The Impact of Gut Microbiota Disorders on the Blood–Brain Barrier

Abstract

The gut microbiota is symbiotic with the human host and has been extensively studied in recent years resulting in increasing awareness of the effects of the gut microbiota on human health. In this review, we summarize the current evidence for the effects of gut microbes on the integrity of the cerebral blood–brain barrier (BBB), focusing on the pathogenic impact of gut microbiota disorders. Based on our description and summarization of the effects of the gut microbiota and its metabolites on the nervous, endocrine, and immune systems and related signaling pathways and the resulting destruction of the BBB, we suggest that regulating and supplementing the intestinal microbiota as well as targeting immune cells and inflammatory mediators are required to protect the BBB.

Keywords:

• microbiota
• metabolites
• blood–brain barrier

Introduction

Under physiological conditions, the intestinal microbiota and the human body are in a mutually beneficial relationship. The human body provides a protected environment and nutrients for the microbiome,¹ while the microbes participate in the metabolism, digestion and absorption of substances. The gut microbiota is constantly changing but maintained in a relatively stable state, participating in various physiological processes. Gut microbes dysbiosis can cause dysfunction of many organs, including the brain.² The blood–brain barrier (BBB) is important for protection of the brain and preventing microbes and toxins from entering the central nervous system (CNS). Many studies have shown that gut microbiota disorders are related to the destruction of the BBB,^3–5 although the mechanism remains to be clarified.

The Composition and Function of the Blood–Brain Barrier

The BBB refers to the barrier between the plasma and brain cells formed by the brain capillaries and glial cells and the barrier between the plasma and cerebrospinal fluid formed by the choroid plexus. The BBB consists of brain microvascular endothelial cells (BMEC), the perivascular foot of astrocytes, a basement membrane (BM) and pericytes (PCs). In July 2001, the National Institute of Neurology and Stroke (NINDS) proposed the concept of a neurovascular unit (NVU) to emphasize the dynamic interaction between the BBB, neurons, the extracellular matrix (ECM), and microglia. The components of this unit work together to regulate the structure and function of the BBB.^6,7

BMECs are held together by tight junction (TJ) proteins to form an endothelial cell barrier, which includes claudin, occludin, and zonula occludens (ZO) and junctional adhesion molecules (JAMs) linked to the cytoskeleton.^8,9 BMECs overlap and occlude adjacent vascular endothelial cells (ECs) to form a high-resistance barrier to molecules and metal ions that strictly controls the paracellular transport of solutes and liquids.^10,11 The EC barrier effectively prevents the passage of macromolecular substances through the EC junctions and maintain the ion homeostasis in the brain.¹² TJs are particularly important for BBB function, and their loss can greatly increase its permeability.^13,14 Cerebrovascular ECs have a very low transcytosis rate due to the lack of plasma membrane vesicles.^15–17 PCs protrude from the surface of ECs, spanning multiple EC bodies, and wrapping around the capillary endothelium.¹⁸ The contractile proteins in PCs regulate the diameter of capillaries,¹⁹ regulating transcytosis and immune cell transport across the BBB.

ECs are also surrounded by a continuous BM, which is composed of collagen, laminin, nestin, heparin sulfate proteoglycan and other glycoproteins. The ECM is secreted by EC, PCs and astrocytes. The BM provides binding sites for signal transduction of many cytokines (VEGF, Wnt, etc.) and forms the second part of the BBB, which can be destroyed by matrix metalloproteinases (MMPs).²⁰

Astrocytes are the most abundant glial cell type and play an important role in neurovascular regulation.²¹ The perivascular foot of astrocytes surrounds approximately 85% of the surface of the brain capillaries outside the BM, forming another physical, transport, and metabolic barrier. It also communicates with neurons to establish endothelial neuronal connections,²² which transmit neural signals to the local vasculature, affecting barrier physiology by altering arteriolar expansion and blood flow.^23,24 (see Figure 1)

Figure 1 The blood–brain barrier is composed of brain microvascular endothelial cells, pericytes, the continuous basement membrane and the perivascular feet of astrocytes, preventing the entry of harmful substances into the brain tissue. The blood–brain barrier interacts with extracellular matrices, neurons, and microglia, forming neurovascular units, which regulate the structure and function of the blood–brain barrier.

