The progress of gut microbiome research related to brain disorders

The gut microbiota has been shown to play critical roles in the pathogenesis of depression and anxiety-like behavior [138, 139]. Abnormal HPA hyperactivity in response to stress is associated with depressive episodes. Sudo et al. found that the HPA stress response is hyperactive in germ-free mice, but this hyperactivity can be reversed by Bifidobacterium infantis [121]. Plasma ACTH and corticosterone levels have been observed to be higher in GF mice than SPF mice when responding to stress. However, Diaz et al. revealed that GF mice exhibit increased plasma levels of tryptophan and serotonin compared with SPF mice [61], which is correlated with increased motor activity and reduced anxiety. Neufeld et al.’s results also demonstrated that GF mice exhibit anxiolytic behavior via increased expression of brain-derived neurotrophic factor (BDNF) and reduced serotonin 1A receptor levels in the hippocampus [122]. Anxiety and depression are typical complications in irritable bowel syndrome (IBS) patients, who show a higher prevalence of these conditions than healthy controls [140]. In addition to anxiety and depression, IBD patients exhibit mild verbal memory dysfunctions. The VFA isovaleric acid has been regarded as an important mediator of depression [112]. Gut-derived isovaleric acid can cross the BBB and interfere with synaptic neurotransmitter release. Isovaleric acid is positively correlated with saliva cortisol, which is strongly associated with depression in boys [141]. Bravo et al. have shown that GABA receptor expression in different brain areas can be altered by chronic treatment with Lactobacillus rhamnosus. This treatment further reduces stress-induced corticosterone levels and depression-like behaviors [119]. Stress-induced reduction of hippocampal neurogenesis can also be prevented by a probiotic combination of Lactobacillus helveticus strain R0052 and Bifidobacterium longum strain R0175 [142]. De Theije et al. showed that the effect of probiotics is highly strain specific. The administration of Campylobacter jejuni promotes depressive- or stress-like behaviors, while Bifidobacterium strains exert a decreasing effect on these behaviors [143].

ASDs are characterized by abnormal communication and social behaviors, which begin in early childhood neurodevelopment. Gut problems or a history of gastrointestinal perturbations such as infection and anti- or pro-biotic intake in early childhood contributes to the disease development [144‐146]. Gastrointestinal discomfort in ASD patients is usually thought to have a neurologic rather than a gastroenteric cause [147, 148]. However, recent studies have revealed that gut microflora alteration-related gastrointestinal symptoms are associated with mucosal inflammation [148, 149]. Anti-inflammatory and SCFA-synthesizing species such as Faecalibacterium species are decreased in ASD patients compared to controls [150]. Oral administration of Bacteroides fragilis (a commensal bacterium) can correct intestinal epithelial permeability caused by dysbiosis, leading to the modulation of several metabolites and ameliorated symptoms of ASD [114]. The Firmicutes/Bacteroides ratio and the composition of Fusobacteria or Verrucomicrobia are associated with ASDs. Lower levels of Bifidobacteria species, mucolytic bacteria, and Akkermansia muciniphila are found in children with ASD, while Lactobacillus, Bacteroides, Prevotella, and Alistipes are present at higher levels [145, 151, 152]. However, Desulfovibrio species, Bacteroides vulgatus, and Clostridia are over-represented in children with ASD with gastrointestinal symptoms compared with normal subjects with similar GI complaints [153, 154]. One hypothesis regarding the reoccurrence of ASD symptoms after oral vancomycin treatments is that Clostridia undergo spore formation to avoid eradication [145]. Although there is little direct causal evidence that the microbiota can cure ASD, the use of probiotic strains of Lactobacillus species and Bifidobacterium species has been shown to ameliorate gastrointestinal symptoms in children with ASD [119, 155]. Hsiao et al.’s research suggested that oral treatment with Bacteroides fragilis in a mouse model altered the microbial composition, improved gut permeability, and reduced defects in communicative and anxiety-like behaviors [114].

