Gut Permeability Pathway The microbiota and its metabolite products modulate the function and integrity of the gut epithelium barrier. Therefore, a change in gut microbial diversity can influence the gut barrier integrity, potentially resulting in the “leaky gut” condition. Indeed, an impaired gut barrier can increase the levels of gut microbial components (e.g., lipopolysaccharide (LPS)) in the blood; trigger the hypothalamic–pituitary–adrenal (HPA) axis; and stimulate immune responses, producing cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-4. These immune cytokines can circulate and cross the blood–brain barrier (BBB), inducing systemic and CNS inflammation. The serum level of LPS was found to be significantly increased in ASD individuals compared to healthy controls. This may be linked to a worse social communication score, which has been noticed in ASD patients. In physiological states, LPS can enter the brain, possibly through a lipoprotein transport mechanism, and elicit neural impairment, behavioral alteration, and neuroinflammation by triggering the Nuclear Factor Kappa B (NF-kB) signalling pathway, which is related to microglia stimulation and neuronal cell loss. The everyday injection of pregnant rats with lipopolysaccharide (LPS) resulted in ASD-like behavior in offspring, involving hyperlocomotion and social defects. Multiple findings have suggested that ASD patients have abnormal intestinal permeabilities ranging from 43% to 76%, both with and without gastrointestinal symptoms. Moreover, intestinal permeability was reported in 9 out of 21 autistic children, but not in 40 non-autistic children [26]. De Magistris and colleagues found that ASD individuals and their first-degree relatives had 36.7% and 21.2% altered gut permeabilities, respectively, while ordinary people had only 4.8%. In accordance with previous studies, a significant decrease in the mRNA levels of occludin and zonulin was observed in male BTBR mice (a mouse model of idiopathic autism). Occludin and zonulin are intestinal permeability-modulating proteins that are associated with the maintenance of intestinal permeability. Interestingly, intestinal permeability was found to be considerably reduced in autism patients who were on a gluten-free, casein-free diet. In comparison with the above-mentioned studies, others have shown no changes in gut permeability in autistic children, demonstrating that the disruption of the intestinal barrier is not always a symptom of autism, but this primarily occurred in ASD children with intestinal abnormality. Immune System Pathway Immunological pathways have a vital function in the bidirectional connection between the microbiota, gut and brain, allowing the gut and brain to influence each other. Gut microbial composition is an essential part of regulating immune hemostasis, since gut mucosal surfaces are constantly exposed to beneficial and pathogenic microorganisms and can trigger an immunological response. In addition, the mucosal surface layers of the gut contain different types of immune cells involving gut-associated lymphoid tissue (GALT). GALT utilizes lymphocytes to produce immunoglobulins (IgA). IgA can modify the innate immune response once microbial cells come into contact with dendrites in the ENS. In some studies, a high level of IgA was recognized in ASD patients. Different inflammatory signs have been found in ASD individuals. For example, elevated levels of tumor necrosis factor (TNF) and pro-inflammatory cytokines such as interferon (IFN), IL-1b, IL-6, IL-8, and IL 12p4 were found in the brains of ASD children compared to controls. Moreover, the brains of ASD patients revealed a pattern of triggering immunological responses involving the activation of microglial cells, which are responsible for eliminating pathogens. The defect in the immune system in autistic patients has been connected with the alteration of the gut microbial composition. For example, germ-free mice show a higher microglia density in various brain areas than mice grown in a specific pathogen-free (SPF) environment. Additionally, atypical social avoidance behavior and low immune response against virus infection were noticed in these GF mice. Both microglia defects and ASD-linked symptoms were improved following the supplementation of germ-free mice with microbial SCFAs. This study proposed that the gut microbiota can indirectly affect the innate immune system, which can modify the circulating levels of pro-inflammatory and anti-inflammatory cytokines that directly impact microglia homeostasis. Moreover, in the Hsiao et al. study, an increased level of IL-6 was detected in the adult offspring of a maternal immune activation (MIA) mouse model. Interestingly, the supplementation of MIA offspring with Bacteroides fragilis NCTC 9343 restored microbiota composition, IL-6 levels, and the integrity of the intestinal permeability. Several cytokines, including IL-6, were found to adjust the tight junction transcription level and intestinal barrier integrity by modulating the levels of CLDN 8 and 15. Therefore, this report proposes that the B. fragilis-mediated restoration of IL-6 levels might underpin the role of IL-6 in gut permeability. The Metabolic Pathway The gut microbiota generates various metabolites that can travel across the systemic circulation and contact the host immune cells, impact the metabolism, and/or influence the ENS and afferent signaling pathways of the vagus nerve that send signals directly to the CNS. The metabolites that are derived from the microbiota include multiple products, such as short-chain fatty acids (SCFAs), phenolic compounds, and free amino acids (FAAs). Butyric acid (BA), propionic acid (PAA), and acetic acid (AA) are all types of short-chain fatty acids that result from the anaerobic fermentation of indigestible carbohydrates. SCFAs play a vital function in the body such as in the homeostasis of energy, in the enhancement of glucose metabolism, in lowering body weight, and in reducing the chance of colon cancer. Additionally, SCFA is implicated in the regulation of the immune response by modulating the secretion