This multilayer membrane structure of brain capillaries constitutes a protective barrier for brain tissue. The BBB serves as a key regulator of the entry of nutrients from blood sources and compounds required for brain health. It also prevents the entry of potentially harmful molecules and cells into the brain, and maintains brain homeostasis and a suitable microenvironment for neuronal growth.^1,25,26

The Effects of Blood–Brain Barrier Disruption on the Brain

Destruction of the BBB is involved in a variety of acute and chronic CNS diseases and neuropsychiatric disorders, such as encephalomyelitis, multiple sclerosis (MS), Alzheimer’s disease and schizophrenia. High levels of superoxide,²⁷ activation of MMPs,²⁸ and upregulation of inflammatory mediators in the CNS can cause damage to the BBB. Degradation of the BBB leads to increased permeability and leakage, resulting in the recruitment of immune cells to the CNS²⁹ and the occurrence of neuroinflammation.³⁰

The Impact of Gut Microbiota Disorders on the BBB

Gut Microbiota Disorders Associated with the BBB

The human body contains about 100 trillion microbes from approximately 1000 species, of which the intestinal microbiota accounts for more than 90%, mainly comprising Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria.³¹ These microbes play important roles in many physiological functions, such as metabolism,^32,33 nutrient absorption in the gut, synthesis of beneficial bioactive molecules,³⁴ regulation of neurotransmitters, maintenance of the integrity and function of barriers,^35,36 and the immune system.³⁷ Under normal circumstances, intestinal microbes are combined in a certain proportion to form an ecological balance. When the internal and external environment changes, this balance is broken, resulting in gut microbiota disorders. Diet, infection and oral antibiotics can change the structure of intestinal microflora.^38–40 The disorder of intestinal microbes may promote the occurrence of some diseases. Studies have confirmed that intestinal microbes disorders are associated with many CNS diseases, including Alzheimer’s disease (AD),⁴¹ Parkinson’s disease (PD),⁴² and amyotrophic lateral sclerosis.⁴³ Thus, intestinal microbes disorders are is considered to be the important cause of dementia.^44,45

A growing number of studies suggest that gut microbes have an important influence on BBB integrity. In 2014, Braniste et al found that different regions of the brain in germ-free (GF) mice (including the frontal cortex, hippocampus and striatum) displayed increased BBB permeability compared to pathogen-free (PF) mice. The increased BBB permeability was associated with decreased expression of occludin and claudin-5. After fecal transplantation from PF mice or administration of short-chain fatty acid (SCFA)-producing bacteria to GF mice, the expression of TJs increased, and the integrity of the BBB was restored. This indicated the establishment of communication between the gut microbiota and the BBB during embryonic development, which persists throughout life.³ Fröhlich et al reported that induced intestinal dysbiosis induced in mice through the administration of antibiotics resulted in reduced expression of TJ proteins in the hippocampus, without a reduction in the prefrontal cortex and hypothalamus.⁴⁶

These findings indicate the importance of the gut microbiota in the BBB, although the mechanism remains unclear. Current research suggests that the gut microbiota regulate the BBB through a variety of pathways, including the vagus and sympathetic nerves,⁴⁷ immune⁴⁸ and endocrine systems,⁴⁹ and intestinal microbial metabolites such as SCFAs and lipopolysaccharides (LPS).^50,51 (see Figure 2)

Figure 2 Pathways of the effects of gut microbiota on the blood–brain barrier. The gut microbiota can affect the structure and function of the blood–brain barrier through various pathways, such as their metabolites, and the nervous, endocrine, and immune systems.

Microorganisms and Their Metabolites

Microorganisms in the gut communicate with the brain via mechanical stimulation of intestinal mucosal cells, causing local inflammation by producing toxins, receptor-mediated signaling, and increasing intestinal permeability.^52,53 When the intestinal flora is in disorder, some pathogenic bacteria in the gastrointestinal tract can directly stimulate intestinal chromaffin cells to release serotonin, which can activate endogenous afferents and cause nerve reflex, thereby enhancing the release of chloride and water to the intestinal cavity, thus stimulating intestinal motility.⁵⁴ The gut contains numerous bacteria, which produce LPS, and convert dietary components into a series of metabolites such as SCFAs, trimethylamine and serotonin. These metabolites regulate homeostasis, maintain BBB integrity and affect brain function.⁵⁵ The gut-vascular barrier (GVB) prevents the entry of bacteria into the bloodstream from the intestines. Following destruction of the GVB, bacteria and their toxic metabolites enter the bloodstream, causing an inflammatory response.