Pain is common in the general population, and the microbiota has shown to impact several types of pain, including spinal visceral pain in IBS [138, 156] and small intestinal bacterial overgrowth (SIBO) [157] as well as migraine [158] and sometimes headache [159]. Administration of antibiotics may induce gut dysbiosis and alter host-bacterial interactions, leading to colonic sensory and motor changes in a mouse model. These effects can be reflected by nociceptive markers such as CB2 and TLR7 [160]. Amaral et al. showed that inflammatory hypernociception induced by LPS, TNF-α, IL-1beta, and the chemokine CXCL1 is reduced in germ-free mice. This effect was similar to the induction by prostaglandins and dopamine [161]. It has been revealed by meta-analyses that Helicobacter pylori infection is associated with migraine [162]. Faraji et al. performed a randomized blinded clinical trial that demonstrated improvement of migraine associated with H. pylori eradication [158]. A recent study revealed that gut microbiota dysbiosis-induced chronic migraine-like pain are accomplished by up-regulating TNF-α level [163].

On the other hand, visceral pain can be effectively alleviated by probiotic treatment in animal models. Rousseaux et al. found that analgesic functions could be achieved by taking specific Lactobacillus strains orally. The resulting morphine-like effect was shown to be mediated by enhanced expression of intestinal epithelial μ-opioid and cannabinoid receptors [164]. Probiotic B. infantis 35624 and Lactobacillus farciminis were shown to exert visceral antinociceptive effects by altering central sensitization in rat models [165, 166]. L. farciminis can also attenuate stress-induced Fos expression in the spine, which explains the antinociceptive effect of this probiotic. In a clinical-alimentary study, O’Mahony et al. treated patients with probiotics and found that Lactobacillus salivarius UCC4331 significantly reduced pain and discomfort during the administration phase for one week, while B. infantis 35624 reduced pain and discomfort scores during both the administration and washout phases [167].

PD is a typical neurodegenerative disorder affecting more than 1% of the population over 65 years of age [172]. PD is thought to be caused by the interaction between environmental and genetic risk factors. This neuropathology is characterized by motor deficits and non-motor symptoms (NMS), which ultimately have an impact on quality of life [173].

Recently, the gut microflora has drawn increasing attention with respect to how it may be implicated in PD [173]. A variety of enteral dysfunctions are associated with PD, such as SIBO, malnutrition, H. pylori infection, and constipation [174]. In terms of the role of GI tracts pathology in PD, a higher frequency of α-synuclein detection is found in the patients than in controls from many researches [175]. Animal studies validated that resection of vagus nerve can stop transmission of α-synuclein from gastro intestine to the CNS [66, 176]. Bowel inflammation can also trigger neuroinflammation to promote dopaminergic neuronal loss in the rodent substantia-nigra tissue [177]. Forsyth and colleagues found that “leaky guts” and bowel inflammation affect the progression of PD [178]. The increased colonic permeability was found to be correlated with increased α-synuclein and E. coli accumulation in the sigmoid of PD patients [179].

Counts of butyrate-producing and anti-inflammatory bacterial genera such as Blautia, Coprococcus, and Roseburia are significantly lower in PD patients, while those of LPS-producing genera Oscillospira and Bacteroides are significantly higher [180]. The pro-inflammatory genus Ralstonia is significantly abundant in PD patient mucosae, implying that the inflammatory gut barrier is pathogenic. Scheperjans et al. revealed that the altered intestinal microbiota and the related motor phenotype could be applied to the diagnosis of PD [181]. The relative abundance of Prevotellaceae is significantly reduced in PD and has been validated as a very sensitive biomarker for PD diagnosis. A model based on multiple bacterial families and constipation status was shown to present 90.3% specificity in PD diagnosis. Ingestion of fermented milk for 4 weeks was proven to be effective in improving PD complications such as constipation [182]. Two studies have shown that anti-TNF therapy and immunosuppressants reduced PD risk [183, 184]. However, there is very limited clinical evidence of the beneficial effects of probiotics in the treatment of PD and further evidences are needed.

