Gut Microbial and Metabolic Profiling Reveal the Lingering Effects of Infantile Iron Deficiency Unless Treated with Iron.

SCOPE: Iron deficiency (ID) compromises the health of infants worldwide. Although readily treated with iron, concerns remain about the persistence of some effects. Metabolic and gut microbial consequences of infantile ID were investigated in juvenile monkeys after natural recovery (pID) from iron deficiency or post-treatment with iron dextran and B vitamins (pID+Fe). METHODS AND RESULTS: Metabolomic profiling of urine and plasma is conducted with 1 H nuclear magnetic resonance (NMR) spectroscopy. Gut microbiota are characterized from rectal swabs by amplicon sequencing of the 16S rRNA gene. Urinary metabolic profiles of pID monkeys significantly differed from pID+Fe and continuously iron-sufficient controls (IS) with higher maltose and lower amounts of microbial-derived metabolites. Persistent differences in energy metabolism are apparent from the plasma metabolic phenotypes with greater reliance on anaerobic glycolysis in pID monkeys. Microbial profiling indicated higher abundances of Methanobrevibacter, Lachnobacterium, and Ruminococcus in pID monkeys and any history of ID resulted in a lower Prevotella abundance compared to the IS controls. CONCLUSIONS: Lingering metabolic and microbial effects are found after natural recovery from ID. These long-term biochemical derangements are not present in the pID+Fe animals emphasizing the importance of the early detection and treatment of early-life ID to ameliorate its chronic metabolic effects.

Symposium review: Microbial endocrinology-Why the integration of microbes, epithelial cells, and neurochemical signals in the digestive tract matters to ruminant health.

The union of microbiology and neurobiology, which has been termed microbial endocrinology, is defined as the study of the ability of microorganisms to produce and respond to neurochemicals that originate either within the microorganisms themselves or within the host they inhabit. It serves as the basis for an evolutionarily derived method of communication between a host and its microbiota. Mechanisms elucidated by microbial endocrinology give new insight into the ways the microbiota can affect host stress, metabolic efficiency, resistance to disease, and other factors that may prove relevant to the dairy industry.

Stressor exposure disrupts commensal microbial populations in the intestines and leads to increased colonization by Citrobacter rodentium.

The gastrointestinal tract is colonized by an enormous array of microbes that are known to have many beneficial effects on the host. Previous studies have indicated that stressor exposure can disrupt the stability of the intestinal microbiota, but the extent of these changes, as well as the effects on enteric infection, has not been well characterized. In order to examine the ability of stressors to induce changes in the gut microbiota, we exposed mice to a prolonged restraint stressor and then characterized microbial populations in the intestines using both traditional culture techniques and bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). Exposure to the stressor led to an overgrowth of facultatively anaerobic microbiota while at the same time significantly reducing microbial richness and diversity in the ceca of stressed mice. Some of these effects could be explained by a stressor-induced reduction in the relative abundance of bacteria in the family Porphyromonadaceae. To determine whether these alterations would lead to increased pathogen colonization, stressed mice, as well as nonstressed controls, were challenged orally with the enteric murine pathogen Citrobacter rodentium. Exposure to the restraint stressor led to a significant increase in C. rodentium colonization over that in nonstressed control mice. The increased colonization was associated with increased tumor necrosis factor alpha (TNF-alpha) gene expression in colonic tissue. Together, these data demonstrate that a prolonged stressor can significantly change the composition of the intestinal microbiota and suggest that this disruption of the microbiota increases susceptibility to an enteric pathogen.

Elucidation of the mechanism by which catecholamine stress hormones liberate iron from the innate immune defense proteins transferrin and lactoferrin.