Lipopolysaccharide

LPS is the main component of the cell wall of Gram-negative bacteria, which is lysed and shed after bacterial death. Gut microbiota disorders can lead to increased LPS release, increasing intestinal permeability, and affecting intestinal and general health.^56,57 LPS activates gastrointestinal immune cells to release inflammatory cytokines from the gut. In vitro experiments, Kacimi et al studied the model of endothelial cell death only in the presence of microglia and found that LPS induced the death of microglia rather than EC. However, when microglia were co-cultured with EC, LPS increased EC death. Furthermore, inhibiting microglial activation can prevent injury to the EC. Thus, it has been proposed that LPS disrupts the BBB by activating microglia to damage EC.⁵⁸ Singh et al demonstrated the interaction of EC with LPS and the main component of the cell wall of Gram-positive bacteria, lipoteichoic acid (LTA) in vitro. They found that the toxins did not cross the endothelial barrier, but the mRNA levels of ZO-1, occludin and JAM were suppressed, while mRNA levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1 β) were increased. These findings indicated that disruption of the BBB and release of pro-inflammatory cytokines induce neuroinflammation.⁵⁹ In addition, LPS affects adhesion proteins and membrane transporters, as well as the basal lamina, and extracellular matrix.⁶⁰ LPS also activates Toll-like receptor 4 on microglia to release inflammatory cytokines and chemokines in the CNS,⁶¹ and enhances neuronal apoptosis,^62,63 thereby affecting the BBB and CNS.

Short-Chain Fatty Acids

SCFAs, which are produced mainly by digestion of dietary fiber by the beneficial intestinal microbes, are biologically active molecules that can pass the BBB.^64,65

SCFAs act as signaling molecules by binding to G protein-coupled receptors (GPCRs).⁶⁶ SCFAs stimulate GPCRs, the free fatty acid receptors (FFAR2 or FFAR3), on intestinal epithelial cells and brain EC,^67–71 protecting the BBB from oxidative stress.⁶⁷ SCFAs can enter the interior of cells and inhibit histone deacetylase (HDAC), thus activating gene expression.⁷² HDAC blocks the transcription of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF).⁷³ In addition, SCFAs stimulate synapse and outer membrane vesicles (OMV) for exocytosis.⁷⁴ OMVs, which encapsulate some bioactive molecules secreted by bacteria, enter the systemic circulation and cross the BBB to induce an inflammatory response.⁷⁵ Erny et al observed that both GF and temporary or partial eradication of the host microbiota resulted in microglial defects that partially restored microglial features by recolonization with a complex microbiota. It was also found that mice lacking the SCFA receptor FFAR2 mirrored the microglial defects found under GF conditions, suggesting that SCFAs regulate microglial homeostasis.⁷⁶

Neural Pathway

The neural pathway is mediated by the interaction of the gut microbiota with the central, autonomic, and enteric nervous systems,⁴⁵ and participates in normal neurological functions, such as the development and formation of neurons and myelin,⁷⁷ the development of the neurotransmitter system,⁷⁸ neural signaling and BBB integrity.

Microbial populations can regulate the CNS development and affect the expression and signaling of amygdala transcriptional genes.⁷⁹ Significant amygdalar and hippocampal expansion was observed in GF mice compared to that in the conventional colonization mice. The basolateral amygdala pyramidal neurons of GF mice had more stubby and mushroom spines, while the ventral hippocampal pyramidal neurons were shorter and less branched,⁸⁰ with decreased BDNF expression in the hippocampus.⁸¹

Metabolites produced by the gut microbes act locally on the intestinal neurons that innervate the gut and are transmitted to the brain via neural signals. Metabolites also activate Toll-like receptors present in intestinal epithelial cells,^82,83 producing intestinal inflammatory responses and increasing apoptosis and loss of intestinal neurons,⁸⁴ thus affecting the function of the enteric and central nervous systems.

