inconsistent with a remarkably low incidence of Gram negative infections observed in this child and other children with IRAK4-deficiency. This unpredicted clinical phenotype was validated by demonstrating that IRAK4-deficient mice had a similar resistance to infection with Gram negative S. typhimurium as wildtype mice. A number of immunity genes, such as chemokines, were expressed at normal levels in human IRAK4-deficient monocytes, indicating that particular IRAK4-independent elements within the repertoire of TLR4-induced responses are expressed. CONCLUSIONS: Sufficient defence to Gram negative immunity does not require IRAK4 or a robust, 'classic' inflammatory and immune response.

Immunosuppressive Treatment Alters Secretion of Ileal Antimicrobial Peptides and Gut Microbiota, and Favors Subsequent Colonization by Uropathogenic Escherichia coli.

BACKGROUND: Transplant recipients are treated with immunosuppressive (IS) therapies, which impact host-microbial interactions. We examined the impact of IS drugs on gut microbiota and on the expression of ileal antimicrobial peptides. METHODS: Mice were treated for 14 days with prednisolone, mycophenolate mofetil, tacrolimus, a combination of these 3 drugs, everolimus, or water. Feces were collected before and after treatment initiation. Ileal samples were collected after sacrifice. Fecal and ileal microbiota were analyzed by pyrosequencing of 16S rRNA genes and enumeration of selected bacteria by culture, and C-type lectins were assessed in ileal tissues by reverse transcriptase-quantitative polymerase chain reaction. RESULTS: Prednisolone disrupted fecal microbiota community structure, decreased Bacteroidetes, and increased Firmicutes in the feces. Prednisolone, tacrolimus, and mycophenolate mofetil modified fecal microbiota at the family level in each experimental replicate, but changes were not consistent between the replicates. In ileal samples, the genus Clostridium sensu stricto was dramatically reduced in the prednisolone and combined IS drug groups. These modifications corresponded to an altered ileal expression of C-type lectins Reg3γ and Reg3ß, and of interleukin 22. Interestingly, the combined IS treatment enabled a commensal Escherichia coli to flourish, and dramatically increased colonization by uropathogenic E. coli strain 536. CONCLUSIONS: IS treatment alters innate antimicrobial defenses and disrupts the gut microbiota, which leads to overgrowth of indigenous E. coli and facilitates colonization by opportunistic pathogens.

An antibiotic-altered microbiota provides fuel for the enteric foe.

Antibiotic therapies disrupt the intestinal microbiota and render the host susceptible to enteric infections. A recent report by Ng et al. explores the ability of two intestinal pathogens (Salmonella enterica serovar Typhimurium and Clostridium difficile) to use this disruption to their advantage and consume host carbohydrates that would otherwise be unavailable in the presence of a normal gut microbiota.

Perinatal antibiotic treatment affects murine microbiota, immune responses and allergic asthma.

There is convincing evidence from recent human and animal studies that suggests the intestinal microbiota plays an important role in regulating immune responses associated with the development of allergic asthma, particularly during early infancy. Although identifying the mechanistic link between host-microbe interactions in the gut and lung mucosal tissues has proved challenging, several very recent studies are now providing significant insights. We have shown that administering vancomycin to mice early in life shifts resident gut flora and enhances future susceptibility to allergic asthma. This effect was not observed in mice given another antibiotic, streptomycin, nor when either antibiotic was administered to adult mice. In this addendum, we further analyze the link between early life administration of vancomycin and future susceptibility to asthma and describe how specific immune cell populations, which have been implicated in other asthma-related microbiota studies, are affected. We propose that shifts in gut microbiota exacerbate asthma-related immune responses when they occur shortly after birth and before weaning (perinatal period), and suggest that these effects may be mediated, at least in the case of vancomycin, by elevated serum IgE and reduced regulatory T cell populations.

The Salmonella enterica PhoP directly activates the horizontally acquired SPI-2 gene sseL and is functionally different from a S. bongori ortholog.

To establish a successful infection within the host, a pathogen must closely regulate multiple virulence traits to ensure their accurate temporal and spatial expression. As a highly adapted intracellular pathogen, Salmonella enterica has acquired during its evolution various virulence genes via numerous lateral transfer events, including the acquisition of the Salmonella Pathogenicity Island 2 (SPI-2) and its associated effectors. Beneficial use of horizontally acquired genes requires that their expression is effectively coordinated with the already existing virulence programs and the regulatory set-up in the bacterium. As an example for such a mechanism, we show here that the ancestral PhoPQ system of Salmonella enterica is able to regulate directly the SPI-2 effector gene sseL (encoding a secreted deubiquitinase) in an SsrB-independent manner and that PhoP plays a part in a feed-forward regulatory loop, which fine-tunes the cellular level of SseL. Additionally, we demonstrate the presence of conserved cis regulatory elements in the promoter region of sseL and show direct binding of purified PhoP to this region. Interestingly, in contrast to the S. enterica PhoP, an ortholog regulator from a S. bongori SARC 12 strain was found to be impaired in promoting transcription of sseL and other genes from the PhoP regulon. These findings have led to the identification of a previously uncharacterized residue in the DNA-binding domain of PhoP, which is required for the transcriptional activation of PhoP regulated genes in Salmonella spp. Collectively our data demonstrate an interesting interface between the acquired SsrB regulon and the ancestral PhoPQ regulatory circuit, provide novel insights into the function of PhoP, and highlight a mechanism of regulatory integration of horizontally acquired genes into the virulence network of Salmonella enterica.

Salmonella SPI-1-mediated neutrophil recruitment during enteric colitis is associated with reduction and alteration in intestinal microbiota.

