Genetically engineered Lactococcus lactis strain constitutively expresses GABA-producing genes and produces high levels of GABA.

GABA is an inhibitory neurotransmitter of the central nervous system that impacts physical and mental health. Low GABA levels have been documented in several diseases, including multiple sclerosis and depression, and studies suggest that GABA could improve disease outcomes in those conditions. Probiotic bacteria naturally produce GABA and have been engineered to enhance its synthesis. Strains engineered thus far use inducible expression systems that require the addition of exogenous molecules, which complicates their development as therapeutics. This study aimed to overcome this challenge by engineering Lactococcus lactis with a constitutive GABA synthesis gene cassette. GABA synthesizing and transport genes (gadB and gadC) were cloned onto plasmids downstream of constitutive L. lactis promoters (P2, P5, and P8s) of different strengths and transformed into L. lactis. Fold increase in gadCB expression conferred by these promoters (P2, P5, and P8s) was 322, 422, and 627, respectively, compared to the unmodified strain (P = 0.0325, P8s). GABA synthesis in the highest gadCB expressing strain, L. lactis P8s-GAD, was dependent on media supplementation with glutamic acid and significantly higher than the unmodified strain (P < 0.0001, 125 mM, 200 mM glutamic acid). Lactococcus lactis-P8s-GAD is poised for therapeutic testing in animal models of low-GABA-associated disease.

Gut microbiota in multiple sclerosis and animal models.

Multiple sclerosis (MS) is a chronic central nervous system (CNS) neurodegenerative and neuroinflammatory disease marked by a host immune reaction that targets and destroys the neuronal myelin sheath. MS and correlating animal disease models show comorbidities, including intestinal barrier disruption and alterations of the commensal microbiome. It is accepted that diet plays a crucial role in shaping the microbiota composition and overall gastrointestinal (GI) tract health, suggesting an interplay between nutrition and neuroinflammation via the gut-brain axis. Unfortunately, poor host health and diet lead to microbiota modifications that could lead to significant responses in the host, including inflammation and neurobehavioral changes. Beneficial microbial metabolites are essential for host homeostasis and inflammation control. This review will highlight the importance of the gut microbiota in the context of host inflammatory responses in MS and MS animal models. Additionally, microbial community restoration and how it affects MS and GI barrier integrity will be discussed.

Farnesol brain transcriptomics in CNS inflammatory demyelination.

BACKGROUND: Farnesol (FOL) prevents the onset of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). OBJECTIVE: We examined the transcriptomic profile of the brains of EAE mice treated with daily oral FOL using next-generation sequencing (RNA-seq). METHODS: Transcriptomics from whole brains of treated and untreated EAE mice at the peak of EAE was performed. RESULTS: EAE-induced mice, compared to naïve, healthy mice, overall showed increased expression in pathways for immune response, as well as an increased cytokine signaling pathway, with downregulation of cellular stress proteins. FOL downregulates pro-inflammatory pathways and attenuates the immune response in EAE. FOL downregulated the expression of genes involved in misfolded protein response, MAPK activation/signaling, and pro-inflammatory response. CONCLUSION: This study provides insight into the molecular impact of FOL in the brain and identifies potential therapeutic targets of the isoprenoid pathway in MS patients.

Gut microbiome-modulated dietary strategies in EAE and multiple sclerosis.

Over the last few decades, the incidence of multiple sclerosis has increased as society's dietary habits have switched from a whole foods approach to a high fat, high salt, low dietary fiber, and processed food diet, termed the "Western diet." Environmental factors, such as diet, could play a role in the pathogenesis of multiple sclerosis due to gut microbiota alterations, gut barrier leakage, and subsequent intestinal inflammation that could lead to exacerbated neuroinflammation. This mini-review explores the gut microbiome alterations of various dietary strategies that improve upon the "Western diet" as promising alternatives and targets to current multiple sclerosis treatments. We also provide evidence that gut microbiome modulation through diet can improve or exacerbate clinical symptoms of multiple sclerosis, highlighting the importance of including gut microbiome analyses in future studies of diet and disease.

The immunomodulatory roles of the gut microbiome in autoimmune diseases of the central nervous system: Multiple sclerosis as a model.