AD, which is the most common degenerative disease, is characterized by a decline in cognitive skills, including memory, language, and problem solving, eventually resulting in dementia [185]. AD is pathologically characterized by neuroinflammation, accumulation of beta-amyloid (Aβ) plaque, and neurofibrillary tau tangles in the brain. Aβ (Aβ40 or Aβ42) are cleaved products from the amyloid precursor protein (APP). The Aβ polymerizes into fibrils in the CNS by self-aggregation and further triggers inflammation and neurotoxicity. Recent studies have demonstrated the role and impact of microbial dysfunction and infection on the aetiology of AD, especially the activation of neuroinflammation and the formation of amyloids.

Neuroinflammation factors, including IL-1beta, IL-6, and TNF-alpha, have been observed in AD patients [186, 187]. Gareau et al.’s work implied that memory loss and dysfunction are exacerbated by infection under exposure to acute stress [129]. Released LPS triggers the inflammation and promotes amyloid fibrillogenesis in the brain [188]. The microglia can recognize amyloid molecules by TLR4 and TLR2 for clearance. Interestingly, signaling of myeloid differentiation primary response 88 (MyD88) from microglial TLR2 is responsible for activation of TNF-α and nuclear factor kappa B (NF-κB) which also induces the formation of Aβ by promoting α-secretase and β-secretase, respectively [189].

As a generic term, amyloid denotes any insoluble, aggregation-prone, and lipoprotein-rich deposit that resembles carbohydrate starches [190]. Bacterial metabolites and products have recently been shown to worsen AD. In AD patients, bacteria-derived amyloids (curli, tau, Aβ, α-syn, and prion) can function as initiators to cross-seed and aggregate host amyloids [191]. Amyloids such as CsgA and Aβ42 can exhibit cerebral deposition and trigger a cascade of AD-related pathological events despite their dissimilarity of sequences [192]. It is reported that chronic H. pylori could trigger the release of both inflammatory mediators and amyloids in AD patients [193, 194]. H. pylori filtrate was shown to have the ability to induce the hyperphosphorylation of tau protein in a cell model [195]. E. coli has been reported to produce extracellular amyloids known as curli fibres, a major subunit of CsgA. Other amyloids produced by microbes include CsgA produced by Salmonella spp., FapC by Pseudomonas fluorescens, MccE492 by Klebsiella pneumonia, phenol-soluble Modulins by Staphylococcus aureus, TasA by Bacillus subtills, and Chaplins by Streptomyces coelicolor [190, 196, 197]. Harach et al. found a remarkable shift in the gut microbiota in fecal samples from an APP-transgenic mouse and reported that the microbiota contributed to the development of this neurodegenerative diseases. Intestinal germ-free APP transgenic mice were found to have a reduction of cerebral amyloid pathology compared to controls [198]. More recently, Chlamydia pneumoniae infection in astrocytes has been demonstrated to be involved in the generation of β-amyloid, which promotes AD [199]. Ingestion of probiotics has been reported to be beneficial in AD. Azm S et al. demonstrated that Lactobacilli and Bifidobacteria were effective in ameliorating memory and learning dysfunction in a β-amyloid-injected rodent model [200]. Wang and Liang et al. revealed that Lactobacillus fermentum NS9 and Lactobacillus helveticus NS8 alleviated ampicillin-induced spatial memory impairment and improved the spatial memory of chronic restraint stress [201, 202]. In a randomized clinical trial of probiotic treatment conducted in 60 AD patients, Akbari et al. showed that after 12 weeks of administration of Lactobacillus and Bifidobacterium species via fermented milk, mini-mental state examination (MMSE) scores improved significantly. Improvements in glucose and lipid metabolism were also observed, which were thought to enhance the cognitive assessment scoring [203]. The elimination of Helicobacter pylori by triple eradication therapy results in improvement of cognition parameters in AD patients [204]. It is promising that the use of pro- or antibiotics could be future therapeutic agents for AD. It has been demonstrated in a recent study that exercise and probiotics could reduce Aβ plaques in the hippocampus, improve cognitive performance, and finally, attenuate the development of Alzheimer's disease in the mouse model [205].