Gastrointestinal infections involve an interactive tripartite relationship between the invading pathogen, the host, and the host's resident intestinal microbiota. To characterize the host inflammatory response and microbiota alterations during enteric salmonellosis, C57BL/6 mice were pre-treated with a low dose of streptomycin (LD model) and then infected with S. typhimurium strains, including mutants in the two Type III secretion systems, SPI-1 and SPI-2 (invAmut and ssaRmut, respectively). Cecal colonization and inflammation in the LD model were evaluated to assess infection success and progression, and compared to the traditional high dose (HD) model. Perturbations to the microbial community in the LD model were assessed via evaluation of total microbial numbers, the proportion of intestinal γ-Proteobacteria and tRFLP analysis. In the LD model, consistently high colonization by the parental strain (WT) and invAmut S. typhimurium was associated with significant intestinal pathology. However, microbial community profiles were more similar both in numbers and composition between mice infected with the mutant strains, than with the WT strain. Consequently, significant infection-induced inflammation did not always produce similar microbiota perturbations. Large numbers of luminal neutrophils were observed in the ceca of WT-infected, but not in invAmut or ssaRmut infected mice. Neutrophils were thus implicated as a potential mediator of microbiota perturbations during WT enteric salmonellosis. These studies offer a new model of S. typhimurium-induced intestinal disease that retains the three participants of the disease process and further defines the role of virulence factors, the host microbiota, and inflammation in S. typhimurium-induced intestinal disease.

Multiple seropathotypes of verotoxin-producing Escherichia coli (VTEC) disrupt interferon-gamma-induced tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1.

Verotoxin-producing Escherichia coli (VTEC) O157:H7 inhibits interferon-gamma-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1 in epithelial cells, independent of Verotoxins and the locus of enterocyte effacement pathogenicity island. Although E. coli O157:H7 is the major cause of disease in humans, non-O157:H7 VTEC also cause human disease. However, the virulence properties of non-O157:H7 VTEC are less well characterized. The aims of this study were to define the ability of VTEC strains of differing seropathotypes (classified as A-E) to inhibit interferon-gamma stimulated Stat1-phosphorylation and to further characterize the bacterial-derived inhibitory factor. Confluent T84 and HEp-2 cells were infected with VTEC strains (MOI 100:1, 6h, 37 degrees C), and then stimulated with interferon-gamma (50 ng/mL) for 0.5h at 37 degrees C. Whole-cell protein extracts of infected cells were collected and prepared for immunoblotting to detect tyrosine phosphorylation of Stat1. The effects of E. coli O55 strains, the evolutionary precursors of VTEC, on Stat1-tyrosine phosphorylation were also determined. The effects of isogenic mutants of O-islands 47 and 122 were tested to determine the role of genes encoded on these putative pathogenicity islands in mediating VTEC inhibition of the interferon-gamma-Stat1 signaling cascade. To evaluate potential mechanism(s) of inhibition, VTEC O157:H7-infected cells were treated with pharmacological inhibitors, including, wortmannin and LY294002. Relative to uninfected cells, Stat1-tyrosine phosphorylation was significantly reduced after 6h infection of both T84 and HEp-2 cells by VTEC strains of all five seropathotypes. E. coli O55 strains, but not enteropathogenic E. coli (EPEC), also caused inhibition of Stat1-tyrosine phosphorylation, suggesting that this effect was acquired early in the evolution of VTEC. Stat1-activation did not recover in epithelial cells infected with isogenic mutants of O-islands 47 and 122, indicating that the inhibitory factor was not contained in these genomic regions. Stat1-phosphorylation remained intact when VTEC-infected cells were treated with wortmannin (0-100 nM), but not by treatment with the more specific PI3-kinase inhibitor, LY294002. Inhibition of interferon-gamma stimulated Stat1-tyrosine phosphorylation by VTEC of multiple seropathotypes indicates the presence of a common inhibitory factor that is independent of bacterial virulence in humans. The results of treatment with wortmannin suggest that the bacterial-derived inhibitory factor employs host cell signal transduction to mediate inhibition of Stat1-activation.

Modulation of chloride secretory responses and barrier function of intestinal epithelial cells by the Salmonella effector protein SigD.

The Salmonella effector protein SigD is an inositol phosphate phosphatase that inhibits phosphatidylinositol 3-kinase-dependent signaling. Because epidermal growth factor (EGF) inhibits chloride secretion via phosphatidylinositol 3-kinase, we explored whether Salmonella infection might modify the inhibitory effect of EGF. As expected, EGF inhibited chloride secretion induced by carbachol in T(84) epithelial cells. Infection with wild-type (WT) but not sigD(-) mutant S. typhimurium SL1344 decreased CCh-stimulated chloride secretion. Moreover, WT but not sigD(-) Salmonella reduced the inhibitory effect of EGF on carbachol-stimulated chloride secretion. Complementation of sigD restored the ability of mutant Salmonella to reverse the inhibitory effect of EGF. EGF-induced EGF receptor phosphorylation was similar in cells infected with either WT or mutant Salmonella, and neither WT nor sigD(-) Salmonella altered recruitment of the p85 subunit of phosphatidylinositol 3-kinase to EGF receptor, implying that SigD acts downstream of these signaling events. Furthermore, transepithelial resistance fell more rapidly in cells infected with WT vs. sigD(-) Salmonella, indicating an early role for SigD in reducing barrier function, perhaps via activation of protein kinase C. We conclude that the Salmonella bacterial effector protein SigD may play critical roles in the pathogenesis of disease caused by this microorganism.