The gut-associated lymphoid tissue is a primary activation site for immune responses to infection and immunomodulation. Experimental evidence using animal disease models suggests that specific gut microbes significantly regulate inflammation and immunoregulatory pathways. Furthermore, recent clinical findings indicate that gut microbes' composition, collectively named gut microbiota, is altered under disease state. This review focuses on the functional mechanisms by which gut microbes promote immunomodulatory responses that could be relevant in balancing inflammation associated with autoimmunity in the central nervous system. We also propose therapeutic interventions that target the composition of the gut microbiota as immunomodulatory mechanisms to control neuroinflammation.

Farnesol induces protection against murine CNS inflammatory demyelination and modifies gut microbiome.

Farnesol is a 15­carbon organic isoprenol synthesized by plants and mammals with anti-oxidant, anti-inflammatory, and neuroprotective activities. We sought to determine whether farnesol treatment would result in protection against murine experimental autoimmune encephalomyelitis (EAE), a well-established model of multiple sclerosis (MS). We compared disease progression and severity in C57BL/6 mice treated orally with 100 mg/kg/day farnesol solubilized in corn oil to corn-oil treated and untreated EAE mice. Farnesol significantly delayed the onset of EAE (by ~2 days) and dramatically decreased disease severity (~80%) compared to controls. Disease protection by farnesol was associated with a significant reduction in spinal cord infiltration by monocytes-macrophages, dendritic cells, CD4+ T cells, and a significant change in gut microbiota composition, including a decrease in the Firmicutes:Bacteroidetes ratio. The study suggests FOL could protect MS patients against CNS inflammatory demyelination by partially modulating the gut microbiome composition.

Microbiome Methods in Experimental Autoimmune Encephalomyelitis.

Microbiome composition studies are increasingly shedding light on animal models of disease. This paper describes a protocol for analyzing the gut microbiome composition prior to and after the induction of mice to experimental autoimmune encephalomyelitis (EAE), the principal animal model of the human neuroinflammatory demyelinating disease multiple sclerosis (MS). We also address and provide data assessing the impact of mice reared in different animal facilities on EAE induction. Furthermore, we discuss potential regulators of the gut-microbiome-brain axis (GMBA) in relation to neuroinflammation and implications on demyelinating disease states. Our results suggest that mice reared in different animal facilities produce different levels of EAE induction. These results highlight the importance of accounting for consistent environmental conditions when inducing EAE and other animal models of disease. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Study of the composition of the gut microbiome in the neuroinflammatory model of experimental autoimmune encephalomyelitis Basic Protocol 2: Experimental procedures for DNA extraction and microbiome analysis.

Exploring the Gut-Brain Axis for the Control of CNS Inflammatory Demyelination: Immunomodulation by Bacteroides fragilis' Polysaccharide A.

The symbiotic relationship between animals and their resident microorganisms has profound effects on host immunity. The human microbiota comprises bacteria that reside in the gastrointestinal tract and are involved in a range of inflammatory and autoimmune diseases. The gut microbiota's immunomodulatory effects extend to extraintestinal tissues, including the central nervous system (CNS). Specific symbiotic antigens responsible for inducing immunoregulation have been isolated from different bacterial species. Polysaccharide A (PSA) of Bacteroides fragilis is an archetypical molecule for host-microbiota interactions. Studies have shown that PSA has beneficial effects in experimental disease models, including experimental autoimmune encephalomyelitis (EAE), the most widely used animal model for multiple sclerosis (MS). Furthermore, in vitro stimulation with PSA promotes an immunomodulatory phenotype in human T cells isolated from healthy and MS donors. In this review, we discuss the current understanding of the interactions between gut microbiota and the host in the context of CNS inflammatory demyelination, the immunomodulatory roles of gut symbionts. More specifically, we also discuss the immunomodulatory effects of B. fragilis PSA in the gut-brain axis and its therapeutic potential in MS. Elucidation of the molecular mechanisms responsible for the microbiota's impact on host physiology offers tremendous promise for discovering new therapies.

Protection Conferred by Drinking Water Administration of a Nanoparticle-Based Vaccine against Salmonella Enteritidis in Hens.