Vagus Nerve

The vagus nerve is an important pathway by which the intestinal microbiota communicates with the brain. The gut microbiota can act on the intestinal neurons and communicate with the brain by altering the vagus signals to stimulate anti-inflammatory reflexes, release mediators such as acetylcholine, and interact with immune cells to reduce inflammation.⁴⁷ Stimulation of the vagus nerve reduces the co-localization of neutrophils and endothelial cell adhesion molecule (ICAM)-1 induced by LPS stimulation, as well as decreasing gene expression of hypothalamic inflammatory mediators (IL-6, CXCL-1, ICAM-1) and attenuating inflammatory responses in brain areas.⁸⁵ In the rat model of ischemic stroke, non-invasive vagus nerve stimulation was also observed to reduce BBB leakage in the lesion area, improve TJ levels, and reduce MMP-2/9 expression to protection the integrity of the BBB.⁸⁶

Neurotransmitters

Neurotransmitters are neurochemicals that transmit neural signals between synapses. Intestinal microbes are symbiotic with the human body to secrete neurotransmitters (GABA, 5-HT, catecholamine, and histamine).⁸⁷ Elevated enteric neurotransmitters may be involved in the pathological process of abnormal excitation of intestinal nerve cells. While being transported to the brain through the blood circulation and neural channels, they may also activate the vagal nerve chemoreceptor by paracrine, and finally stimulate anti-inflammatory reflex.^47,88 By controlling the function of the BBB, neurotransmitters regulate the transmission of information between the periphery and the brain.^89,90 Gut dysbiosis changes the production of 5-HT, which is also called serotonin. Changes in the local concentration of 5-HT are transmitted to the brain along the gut-brain axis, affecting CNS signaling.⁹¹ The attenuation of pro-inflammatory factors and the elevation of tryptophan and the serotonergic precursor was observed in rats following treatment with bifidobacteria.⁹² The gut microbes can convert histidine to histamine. Histamine affects both anti-apoptotic proteins and autophagy proteins through the heat shock response pathway, and induces microglia anti-inflammation to reduce neuroinflammation as well as motor neuron death.⁹³

Endocrine Pathway

Neuroendocrine-Hypothalamic-Pituitary-Adrenal Axis

The endocrine pathway allows the transfer of humoral factors to mediate bidirectional activity between the gut microbiota and the brain.^94,95 Changes in the structure of the gut microbiota drive the pro-inflammatory state, which leads to increased permeability of the intestinal barrier.⁹⁶ LPS crosses the intestinal epithelial barrier and activates the neuroendocrine-hypothalamic-pituitary-adrenal axis.⁹⁷ As a result, mast cells are activated and corticotropin-releasing hormone (CRH) is released, resulting in increased permeability of the blood–brain barrier. CRH and adrenocorticotropic hormone (ACTH) can also directly activate microglia to release neuroinflammatory mediators and promote neuroinflammation in the brain.⁹⁸ Exposure of neonates to LPS results in increased ACTH and corticosterone production in response to stress and decreased brain glucocorticoid receptor (GR) density.⁹⁹ Bifidobacterium administration increases the diversity of intestinal microbes in maternal separation mice, which not only downregulates intestinal inflammation, but also weakens the excessive stress response of the hypothalamus-pituitary-adrenal axis, thereby affecting brain biochemistry and behavior.¹⁰⁰

Enterogenous Hormones

Intestinal microbes regulate the number and activity of enteroendocrine cells and the synthesis and secretion of biological hormones through local stimulation and production of metabolites.^101,102 Hormones such as leptin, ghrelin, and glucagon-like peptide 1 (GLP-1), which are synthesized in the intestines, regulate energy homeostasis and have protective effects on neurotoxicity induced by toxic microbial metabolites.^103–105 In response to SCFAs, enteroendocrine cells release the neuropeptides GLP-1 and PYY to enhance satiety via the neuroendocrine pathway,^106,107 while the expression is reduced in GF mice.¹⁰⁸ GLP-1 is secreted by intestinal L cells and participates in the regulation of a variety of central nervous system functions.¹⁰⁹ Changes in GLP-1 content are related to changes in gut microbes. After traumatic brain injury (TBI), the number of bacterial species in the faecal microbes of mice decreased significantly.¹¹⁰ Clostridium butyricum (Cb) can produce a large amount of SCFA butyrate in the intestinal tract to stimulate the production of gastrointestinal hormone in the colon.¹¹¹ After supplement of Cb, reduction of inflammatory reaction and intestinal permeability, improvement of neurological dysfunction and BBB injury are observed in TBI mice, which is considered to be related to increased GLP-1 secretion.¹¹²

