Michael T Bailey, PhD - US grants

The NRSA will facilitate my training in the behavioral neurosciences by supporting research on the role of neuroimmune processes in the adaptive response to stressful situations. Specifically, the studies will investigate how individual differences in sympathetic nervous system (SNS) activity contribute to variation in the functioning of the gastrointestinal (gi) system and its vulnerability to infection during stress. Increased SNS activity may lead to disruption of gi homeostasis and subsequent enteric infection by disrupting the protective microflora of the gut, creating a permissive environment for colonization by pathogenic bacteria. These questions will be assessed in infant rhesus monkeys with different behavioral and emotional temperaments, previously shown to be associated with distinct neurochemical profiles. The proposal will test whether gut flora are associated with temperamental attributes, such as emotionally reactive/behaviorally inhibited, in both the baseline and aroused by assessing these monkeys while the mother and subsequent to removal from her at 6 months of age. Finally, I will investigate whether alterations in gi microflora translate into an increased susceptibility to enteric pathogens, such as Shigella, and thus lead to a maladative response to psychological stress. Beyond the specific goals of the project,, this NRSA will allow me to further my graduate training in psychobiological techniques leading to a Ph.D. in Behavioral Neuroscience through the Department of Psychology at the University of Wisconsin.

[unreadable] DESCRIPTION (provided by applicant): Psychological stress is often thought to be immunosuppressive, but it is now known that in some cases stress can enhance certain aspects of the immune response. Despite this knowledge, relatively few studies have focused on stress-induced immunoenhancement. Therefore, the overarching goal of this grant proposal is to identify how stress enhances innate resistance to infectious disease. Social stressors in mice involve physical interactions, making them unique from other types of stressors. The social stressor called social disruption (SDR) has the added characteristic of changing social order and dominance in group-living mice. We have determined that these interactions enhance certain aspects of the immune response. For example, SDR results in the trafficking of CD11b+ myeloid cells from the bone marrow to the spleen, where they are resistant to the suppressive effects of corticosterone and produce significantly higher levels of pro-inflammatory cytokines upon in vitro stimulation with the toll-like receptor (TLR) 4 agonists lipopolysaccharide and lipid A. SDR also increases the capacity of splenic CD11b+ macrophages to kill Escherichia coli in culture. This stress-induced enhancement was also evident in vivo; mice exposed to SDR prior to infection with E. coli cleared the bacteria from the blood and spleen more rapidly than did non-stressed control mice. Because signaling through TLR4 heavily influences the microbicidal activities of macrophages, and because the effects of SDR on CD11b+ cells are dependent upon TLR stimulation, this is an ideal model system to test the hypothesis that social stress enhances resistance to microbial infection by altering TLR4 functioning on CD11b+ cells. Three specific aims have been developed to: 1) test whether SDR enhances TLR4 expression and/or sensitivity on splenic CD11b+ cells, using microbicidal reactive oxygen intermediates (ROI) and nitric oxide (NO) as outcome variables, 2) examine whether this increased reactivity is dependent upon mitogen activated protein (MAP) kinases p38, JNK, and ERK1/2, which can enhance microbicidal activity 3) examine the importance of SDR-enhanced TLR4 and MAP kinase signaling for clearing a bacterial infection. This interdisciplinary approach will provide valuable insight into the complex host-pathogen relationship during quiescent and stressful periods. Defining the stress-induced cellular changes that enhance immune functioning could stimulate new therapeutic approaches to treating infectious diseases. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The inflammatory bowel diseases (IBD, including Crohn's disease and ulcerative colitis) involve disrupted homeostatic interactions between the microbiota and the mucosal immune system as a result of multiple genetic and environmental factors. A major environmental factor that is well recognized to increase the severity of IBD is psychosocial stress. However, the biological mechanisms linking stress to disrupted homeostatic relationships between the microbiota and the mucosal immune system are not well understood. This proposal will test the novel hypothesis that intestinal epithelial cells represent a mechanistic link between stress, alterations of the gut microbiota, and pathogen-induced colitis. Our preliminary data demonstrate that exposing mice to a well characterized and widely used social stressor, called social disruption (SDR) reduces the abundance of commensal Lactobacillus reuteri in the colon. Upon oral challenge with the murine colonic pathogen Citrobacter rodentium, which induces colonic histopathology with similarities to human IBD, mice exposed to SDR (and thus having lower levels of L. reuteri) had a significant increase in pathogen-induced colitis as indicated by a significant increase in colonic histopathology, chemokines (e.g., CCL2), cytokines (e.g., TNF-?), effector molecules (e.g., iNOS), and macrophage infiltration. Importantly, preventing the stressor-induced reduction in L. reuteri by feeding L. reuteri to the mice during stressor exposure abrogated the effects of the stressor on C. rodentium-induced colitis. Like its human homologue (i.e., enteropathogenic E. coli), C. rodentium induces colonic inflammation by colonizing the colonic epithelium. Thus, the use of C. rodentium is an ideal model to determine whether the colonic epithelium is critical in the link between stress, the microbiota, and exacerbation of colitis. This proposal will test the novel hypothesis that when L. reuteri are decreased due to stressor exposure, colonic epithelial cells overproduce chemokines (particularly CCL2) that increase the recruitment of inflammatory macrophages to the colon where they ultimately exacerbate colitis. The first aim will examine the role of CCL2 as a novel primary mechanism (pathway) mediating the effects of stress on colonic inflammation, by assessing the effects of stress-induced alterations of L. reuteri on CCL2 production by colonic epithelial cells and by using CCL2 knockout mice. In the second aim, we will use adoptive transfer experiments to test whether peripheral inflammatory monocytes, which we show are increased in the circulation of stressed mice, traffic to the colon in response to the elevated CCL2, where they can exacerbate colitis through an overproduction of TNF-? and iNOS. Finally, in the third aim, we will determine whether the ability of L. reuteri to affect chemokine production by colonic epithelial cells is dependent upon L. reuteri colonizing the colon, and/or due to the production of immunomodulatory factor(s). This proposal will identify novel mechanism(s) that may lead to new therapeutic targets in the treatment of IBD. PUBLIC HEALTH RELEVANCE: The stress response exacerbates colitis, both in experimental animals and in patients with inflammatory bowel disease, but the biological mechanisms by which this occurs are not well understood. Our studies demonstrate that stressor-induced reduction in commensal Lactobacillus reuteri is involved with the observed increase in colonic histopathology in mice challenged with Citrobacter rodentium during exposure to a well characterized and widely used social stressor. This proposal will test the novel hypothesis that the stressor-induced reduction in L. reuteri leads to an overproduction of chemokines, particularly CCL2, and cytokines by colonic epithelial cells, ultimately resulting in the recruitment of TNF-?-producing inflammatory macrophages that exacerbate colitis.