Salmonellosis remains a major medical and an unmet socioeconomic challenge. Worldwide, more than three million deaths per year are associated with Salmonella enterica serovar Enteritidis infections. Although commercially available vaccines for use in poultry exist, their efficacy is limited. We previously described a method for isolating a heat extract (HE) fraction of the cell surface of S. Enteritidis that contained major antigenic complexes immunogenic in hens naturally infected with the bacterium. One single dose of S. Enteritidis' HE induced protection against lethal salmonellosis in mice. Furthermore, HE encapsulation in nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (PVM/MA), Gantrez AN, improved and prolonged the protection against the disease in mice. We formulated new preparations of Gantrez AN nanoparticles with HE S. Enteritidis and assessed their stability in drinking water and their efficacy in hens after experimental infection. The oral treatment of six-week-old hens with two doses of HE nanoparticles significantly reduced the Salmonella excretion in hens. Due to the effectiveness of the treatment in reducing bacterial excretion, we conclude that HE nanoencapsulation obtained from S. Enteritidis is a viable novel vaccination approach against salmonellosis in farms.

Dysbiosis of the intestinal microbiome as a component of pathophysiology in the inborn errors of metabolism.

Inborn errors of metabolism (IEMs) represent monogenic disorders in which specific enzyme deficiencies, or a group of enzyme deficiencies (e.g., peroxisomal biogenesis disorders) result in either toxic accumulation of metabolic intermediates or deficiency in the production of key end-products (e.g., low cholesterol in Smith-Lemli-Opitz syndrome (Gedam et al., 2012 [1]); low creatine in guanidinoacetic acid methyltransferase deficiency (Stromberger, 2003 [2])). Some IEMs can be effectively treated by dietary restrictions (e.g., phenylketonuria (PKU), maple syrup urine disease (MSUD)), and/or dietary intervention to remove offending compounds (e.g., acylcarnitine excretion with the oral intake of l-carnitine in the disorders of fatty acid oxidation). While the IEMs are predominantly monogenic disorders, their phenotypic presentation is complex and pleiotropic, impacting multiple physiological systems (hepatic and neurological function, renal and musculoskeletal impairment, cardiovascular and pulmonary activity, etc.). The metabolic dysfunction induced by the IEMs, as well as the dietary interventions used to treat them, are predicted to impact the gut microbiome in patients, and it is highly likely that microbiome dysbiosis leads to further exacerbation of the clinical phenotype. That said, only recently has the gut microbiome been considered as a potential pathomechanistic consideration in the IEMs. In this review, we overview the function of the gut-brain axis, the crosstalk between these compartments, and the expanding reports of dysbiosis in the IEMs recently reported. The potential use of pre- and probiotics to improve clinical outcomes in IEMs is also highlighted.

A Gut Feeling: The Importance of the Intestinal Microbiota in Psychiatric Disorders.

The intestinal microbiota constitutes a complex ecosystem in constant reciprocal interactions with the immune, neuroendocrine, and neural systems of the host. Recent molecular technological advances allow for the exploration of this living organ and better facilitates our understanding of the biological importance of intestinal microbes in health and disease. Clinical and experimental studies demonstrate that intestinal microbes may be intimately involved in the progression of diseases of the central nervous system (CNS), including those of affective and psychiatric nature. Gut microbes regulate neuroinflammatory processes, play a role in balancing the concentrations of neurotransmitters and could provide beneficial effects against neurodegeneration. In this review, we explore some of these reciprocal interactions between gut microbes and the CNS during experimental disease and suggest that therapeutic approaches impacting the gut-brain axis may represent the next avenue for the treatment of psychiatric disorders.

The Microbiome as a Therapeutic Target for Multiple Sclerosis: Can Genetically Engineered Probiotics Treat the Disease?

There is an increasing interest in the intestinal microbiota as a critical regulator of the development and function of the immune, nervous, and endocrine systems. Experimental work in animal models has provided the foundation for clinical studies to investigate associations between microbiota composition and function and human disease, including multiple sclerosis (MS). Initial work done using an animal model of brain inflammation, experimental autoimmune encephalomyelitis (EAE), suggests the existence of a microbiota-gut-brain axis connection in the context of MS, and microbiome sequence analyses reveal increases and decreases of microbial taxa in MS intestines. In this review, we discuss the impact of the intestinal microbiota on the immune system and the role of the microbiome-gut-brain axis in the neuroinflammatory disease MS. We also discuss experimental evidence supporting the hypothesis that modulating the intestinal microbiota through genetically modified probiotics may provide immunomodulatory and protective effects as a novel therapeutic approach to treat this devastating disease.

The Gut Microbiome in Multiple Sclerosis: A Potential Therapeutic Avenue.