Immune Pathway

Under normal circumstances, the intestinal microbiota and the host are in a symbiotic state. Following disruption of the intestinal flora, the microorganisms and their metabolites interaction with the host immune system may occur.^91,113 The changes in the composition of the gut microbiota lead to increased intestinal permeability and stimulation of an immune response by the bacteria and their toxic metabolites. The activated immune cells and the secreted immune signal molecules then reach the BBB via the blood circulation. Systemic inflammation and elevated levels of circulating cytokines upregulate adhesion molecules, chemokines, and MMPs in the BBB and the brain,^114,115 and downregulate TJs to increase the permeability of the BMEC layer.¹¹⁶ The disrupted BBB allows the entry of fibrin, which is deposited as insoluble fibrin and activates an immune response.¹¹⁷ Solutes and toxins enter the brain causing increased inflammation and recruitment of leukocytes and macrophages,¹¹⁴ which stimulate the inflammatory signaling of the NVU.¹¹⁸ Thus, intracerebral inflammation and neurodegeneration are exacerbated via a vicious cycle.

T Lymphocytes

Atarashi et al isolated bacterial strains that induced the proliferation of regulatory T cells (Tregs) from human stool samples, suggesting that the intestinal flora can activate Tregs.¹¹⁹ SCFAs also induce the production and differentiation of Tregs by inhibiting HDAC activity.^120,121 In multiple sclerosis, the species abundance of intestinal microbes decreased, with the depletion of Clostridia XIVa and IV Clusters.¹²² Intestinal dysbiosis leads to reduced the content of propionic acid in serum and feces,¹²³ decreased Treg cells and increased helper T cells (Th1 and Th17) in peripheral blood.¹²⁴ Activated T lymphocytes migrate from the periphery to the CNS, secreting cell adhesion molecules and chemokines, and leading to infiltration of the CNS by monocytes and macrophages. In this way, the blood–brain barrier is destroyed.¹²⁵

Microglia

Microglia are innate immune cells of the CNS and play an role in immunological surveillance in the brain by participating in information transfer and clearing cell debris.¹²⁵ The gut microbiota plays an important role in regulating the maturation and function of microglia.⁷⁶ GF mice showed immature gene expression profiles and morphological differences, such as increased microglial cell volume and branching. After colonization with diverse microbial communities, GF mice showed a mature microglial phenotype similar to that of SPF mice. After treatment with antibiotics, microglia isolated from SPF mice showed a cell morphology similar to that in GF mice.⁷⁶ Following destruction of the BBB, microglia are activated by inflammatory substances and oxidative stress. The activated microglia exhibit an amoebic phenotype and mediate phagocytosis from a branching state.¹²⁶ The microglia upregulate a variety of active proteins, including major histocompatibility complex (MHC) I, MHC II, and secrete multiple cytokines and chemokines. Activated microglia can be divided into pro-inflammatory (M1 type) and anti-inflammatory (M2 type) subtypes, which interact with infiltrating T lymphocytes to generate nociceptive or neuroprotective outcomes.¹²⁷

Inflammatory Cytokines

An imbalance in the intestinal flora can lead to increased intestinal permeability. Bacterial cell wall antigens are recognized as patterns and combined to produce pro-inflammatory cytokines. Pro-inflammatory cytokines can be transported to tissues including the brain to initiate inflammatory processes,^128,129 leading to extravasation of leukocytes, upregulation of vascular cell adhesion protein 1 (VCAM-1), ICAM-1 and MMPs,¹³⁰ and disruption of the BBB integrity. Increased BBB permeability allows the entry of pathogens and toxins into the brain. Astrocytes adopt a pro-inflammatory phenotype, releasing IL-1β, IL-6, TNF-α and prostaglandins,¹³¹ which influence crossing of the paracellular and transcellular barrier. In addition, the inflammatory cytokines TNF-α and IL-1β can also induce the expression of CXCL1 and CCL2,¹³² which participate in the recruitment of immune cells to the brain and further promote the inflammatory response.