The ability of catecholamine stress hormones and inotropes to stimulate the growth of infectious bacteria is now well established. A major element of the growth induction process has been shown to involve the catecholamines binding to the high-affinity ferric-iron-binding proteins transferrin (Tf) and lactoferrin, which then enables bacterial acquisition of normally inaccessible sequestered host iron. The nature of the mechanism(s) by which the stress hormones perturb iron binding of these key innate immune defense proteins has not been fully elucidated. The present study employed electron paramagnetic resonance spectroscopy and chemical iron-binding analyses to demonstrate that catecholamine stress hormones form direct complexes with the ferric iron within transferrin and lactoferrin. Moreover, these complexes were shown to result in the reduction of Fe(III) to Fe(II) and the loss of protein-complexed iron. The use of bacterial ferric iron uptake mutants further showed that both the Fe(II) and Fe(III) released from the Tf could be directly used as bacterial nutrient sources. We also analyzed the transferrin-catecholamine interactions in human serum and found that therapeutically relevant concentrations of stress hormones and inotropes could directly affect the iron binding of serum-transferrin so that the normally highly bacteriostatic tissue fluid became significantly more supportive of the growth of bacteria. The relevance of these catecholamine-transferrin/lactoferrin interactions to the infectious disease process is considered.

Campylobacter jejuni infection increases anxiety-like behavior in the holeboard: possible anatomical substrates for viscerosensory modulation of exploratory behavior.

The presence of certain bacteria in the gastrointestinal tract influences behavior and brain function. For example, challenge with live Campylobacter jejuni (C. jejuni), a common food-born pathogen, reduces exploration of open arms of the plus maze, consistent with anxiety-like behavior, and activates brain regions associated with autonomic function, likely via a vagal pathway. As yet, however, little is known regarding the interface of immune sensory signals with brain substrates that mediate changes in behavioral states. To address this issue, we challenged mice with either C. jejuni or saline, and 7-8h later assessed anxiety-like behavior using the open holeboard, and used immunohistochemical detection of the protein c-Fos as an activation marker in the brain. C. jejuni treatment was associated with increased avoidance of the center regions of the holeboard, compared to saline-treated controls. Exposure to the holeboard induced activation in multiple brain regions previously implicated in anxiety-like behavior, including the lateral septum (LS), paraventricular (PVN) and dorsomedial hypothalamic nuclei (DMH), basolateral and central nuclei of the amygdala (BLA, CEA), bed nucleus of the stria terminalis (BST) and periaquiductal grey (PAG), compared to homecage controls. In C. jejuni-treated animals c-Fos induction also occurred in autonomic regions, as previously reported. The PVN, BLA, parts of the BST, medial prefrontal (mPFC) and anterior cingulate responded to both C. jejuni treatment and the holeboard, suggesting a role for these regions in the enhanced anxiety-like behavior observed. In saline-treated animals, anxiety-like behavior was predicted by activation in the CEA and BLA, whereas in C. jejuni-treated animals, c-Fos expression in the BST predicted the degree of anxiety-like behavior. These findings implicate the PVN, amygdala and BST as interfaces between gastrointestinal pathogenic challenge and brain regions that mediate behavioral responses to stress, and reinforce these nuclei as anatomical substrates by which viscerosensory stimuli can influence behavior.

Induction of anxiety-like behavior in mice during the initial stages of infection with the agent of murine colonic hyperplasia Citrobacter rodentium.

Symptoms of anxiety frequently occur concomitant to the development and persistence of inflammatory bowel disease (IBD) in patients. In the present study, we utilized an animal model of IBD, infection with Citrobacter rodentium, to determine whether the infection per se can drive anxiety-like behavior. Nine-week-old CF-1 male mice were challenged orally with either saline or C. rodentium. Early in the infective process (7-8 h later), mice were tested on a hole-board open field apparatus for anxiety-like behavior measurement. Immediately following behavioral testing, plasma samples were obtained for immune cytokine analysis and colons were excised for histological analysis. In additional animals, vagal ganglia were removed and processed for c-Fos protein detection. Challenge with C. rodentium significantly increased anxiety-like behavior as evidenced by avoidance of the center area and increased risk assessment behavior. Plasma levels of the cytokines IFN-gamma, TNF-alpha and IL-12 were not different. However vagal sensory ganglia from C. rodentium-treated animals evinced significantly more c-Fos protein-positive neurons, consistent with vagal afferent transmission of C. rodentium-related signals from gut to brain. Histological examination of the colon indicated a lack of overt inflammation at the 8 h post-challenge time point, indicating that the differences in behavior were unlikely to follow from inflammation-related stress. The results of the present study demonstrate that infection with C. rodentium can induce anxiety-like symptoms that are likely mediated via vagal sensory neurons.