DESCRIPTION (provided by applicant): Chronic inflammatory diseases affect millions of Americans each year, and have a significant medical, psychosocial, and economic impact on both the patient and on society. Multiple sclerosis is a chronic, degenerative neurological disorder involving immune-mediated inflammatory demyelinating processes, and is significantly exacerbated by comorbid conditions, such as stress and anxiety. Despite this knowledge, the mechanisms by which this occurs are not yet well understood. The studies in this proposal will test the highly novel and integrative hypothesis that the intestinal microbiota are involved in stressor-induced enhancement of systemic inflammation that leads to symptom exacerbation in an animal model of multiple sclerosis. We have made the exciting discovery that the intestinal microbiota are necessary for stressor-induced increases in splenic IL-1? to occur. This is important, because IL-1 plays a central role in the development of many chronic inflammatory diseases, including multiple sclerosis. How the microbiota lead to increased IL-1 during stressor exposure, as well as the effects on chronic inflammatory diseases, is not well understood. During repeated social defeat, commensal microbes can translocate from their primary niche to the interior of the body. Because there is accumulating evidence that neuroendocrine hormones, such as sympathetic nervous system-derived catecholamine hormones, can impact microbial populations, Aim 1 will test whether stressor-induced activation of the sympathetic nervous system leads to translocation of commensal microbiota. As further confirmation that microbial translocation is necessary for stressor-induced immunoenhancement to occur, Aim 2 will test the hypothesis that stressor-induced increases in splenic IL-1? are dependent upon both macrophage pattern recognition receptor signaling and inflammasome formation. Finally, to determine whether the microbiota contribute to stressor-induced exacerbation of a chronic disease, a widely used animal model of multiple sclerosis, namely experimental autoimmune encephalomyelitis (EAE) will be employed. The effects of repeated social defeat on EAE progression will be determined in germfree mice (i.e., mice that have never come into contact with commensal microbes) and conventional mice. By integrating the results of Aims 1, 2, and 3, we will identify novel mechanisms by which stressor exposure can exacerbate a chronic inflammatory disease. These findings would ultimately facilitate the rational design and use of microbiota-targeting therapeutics to treat chronic disease.

? DESCRIPTION (provided by applicant): The pathophysiological mechanisms leading to anxiety and depression are not understood. The prevalence of these disorders is increasing, imposing a significant burden on our health care. Accumulating data suggest that gut microbiota affects the function of the brain and its chemistry. It is likely that multiple mechanisms are involved, including bacterial production of neuroactive molecules or activation of immune pathways. We will seek to study the mechanisms, by which gut bacteria lead to development of anxiety and depression using gnotobiotic murine models. In the R21 phase (Aim 1) we will develop a gnotobiotic model of anxiety and depression by colonizing germ- free mice with stool microbiota from patients with Generalized Anxiety Disorder (GAD) and Major Depressive Disorder (MDD), using samples from our clinical bio bank at McMaster University. We will characterize mouse behavior, microbiota and metabolomic profiles using 16S DNA-based Illumina sequencing and liquid chromatography-mass spectrometry (LC-MS), respectively. As stress has been shown to play a role in depression and anxiety, we will apply a psychosocial stressor to a group of gnotobiotic mice with signs of immune activation. We hypothesize that bacteria communicate with the brain through both neural and immune pathways, which include neuroactive trace amines and inflammatory cytokines, such as IL-1. These specific pathways will be the focus of our research in the R33 phase. Using selected set of microbiota identified in Aim1, we will colonize additional groups of germ free mice and study in detail the underlying mechanisms. The mouse behavior will be correlated with gut, serum and brain neurotransmitters, as well as neuroactive and immunomodulatory metabolites of bacterial origin. We will assess neuronal activation in specific brain areas by immunohistochemistry. We will use pharmacological tools and genetically modified mice to study the exact pathways of microbiota-brain communications. Taking advantage of our novel approach, which enables to culture >92% of human gut bacteria, we will identify and isolate the bacteria associated with anxiety and/or depression-like behavior. To confirm the cause-effect relationship, we will mono or poly-colonize additional groups of germ-free mice with specific bacteria, or group of bacteria linked to anxiety and/or depression, and assess mouse recipient behavior and brain chemistry.