Signaling Pathways

Wnt/β-Catenin Signaling Pathway

The Wnt signaling pathway is activated by binding of the Wnt protein to the N-terminal cysteine rich domain of the Frizzled (FZD) protein family receptors. The FZD protein family forms part of the seven-transmembrane GPCRs family,¹³³ and is responsible for immobilizing Wnt proteins on the cell surface. The signal is transmitted to the Disheveled (DVL) protein in the cell via the C-terminus of the FZD protein.^134–137 DVL binds to the Axin/GSK3/APC complex, thereby inhibiting the degradation of β-catenin (β-cat) in the cytoplasm. Following the increase in cytoplasmic levels, β-cat is transferred into the nucleus and acts as a transcription factor subunit to induce transcription of the target gene, resulting in subsequent cellular responses. Activation of the canonical Wnt/β-catenin pathway has been reported to control BBB differentiation and maturation,^138,139 and play a positive role in the development of BBB by regulating TJ protein expression.¹³⁸ Inactivation of β-cat leads to a significant downregulation of claudin3, upregulation of plasma membrane vesicle-associated proteins and decomposition of the BBB.¹⁴⁰

Nuclear Factor Kappa-B (NF-κB) Signaling Pathway

The NF-κB pathway is a proteinase-dependent receptor signaling pathway that is activated by microbial pathogens, LPS, cytokines, heat shock protein 90 (HSP90) and high mobility group proteins (HMGB1) in the blood. Normally, the NF-κB dimer binds to the Inhibitor of kappa-B (IκB) protein and remains in the cytoplasm. Stimulation of the upstream signals leads to activation of the IκB kinase (IKK) complex and IκB protein phosphorylation. Following ubiquitination, the IκB protein is then targeted for proteasome-dependent degradation and NF-κB is released into the nucleus to activate downstream gene transcription. NF-κB regulates the expression of numerous genes such as cytokines (IL-1β, IL-6, TNF-α, GM-CSF), inflammatory chemokines (RANTES, MCP-1), and adhesion molecules (VCAM-1, ICAM-1, E-selectin), and plays important roles in various aspects of inflammatory and innate immune responses.^141,142 Activation of the NF-κB signaling pathway promotes glial cell activation¹⁴³ and expression of ICAM-1, VCAM-1, IL-6, IL-8 and monocyte chemoattractant protein 1 (MCP-1), which contribute to the destruction of the BBB.¹⁴⁴

c-Jun N-Terminal Kinase (JNK) Signaling Pathway

The c-Jun N-terminal kinase, also known as stress-activated protein kinase, is a member of the mitogen-activated protein kinase (MAPK) family. The JNK signaling pathway is activated by various factors such as cytokines (IL-1, TNF-α), growth factors (EGF, PDGF), GPCRs, and stress, and is involved in cell proliferation, differentiation and apoptosis and other biological processes.¹⁴⁵ The JNK protein is serine/threonine protein kinase encoded by three genes, Jnk1, Jnk2 and Jnk3, and located mainly in the cytoplasm.¹⁴⁶ After stimulation, JNKK1/MKK4/SEK1 or JNKK2/MKK7 mediates JNK activation by phosphorylation of Thr183 and Thr185.¹⁴⁷ The activated JNK is then transported into the nucleus, where it phosphorylates c-Jun and activates the apoptotic signaling pathway. Studies have shown that JNK inhibitors reduce MMP-9 expression, and increase the expression of TJ proteins (ZO-1, claudin-5, occludin), thereby preventing BBB destruction.^148–150

Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) Signaling Pathway

JAK is a non-receptor tyrosine protein kinase (PTK) the substrate of which is STAT. JAK is rapidly recruited and activated by cell surface receptors (eg, interferons, interleukins, and growth factors). Activated JAK phosphorylates the tyrosine residue of the receptor, providing a binding site for proteins containing an SH2 domain. STAT binds to the receptor and then is also phosphorylated by activated JAK. Activated STATs form dimers and enter the nucleus to induce transcription of the target gene. This signaling pathway is known as the JAK-STAT pathway. In an animal model of ischemia/reperfusion injury, Gong et al found that inhibition of JAK/STAT signaling activation increased TJ levels and reduced BBB permeability.¹⁵¹ Chaudhuri et al also demonstrated that activated STAT1 induces IL-6 expression and reduces the expression of claudin-5, ZO-1 and ZO-2 in BMEC. The STAT1 inhibitor fludarabine attenuates this downregulation of claudin-5 and ZO-1 and blocks the migration of monocytes across the BBB.¹⁵²