Activation in vagal afferents and central autonomic pathways: early responses to intestinal infection with Campylobacter jejuni.

Abundant evidence now supports the idea that multiple pathways or mechanisms underlie communication from the immune system to the brain. The presence of a variety of mechanisms suggests that they may each contribute something different to immunosensory signaling. For instance, brain mediated immune signal transduction is dependent upon the presence of circulating mediators whereas peripheral sensory nerves are more likely to be important early on in an infection, prior to elevation of circulating cytokines, or in local infections within the terminal fields of these nerves. To test the hypothesis that local infection in the gut activates vagal sensory neurons, we assessed expression of the neuronal activation marker c-Fos in neurons in the vagal sensory ganglia and in the primary sensory relay nucleus for the vagus, the nucleus of the solitary tract (nTS) in mice treated orally either with saline or live Campylobacter jejuni (C. jejuni). Male CF1 mice were inoculated orally with either C. jejuni or saline, and c-Fos expression in the vagal sensory neurons and brain 4-12 h later was assessed via immunohistochemistry. Oral inoculation with C. jejuni led to a significant increase in c-Fos expression in neurons bilaterally in the vagal ganglia, in the absence of elevated levels of circulating pro-inflammatory cytokines. C. jejuni treatment activated neurons in the nTS, as well as in brain regions associated with primary viscerosensory pathways and the central autonomic network. These findings provide evidence that peripheral sensory neurons contribute an early signal to the brain regarding potential pathogens.

Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa.

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.

Brain response to cecal infection with Campylobacter jejuni: analysis with Fos immunohistochemistry.

Infections with bacterial pathogens can induce increased anxiety-like behaviors in rodents without otherwise noticeable behavioral or physiological symptoms of sickness, as shown with the food-borne pathogen Campylobacter jejuni. This observation implicates the ability of the brain to sense, and respond to, such an infection. We tested our hypothesis that intestinal infection with the gram-negative bacterium C. jejuni leads to activation of certain brain regions that process gastro-intestinal sensory information. The induction of c-Fos protein as a marker for neuronal activation was assessed in the brains of mice inoculated orally with live C. jejuni, as compared to saline-treated controls. Upon colonization of the intestines, C. jejuni activated visceral sensory nuclei in the brainstem (the nucleus of the solitary tract and the lateral parabrachial nucleus) both one and two days after the oral challenge. In addition, increased c-Fos expression occurred in the hypothalamic paraventricular nucleus on the second day. This neural response occurred in the absence of measurable systemic immune activation, as serum levels of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 were undetectable and/or unchanged. These findings support the notion that information about infection with C. jejuni in the gut is indeed relayed to the visceral sensory structures in the brain. The brain responses observed could contribute to changes in behavior observed after infection.

Involvement of enterobactin in norepinephrine-mediated iron supply from transferrin to enterohaemorrhagic Escherichia coli.

Exposure of bacteria to members of the stress-associated family of catecholamine hormones, principally norepinephrine, has been demonstrated to increase both growth and production of virulence-related factors. Mutation of genes for enterobactin synthesis and uptake revealed an absolute requirement for enterobactin in norepinephrine-stimulated growth of Escherichia coli O157:H7. The autoinducer produced by norepinephrine-stimulated E. coli could not substitute for enterobactin. We also demonstrate that norepinephrine promotes iron shuttling between transferrin molecules, thereby enabling the bacterial siderophore enterobactin to more readily acquire iron for growth. These results suggest one of the possible mechanisms by which the hormonal output of stress may affect enterohaemorrhagic E. coli pathogenicity.

Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia.

The incidence of nosocomial pneumonia (NP) among injured patients is substantial. We hypothesize that traumatic injury induces alterations in local organ effector cell function that may predispose the lungs of injured patients to infection. It is the objective of this study to compare the systemic and alveolar effector cell response to injury and assess the relationship these have to the development of NP. Peripheral blood and bronchoalveolar lavage fluid (BAL) were collected from 10 elective surgery patients (controls) and 16 multitrauma patients at 12, 36, and 60 h post-injury. Systemic and alveolar levels of IL-8 and IL-10 were measured. CD11b expression on peripheral blood neutrophils (PBN) and alveolar neutrophils (AN) and HLA-DR expression on peripheral blood monocytes (PBM) and alveolar macrophages (AM) were measured. Alveolar levels of IL-8 and IL-10 were significantly higher than systemic levels after injury. HLA-DR expression was reduced on both PBM and AM after injury but was lowest on the AM. Six of 16 patients (38%) developed NP (NP+). There were no differences in cytokine levels (IL-8 and IL-10) or effector cell phenotype (CD11b and HLA-DR expression) within the systemic circulation of NP+ and NP- patients. In contrast, NP+ and NP- patients differed significantly in alveolar cytokine levels and alveolar effector cell phenotype. IL-8 was significantly higher in BAL form NP+ patients at all time points after injury. Furthermore, alveolar levels of IL-10 decreased in NP- patients but increased in NP+ patients. In all patients, AM HLA-DR expression was significantly reduced from normal controls 12 h post-injury. In NP-patients, AM HLA-DR expression returned to normal 60 h post-injury, whereas in NP+ patients, expression remained suppressed. These findings identify distinct trends in local organ cytokine production and alterations in effector cell phenotype that precede NP. The persistence of reduced HLA-DR expression amidst increasing levels of IL-10 in NP+ patients suggest that cell-mediated immune function is being suppressed. As such, local organ immunosuppression may be responsible for the development of nosocomial pneumonia in injured patients.

Systemic and pulmonary effector cell function after injury.

OBJECTIVE: Traumatic injury initiates a complex inflammatory response that is associated with end-organ dysfunction, immunosuppression, and the development of nosocomial infection. We hypothesize that the lungs of injured patients experience a unique inflammatory response to traumatic injury in which the ability of alveolar effector cells to respond to a bacterial challenge is impaired. DESIGN: Prospective, longitudinal comparative study. SETTING: The surgical intensive care unit of an ACS level I trauma center. PATIENTS: Forty consecutive multiple trauma patients requiring mechanical ventilation. MEASUREMENT: Blood and bronchoalveolar lavage fluid were collected on admission, 24, and 48 hrs postinjury. Interleukin (IL)-6, IL-8, and IL-10 were measured in each sample initially and after lipopolysaccharide stimulation by using an ex vivo model of whole blood and bronchoalveolar lavage fluid cellular contents. Five patients who underwent elective surgery formed a control group. MAIN RESULTS: Systemic and alveolar levels of IL-6, IL-8, and IL-10 increase dramatically after severe injury. Levels of IL-6 and IL-8 in trauma bronchoalveolar lavage fluid are significantly greater than those of the systemic circulation. Whereas whole blood up-regulates production of IL-6 and IL-8 in response to lipopolysaccharide, bronchoalveolar lavage fluid cellular contents do not. In contrast, bronchoalveolar lavage fluid and whole blood from injured patients contain similar amounts of IL-10 and both up-regulate IL-10 production in response to lipopolysaccharide. CONCLUSION: The lungs of injured patients experience a profound proinflammatory response to injury more severe than that of the systemic circulation. Within this setting, the ability of alveolar effector cells to respond to a bacterial challenge is diminished compared with that of systemic cells. As such, alveolar effector cell function after injury seems to be impaired, possibly explaining the high frequency of pulmonary infection among these patients.