ALS is a fatal neurodegenerative disorder that affects the brain and spinal cord neurons and typically results in death. Most ALS patients die within 3 to 5 years due to respiratory paralysis [206]. The pathogenesis of ALS is proposed to be outcomes of genetic-environmental interactions. Studies on interactions between innate immune response and LPS have provided essential evidences of ALS pathogenesis [207‐209]. More than a decade ago, it was proposed that gut-derived neurotoxins, including tetanus and botulinum toxins produced by Clostridia species, cause ALS [210, 211]. “Leaky gut” is also likely to be responsible for ALS [212]. More recently, Wu’s ALS mouse model revealed that the tight junction structure was damaged and that gut permeability was increased. Gut dysbiosis is also found in ALS mice, particularly in terms of reduced levels of butyrate-producing bacteria, including Butyrivibrio fibrisolvens and E. coli [213]. In the gut of ALS patients, butyrate-producing Oscillibacter, Anaerostipes, and Lachnospira counts were found to be reduced, while that of glucose-metabolizing Dorea was significantly increased [214]. Brenner et al. found the richness of OTUs to be significantly higher in an ALS group compared with a control group. However, the relative ratio of Bacteroidetes/Firmicutes did show a significant difference between ALS patients and controls [215]. Supplementation of the diet with 2% butyrate in drinking water in an ALS mouse model resulted in improved gut integrity and survival [216]. Mazzini et al. revealed unique bacterial profiles in ALS patients compared to the controls. Higher E. coli and Enterobacteria abundance, and lower Clostridium was found in ALS participants [217]. These results imply that anti-inflammatory SCFAs produced by gut microbiota are potential therapeutic agents affecting ALS progression. Mazzini et al. further conducted a clinical trial of bacteriotherapy aimed at understanding the effect of Lactobacillus strains [217]. However, more direct evidence and results are needed to clarify how the gut microbiota improves or aggravates ALS.

MS is a type of autoimmunity-induced neurodegenerative disease in the spinal column and CNS. The environmental factors contribute profoundly to pathogenesis of this demyelinating disease, including obesity, smoking, viruses, and vitamin D [218]. Alterations in the microbiome and prevalence of “leaky gut” have been found in MS patients and experimental autoimmune encephalomyelitis (EAE) animal, the most commonly used inflammatory demyelinating disease model [219, 220]. The immunological changes in EAE are characterized by increasing proinflammatory cell infiltration and impaired Treg function [221]. The favorable gut microbiota can regulate permeability of BBB, limit astrocyte pathogenicity, and activate microglia [222]. In the EAE model, the Bifidobacterium and lactic acid-producing bacteria such as of Lactobacillus are able to reduce severity of EAE symptoms [223, 224]. The abundance of Archaea is high in MS while Firmicutes and Bacteroidetes phyla are lower or even depleted [218]. The Bacteroides and Clostridia species are especially responsible for the induction of FoxP3+ Tregs in suppression of inflammation [222]. One small longitudinal study found that the gut microbiota profiles, especially Fusobacteria were associated with future relapse risk in MS [225]. Psuedomonas, Haemophilus, Blautia, and Dorea genera were detected to be increased in MS patients, while Parabacteroides, Adlercreutzia and Prevotella genera were much lower [219]. Clostridiales order were restored after MS treatment with glatiramer acetate including Bacteroidaceae, Faecalibacterium, Ruminococcus, Lactobacillaceae, and Clostridium [226]. Ochoa-Reparaz et al. unraveled that oral administration of antibiotics can delay the pathogenesis of EAE. However, intraperitoneal injection did not significantly impact the outcomes [227]. This implies changes in intestinal microbiota ecosystem are associated with MS development. Other studies also validated that probiotic treatment and fecal microbial transplantation can achieve similar effect [228, 229].