Toll-Like Receptors (TLRs) Signaling Pathway

TLRs, which were the first pattern-recognition receptors (PRRs) to be discovered, recognize pathogen-associated molecular patterns (PAMPs) on the surface of pathogenic microbes to initiate an innate immune response. TLRs are a type I transmembrane protein receptor composed of an intracellular segment, a transmembrane region, and an extracellular segment. The extracellular domain directly recognizes and binds to pathogens or their products, activating signaling pathways, and inducing expression of certain immune effector molecules. The TLR signaling pathway is divided into two main pathways: the myeloid differentiation factor 88 (MyD88)-dependent pathway and the MyD88-independent pathway. The MyD88-dependent pathway mediates NF-κB activation and produces cytokines, while the MyD88-independent pathway induces the production of type I interferon (IFN).¹⁵³ When the intestinal epithelium is damaged, LPS enters the blood, causing peripheral immune activation. TLR4, which is expressed in various cell types in the CNS,¹⁵⁴ induces immune activation and neuroinflammation in the brain. The mutual interaction between TLR2 and TLR4 affects brain health.¹⁵⁵ Mayerhofer et al demonstrated that the TLR2 agonist LTA increases the circulating levels of cytokines (TNF-α, IL-6, IFN-γ, etc.) and cytokine mRNA expression in the brain in mice, and is also involved in transcriptional downregulation of TJ proteins (claudin 5, occludin) in the brain.¹⁵⁵ In a model that mimics the human BBB, Paradis et al showed that TLR4 increases monocyte migration and stimulates the migration of monocytes across the BBB in response to CCL19.¹⁵⁶

Nucleotide-Binding Oligomerization Domain-Like Receptor (NLR) Signaling Pathway

NLRs belong to the family of intracellular PRRs. The NLR proteins have a central oligomerization domain, NOD,¹⁵⁷ which plays an important role in self-oligomerization and activation. The C-terminus of the leucine-rich repeat is responsible for sensing upstream ligand signals and modulating NLR activity.^158–160 The N-terminus of the effector domain activates the downstream signaling pathway by interacting with downstream proteins.¹⁶¹ NLRs participate in the activation of multiple signaling pathways involved in processes such as autophagy, signal transduction, and inflammatory body formation.¹⁶² This results in activation of the NF-κB signaling pathway, the production and release of pro-inflammatory cytokines (interleukins, chemokines), and initiation of innate and adaptive immune responses. Using LPS and muramyl dipeptide (MDP) to induce the expression of inflammasomes (NOD2, NLRP3 and caspase-1) and cytokines in human cerebral ECs, Nagyőszi et al showed that NLRs and inflammasomes can be activated in brain ECs.¹⁶³ Ge et al found that caspase-1 inhibitors prevent the apoptosis of damaged BMECs by inhibiting the expression of caspase-1 and pro-inflammatory cytokines, thereby reducing BBB damage after TBI.¹⁶⁴

Potential Therapeutic Tools for Promotion and Restoration of BBB Integrity

Current studies have indicated that the gut microbiota and brain perform complex bidirectional activities via the gut-brain-axis. However, the mechanisms by which the intestinal microbes affects the BBB and brain health of the host remain to be clarified. Supplementation of probiotics, prebiotics, synbiotics, and transplantation of fecal microbes may reduce the entry of harmful metabolites into the systemic circulation, contributing to the integrity of the gut and BBB.¹⁶⁵ Lactobacillus plantarum MTCC 9510 supplementation improved the intestinal and BBB integrity and reduced the abundance of Enterobacteriaceae.¹⁶⁶ Patients receiving probiotic treatment exhibited reduced serum C-reactive protein levels, improved insulin metabolism, and increased in Mini-Mental State Examination (MMSE) scores.^167,168 In addition, further studies of therapies targeting immune cells and peripheral cytokines that may also reduce inflammation-induced BBB hyperpermeability and prevent the entry of harmful substances into the brain are warranted.

Conclusions

The gut microbiota affects the activity of the brain through a variety of different mechanisms. The brain is influenced by the interaction of the gut microbiota with intestinal cells, metabolite production by microbes, secretion of gut hormones, and changes in neural and immune signaling and blood circulation to the BBB. Further studies of the correlation of the specific changes in the number and type of species in the gut microbiota and signaling via the gut-brain axis at the molecular level are required to fully elucidate the mechanism by which the microbes influence the brain. Therefore, corresponding strategies aimed at controlling the gut microbiota are implicated in the prevention and treatment of certain neurological and psychological illnesses.

Disclosure

The authors have declared that they do not have competing interests for this work.