Recently, there has been a substantial increase in the number of studies focused upon connecting the gut microbiome with cases of central nervous system (CNS) autoimmunity. Multiple sclerosis (MS) is a neurodegenerative autoimmune disorder of the CNS. Recent experimental and clinical evidence suggests the presence of microbial imbalances in the gut of MS sufferers. The gut microbiome is defined as the summation of all the microbial entities as well as their genes, proteins, and metabolic products in a given space and time. Studies show the MS gut microbiome as having general alterations in specific taxa, some associated with the promotion of inflammatory cytokines and overall inflammation. In conjunction with these findings, experimental models of the disease have reported that T regulatory (Treg) cells have deficits in their function as a result of the aberrant gut microbiota composition. The findings suggest that the interactions between the host and the microbiota are reciprocal, although more extensive work is required to confirm this. Moreover, evidence indicates that changes in microbiota composition may result in imbalances that could result in disease, with the gut as a potential novel therapeutic avenue. By understanding the biological effects of aberrant gut microbiome composition, it is possible to contemplate current therapeutic options and their efficacy. Ultimately, more research is necessary in this field, but targeting the gut microbiota may lead to the development of novel therapeutic strategies.

Progress and perspectives in plant sterol and plant stanol research.

Current evidence indicates that foods with added plant sterols or stanols can lower serum levels of low-density lipoprotein cholesterol. This review summarizes the recent findings and deliberations of 31 experts in the field who participated in a scientific meeting in Winnipeg, Canada, on the health effects of plant sterols and stanols. Participants discussed issues including, but not limited to, the health benefits of plant sterols and stanols beyond cholesterol lowering, the role of plant sterols and stanols as adjuncts to diet and drugs, and the challenges involved in measuring plant sterols and stanols in biological samples. Variations in interindividual responses to plant sterols and stanols, as well as the personalization of lipid-lowering therapies, were addressed. Finally, the clinical aspects and treatment of sitosterolemia were reviewed. Although plant sterols and stanols continue to offer an efficacious and convenient dietary approach to cholesterol management, long-term clinical trials investigating the endpoints of cardiovascular disease are still lacking.

The Gut Microbiome and Multiple Sclerosis.

The microbiome can be defined as the sum of the microbial and host's genome. Recent information regarding this complex organ suggests that in animal models of multiple sclerosis (MS), the composition of the gut microbiome can be altered, giving rise to both the effector and regulatory phases of central nervous system (CNS) demyelination. Experimental findings during the past decade in animal models of MS have provided clear evidence for the significant role of gut microbes in both the effector and regulatory phase of this condition. There is mounting evidence in preliminary human studies suggesting that a dysbiotic MS gut microbiome could affect disease progression. We propose considering the gut microbiome as a key organ for the regulation of tolerance mechanisms and speculate that the gut microbiome is the major environmental risk factor for CNS demyelinating disease. Accordingly, we hypothesize that intervention of the gut microbiome could result in safer novel therapeutic strategies to treat MS.

A bidirectional association between the gut microbiota and CNS disease in a biphasic murine model of multiple sclerosis.

The gut microbiome plays an important role in the development of inflammatory disease as shown using experimental models of central nervous system (CNS) demyelination. Gut microbes influence the response of regulatory immune cell populations in the gut-associated lymphoid tissue (GALT), which drive protection in acute and chronic experimental autoimmune encephalomyelitis (EAE). Recent observations suggest that communication between the host and the gut microbiome is bidirectional. We hypothesized that the gut microbiota differs between the acute inflammatory and chronic progressive stages of a murine model of secondary-progressive multiple sclerosis (SP-MS). This non-obese diabetic (NOD) model of EAE develops a biphasic pattern of disease that more closely resembles the human condition when transitioning from relapsing-remitting (RR)-MS to SP-MS. We compared the gut microbiome of NOD mice with either mild or severe disease to that of non-immunized control mice. We found that the mice which developed a severe secondary form of EAE harbored a dysbiotic gut microbiome when compared with the healthy control mice. Furthermore, we evaluated whether treatment with a cocktail of broad-spectrum antibiotics would modify the outcome of the progressive stage of EAE in the NOD model. Our results indicated reduced mortality and clinical disease severity in mice treated with antibiotics compared with untreated mice. Our findings support the hypothesis that there are reciprocal effects between experimental CNS inflammatory demyelination and modification of the microbiome providing a foundation for the establishment of early therapeutic interventions targeting the gut microbiome that could potentially limit disease progression.