Despite decades of research, the morbidity and mortality of necrotizing enterocolitis (NEC) remain unchanged. An altered intestinal microbiome and intestinal inflammation may predispose prematures to NEC. Probiotics may protect the intestines from NEC, however, they have to be delivered in high numbers with repeated dosing for any effect. We have developed a novel, tunable delivery system in which Lactobacillus reuteri (Lr) administered in a protective biofilm on biocompatable microspheres provides persistent probiotic benefits from just a single dose, greatly reducing experimental NEC. Our long-term goal is to identify novel strategies to protect neonates from NEC. The current overall objective is to identify a novel probiotic-based therapy protective against experimental NEC. Our central hypothesis is that administering Lr in a biofilm state with biocompatible microspheres that can be loaded with beneficial luminal cargo significantly improves its ability to colonize the intestines, reduce intestinal inflammation, and decrease the incidence of NEC. The rationale is that identification of an optimal probiotic delivery system will lead to maximally beneficial treatment of NEC. Elucidating the attributes of Lr that prevent NEC will also provide insight into factors essential for NEC development. We will objectively test our central hypothesis by pursuing the following specific aims: 1) To determine the impact of our Lr formulation in protection of the intestines from NEC. 2) Tuning the beneficial activities (biofilm formation, antimicrobial and anti-inflammatory effects) of Lr to optimize our therapeutic formulation. 3) To determine whether tuning the properties of Lr alters its ability to protect the intestines from NEC. Expected outcomes include identification of the most effective Lr delivery system to reduce NEC, and determination of the relative importance of anti-inflammatory and anti-microbial effects of Lr in protection from NEC. The significance is a better understanding of the effects of probiotics on protection of intestines, allowing the best design of clinical probiotic-based therapies for NEC. This will have a positive impact in terms of providing improved therapeutic interventions for patients at risk of developing NEC, in addition to fundamentally advancing our understanding of the mechanisms by which Lr exerts its beneficial intestinal effects. This research is innovative because it involves a novel probiotic formulation that: (i) requires only a single dose for beneficial effects on NEC and (ii) is a platform by which mechanisms of probiosis can be studied through targeted delivery of prebiotic substrates in well defined microspheres.

PROJECT SUMMARY Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants. Approximately 10% of infants born under 1500 g will develop the disease, and mortality for affected infants is 20-30%. Despite decades of research, the morbidity and mortality of the disease remain generally unchanged. Current treatment and preventive approaches for NEC remain suboptimal, with no FDA-approved therapies for the indication. NEC is the leading cause of death from gastrointestinal disease in premature infants, with an associated cost of $500 million to $1 billion annually for treatment in the US alone. Given the morbidity and mortality associated with the disease and the resulting economic burden, novel approaches for the prevention of NEC are critically needed. Probiotics hold enormous potential for promoting human health and treating disease, including NEC. Current probiotic approaches are inadequate due to the lack of persistence in the gut which necessitates multiple administrations of the probiotic and results in limited efficacy. This creates a safety concern for this vulnerable neonatal population since probiotic administration has been linked to bacteremia. Concern for potential sepsis is one of the major factors limiting widespread use of probiotics by neonatologists to prevent NEC. Scioto Biosciences and their research partners at the Research Institute at Nationwide Children?s Hospital (RINCH) are developing a novel probiotic approach, SB-121, that allows for persistent probiotic benefit from just a single dose well in advance of NEC and the corresponding changes in intestinal permeability, thus dramatically reducing the risk of bacteremia. SB-121 is a biofilm of Lactobacillus reuteri, a persistent community of bacteria, adhered to porous microspheres enabling increased probiotic stability. In a successful Phase I program, Scioto Biosciences met all Phase I milestones resulting in: 1) the production of a stable SB-121 prototype that supports clinical use of the platform; 2) the in vivo demonstration of the effectiveness of SB-121 in preventing NEC; and 3) the identification of optimal SB-121 dosing. Completion of the Phase I program was critical to supporting the proposed Phase II program that will focus on the execution of studies in neonatal pigs. This will inform clinical studies as neonatal pigs are similar in size and intestinal maturity to premature humans. Phase II efforts will include the characterization of the impact of SB-121 on the microbiome and metabolome of healthy full-term and premature pigs (Aim 1) and the assessment of the ability of SB-121 to prevent NEC (Aim 2). Successful execution of the Phase II program will provide critical dose, safety, and efficacy data packages that will be instrumental to the design and execution of a subsequent IND-enabling program, as well as clinical trials supporting the clinical use of SB-121 for the prevention of NEC.