Fernandes et al. revealed that oral Streptococcus mutans is ubiquitous in the atherosclerotic plaques of patients with vascular disease [230]. Apfalter et al. identified traces of Chlamydia pneumoniae in carotid artery atherosclerosis using a nested PCR-based approach [231]. Mitra et al.’s metagenomic study of carotid atherosclerosis plaques showed that 2–16% of the sequencing reads from plaque tissue came from bacteria, including Lactobacillus rhamnosus and Neisseria polysaccharea. The bacterial content is higher in patients with ischemic symptoms. Acidovorax spp. and H. pylori cells were also detected by fluorescence in situ hybridization (FISH) in atherosclerotic tissue [232]. Karlsson et al. sequenced the gut metagenomes of patients with symptomatic atherosclerosis and healthy subjects. The genus Collinsella was observed to be enriched in carotid stenotic patients, whereas Roseburia and Eubacterium were more abundant in healthy controls [233].The gut microbiota has been suggested to transform dietary choline, lecithin, or carnitine into TMAO and, thus, cause vascular atherosclerosis by affecting lipid and hormonal homeostasis [89, 234]. The carnitine-butyrobetaine-trimethylamine-N-oxide pathway has been found to be associated with carotid atherosclerosis [235]. Stimulation of monocyte by LPS can be a critical step of the formation of foam cell in atherosclerotic plaque by either inducing inflammation or triggering LDL uptake [236, 237]. A series of probiotic-based therapy attempts have been in progress, including human trials using L. acidophilus 145, B. longum 913 [238], L. acidophilus and B. bifidum [239] to restore HDL/LDL ratio, and mouse experiments using L. plantarum ZDY04 against TMAO [240].

Benakis et al. showed that infarction-induced ischemic brain injury could be reduced by antibiotic administration. The antibiotic-induced alterations in the gut microbiota could increase regulatory T cells and decrease IL-17+ γδ T cells, which suppress the trafficking of effector T cells after stroke. Interestingly, the gut microbiota-associated infarction model can be transmitted and reproduced by fecal transplantation [241]. Systemic exposure to Porphyromonas gingivalis has been linked to an increased risk of ischemic stroke [242]. Kawato et al. revealed that continual venous injection of Gram-negative bacteria might accelerate stroke in SHRSP (spontaneously hypertensive stroke-prone) rats, probably due to LPS-induced oxidative stress responses [243]. Zhu et al. discovered that TMAO is associated with platelet hyperactivity and thrombosis risk [90]. Their following study validated in a short-term cohort that an omnivorous diet could generate a higher TMAO content in blood than was induced by a vegan diet, which was more likely to promote the formation of blood clots. Their study suggested that the use of low doses of aspirin was helpful to alleviate platelet aggregation caused by TMAO [244]. Yin et al. revealed that opportunistic gut-derived pathogens such as Enterobacter, Megasphaera, Oscillibacter, and Desulfovibrio were enriched in patients experiencing stroke and transient ischemic attack, whereas beneficial genera including Bacteroides, Prevotella, and Faecalibacterium were less abundant. Paradoxically, Yin et al. showed that TMAO levels were lower in patients than in controls [245].

There is no direct causal relationship between the microbiota and AVM onset in humans, while animal models have implied a pivotal role of the microflora in AVM pathophysiology and pathogenesis. Shikata et al. used antibiotics to deplete the gut microbiota in an aneurysm mouse model, and aneurysm induction was drastically reduced due to less macrophage infiltration and cytokine secretion [246]. Cerebral cavernous malformation (CCM) is a common type of vascular malformation resulting in hemorrhagic stroke and seizure [247]. Tang et al. showed that stimulation of TLR4 by bacteria or LPS accelerates CCM formation, whereas the use of germ-free mice and antibiotics use in mice can prevent CCM. The gut microbiome and endothelial TLR4 act as critical stimulants, suggesting their potential use as novel strategies in CCM therapy [248]. Interestingly, Winek et al.’s paradoxical results indicated that a broad-spectrum antibiotic-treated gut microbiota worsens inflammation and stroke in a murine model [249]. However, Evangelo Boumis et al. found that consumption of probiotics such as Lactobacillus rhamnosus may impose a serious risk of infection in patients with special susceptibility factors, and antibiotic prevention should be considered [250]. These findings implied that specific microbiota and antibiotic use result in different outcomes.