of T-cell cytokines. Despite the data being slightly inconsistent, acetate and propionate have been found to be upregulated in individuals with ASD, whereas butyrate was shown to be significantly decreased. PAA can act as a neurotoxin that affects the electron transport chain by inhibiting the formation of nicotinamide adenine dinucleotide (NADH), the primary substrate of the electron transport chain. PAA can also trigger the immune response and change gene expression. Increased levels of PAA have been related to increased severity of ASD for example, in experimental trials, rats treated for eight days with PAA displayed hyperactivity and stereotypy movement. Additionally, PAA-treated rats exhibited significant changes in the composition of brain and plasma phospholipid molecular species. Alterations in brain plasma phospholipid composition, especially throughout development, can theoretically have severe effects on CNS function. In agreement with this study, GI symptoms and modified blood phospholipid profiles have been detected in individuals with ASD. Thus, since phospholipids are the main structural components of many cellular and neuronal membranes, ASD, as a neurodevelopmental disorder, might be related to functional deficits or imbalances in fatty acid metabolism. On the other hand, butyrate was observed to have a positive influence on ASD-related behavior. In addition, butyrate can protect cells from oxidative stress and improve mitochondrial function during physiological stress. Interestingly, butyrate was found to restore the ASD deficiencies introduced by PAA, likely by enhancing the BBB permeability. GF mice colonized with Clostridium tyrobutyricum (butyrate-producing bacteria) or acetate and propionate-producing Bacteroides thetaiotaomicron can improve the expression of occludins, which were found to be associated with the reduced permeability of the BBB. Moreover, p-Cresol and its conjugated derivatives were observed at an elevated rate in the urinary samples of children with ASD. P-Cresol can aggravate ASD severity and gut function because it plays a role in many metabolic processes in the human body. In addition, P-Cresol has been linked with nervous system abnormalities, including raising brain lipid peroxidation, reducing Na(+)-K+ ATPase function, and inhibiting noradrenaline formation. Clostridium difficile is one of the most typical representative microbes and is known for forming p-Cresol. C. difficile can induce the p-hydroxyphenylacetate (p-HPA) enzyme and therefore stimulate the fermentation of tyrosine for the production of p-Cresol. Notably, mice given p-Cresol in drinking water for four weeks exhibited an altered gut microbiota composition and social-behavioral defects. The p-Cresol intervention also decreases the excitability of dopamine neurons in the ventral tegmental area (VTA) of these mice, a circuit implicated in the social reward system. The influence of p-Cresol on behavior was associated with the gut microbial composition, as microbial transplantation from p-Cresol-treated mice to control mice can stimulate behavioral defects. However, microbial transplantation from normal mice to p-Cresol-treated mice was found to restore normal social behaviors. This report suggested that a microbial metabolite such as p-Cresol could provoke ASD-like behavior in mice. Collectively, all these previous studies are consistent with the emerging theory of disruption of excitatory/inhibitory neuronal function in ASD. Neuronal Signaling Pathway The microbiota of the gut can produce molecules such as serotonin (5-hydroxytryptamine, 5-HT), γ-aminobutyric acid (GABA), and acetylcholine, which can act as typical neurotransmitters influencing ENS and CNS activity. Serotonin is one of the essential brain neurotransmitters that have a crucial function in regulating mood and GI activity. About 95% of total serotonin in the human body is formed by enterochromaffin cells (Ecs) in the GI tract, while around 5% of the remaining serotonin is found in the brain. Interestingly, gut microbes such as Escherichia spp., Enterococcus spp., Streptococcus spp., and Candida spp. have been shown to be engaged in the production of serotonin. The production and secretion of 5-HT by Ecs have been suggested to be affected by the gut microbial composition. For example, the depletion of the gut microbiota by antibiotics in mice was found to be associated with impaired learning and elevated depression-like behaviors. This occurred with changes in the levels of CNS 5-HT concentration, as well as with alterations in the mRNA levels of corticotrophin-releasing hormone receptor 1 and the glucocorticoid receptor. Moreover, a positive relationship was detected between the level of 5-HT in the blood and the severity of gastrointestinal symptoms. On other hand, serotonin can also be formed from the essential amino acid tryptophan (Trp) Clostridia spp. stimulates the transformation of tryptophan to 5-HT by raising the mRNA levels of tryptophan hydroxylase1 in Ecs. Reducing tryptophan in the diet indeed seems to increase autistic behavior. Consequently, these studies show that the gut microbiota can have a crucial role in the production and homeostasis of the 5-HT. ​ GABA is an amino acid that functions as the main inhibitory neurotransmitter in the brain. An altered pattern of GABA has been detected as a key feature of the neurophysiology of ASD patients. If the inhibitory GABAergic transmission is altered in individuals with ASD, it can end in an irregular balance of excitation/inhibition in the brain and changes in neural communication, the handling of instructions, and responding performance. Indeed, Bifidobacterium spp. and Lactobacillus spp. have the ability to produce GABA; for example, the colonization of mice with Lactobacillus rhamnosus JB-1 increases the level of GABA receptors in the vagus nerve and decreases stress and depressive behavior. Together, these outcomes emphasize the essential function of the gut microbiota in the communication pathways between the gut microbiota and the brain, suggesting that bacteria may prove to be a beneficial treatment.