Interplay between Human Intestinal Microbiota and Gut-to-Brain Axis: Relationship with Autism Spectrum Disorders

A growing body of scientific reports suggests a relevant key role of human intestinal microbiota (HIM) in maintaining the host’s physiological and mental balance; thus any disturbance in the microbiota diversity and/or concentrations may result in impaired stimulation of the gastrointestinal (GI) system-central nervous system (CNS) bidirectional pathway, termed gut-to-brain axis. Recent data show that HIM composition is significantly unbalanced among a subset of autism spectrum disorder (ASD) subjects, as compared with non-ASD siblings or age-matched control subjects. Several authors claim that specific changes in HIM (diet-based alteration of Bacteroidetes/Firmicutes ratio and death of predominant microbiota after antibiotic treatments, among others) could either trigger or be highly associated events with persistent ASD signs and behaviors. Whether HIM plays a causative or a circumstantial role in ASD severity, then HIM manipulation might be applied as a therapeutic alternative to improve ASD clinical manifestations and behaviors. occurs primarily via neuro-immune and neuroendocrine mechanisms. Those microbial molecules reach brain sites directly or only induce central responses through long-distance neural signaling by vagal and/or spinal afferents. The autonomic nervous system regulates gut functions (motility, secretion, intestinal permeability, and mucosal immune response), which ultimately affect the microbial habitat, thereby modulating microbiota composition and activity.


Introduction
Communication between the gastrointestinal (GI) system and the central nervous system (CNS) occurs constantly and plays a critical role in maintaining the healthy status. That communication engages a bidirectional stimulation system, involving not only the brain and gut cells but endocrine-, immune-, and microbiota-derived components as well, the so-called gut-to-brain axis (GBA) [1,2]. Consequently, impaired communication between both ends of the GBA, associated with or as consequence of disturbance of the GI microbial diversity, has been associated with a negative health outcome later in life [1,3].
Increasing evidence points out that there is a link between alterations of gut microbiota and several disorders of the central nervous system including autism spectrum disorders (ASD) depression, anxiety, irritable bowel syndrome, attention deficit and hyperactivity disorder (ADHD), Parkinson's disease, disorders of mood and affect, and chronic pain [3][4][5]. multiple microbial members. Second, bacterial taxa present in the community are then defined by amplification of the 16S rRNA gene followed by sequencing. Highly similar sequences are grouped into operational taxonomic units (OTUs) or phylotypes, which can be compared to 16S rRNA databases to identify them as accurately as possible. An alternate method identifies community taxa after the total DNA is metagenomically sequenced and compared to reference genomes or gene catalogs. The OTUs can be described in terms of their relative abundance and/or their phylogenetic relationships, while sequenced genomes can be described as relative abundances of its genes and pathways [19].
The human intestinal microbiome is mainly defined by the high abundance of two bacterial phylotypes: Bacteroidetes and Firmicutes. Other phylotypes present at lesser amounts are Proteobacteria, Actinomyces, Fusobacterium, and Verrucomicrobia [14]. The gut microbiome is conformed with nearly 470 phylotypes, more than 1000 bacterial species representing more than 5000 strains, which in turn encode between 5 and 10 million of nonredundant genes (150-fold the number of genes identified in the human genome) [16,17]. Studies on intestinal microbiome in health and disease revealed two microbiome subpopulations, one with high-gene counts and the other with low-gene counts; the first one seems to be associated with a healthy digestive status [20,21].
Every person has a unique microbiome profile; still there is a reduced number of species shared between persons. The aforesaid feature allowed to classify individuals into one of three enterotypes, each one based on the proportions of the three predominant intestinal genera, based on their abundance, Bacteroides, Prevotella, and Ruminococcus. The first two genera represent the Bacteroidetes, and the last one represents the Firmicutes. Enterotype 1 shows predominance of Bacteroides, while enterotypes 2 and 3 were defined by predominance of Prevotella and Ruminococcus, respectively [22].
Alterations of the typical GI microbiota, in number and abundance distribution of distinctive types of microorganisms, and the host's adverse response to such changes have been called as dysbiosis. Thus, dysbiosis with low diversity has been linked particularly with obesity, inflammatory bowel disease, and ASD [16].
There are two ongoing multi-group projects on human microbiome, the Europebased Metagenomics of the Human Intestinal Tract (MetaHIT) and the US-based Human Microbiome Project (HMP). Both of them will allow to define to its finest details the microbiome diversity, at least to species level, their genetic load, and how microbiota interacts with the host [23,24].

Biological role of GI microbiota
The intestinal microbiota maintains a symbiotic relationship with the host. Studies in both humans and mammals have implicated the intestinal microbiome in several physiological processes that are pivotal to the host health, from food digestion and energy homeostasis to immune and neurobehavioral development [25].
The single layer of intestinal epithelial cells, connected by tight junctions, constitutes itself a physical and biochemical barrier that segregates the commensal microbiota organisms to maintain intestinal homeostasis. This occurs through regulation of nutrients, electrolytes, and water absorption, as well as through release of mucins, antimicrobial peptides, and IgA for the prevention of the entry of pathogenic microorganisms [26][27][28]. The interaction between the microbiota and intestinal epithelial cells also promotes tissue restoration in the setting of injury or acute inflammation, thus supporting epithelium integrity. Besides the above statement, the microbiota provides protection against exogenous pathogenic organisms, either through competition for common nutrients and niches or by prompting development and functional maturation of the gut immune system, including gut-associated lymphoid tissue, T-helper 17 cells, inducible regulatory T cells, IgAproducing B cells, and innate lymphoid cells [13].
The HIM microbiota has a considerable input on the metabolomic profile, the complete set of intestinal metabolites, of the host [29]. Specifically, the microbiota is a major source of both circulating organic acids and tryptophan metabolites, which have beneficial effects on the host health ( Table 1) .
Fermentative processes of nondigestible complex carbohydrates, from dietary fiber, by Firmicutes and Bacteroidetes, result in the production of various shortchain fatty acids (SCFAs), such as acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate. These bacteria-derived SCFAs, in a physiological context, may serve as an energy source for enterocytes, stimulate water and sodium absorption, decrease colonic pH, etc. [30].
Hyperproduction or deficiency of SCFAs may also affect the pathogenesis of a diverse range of diseases, from allergies and asthma to neurological diseases [17,29,30]. For example, a diet high in fat and digestible saccharides provokes that majority of nutrients be absorbed in the duodenum, leaving very few substrates for the colonic bacteria, leading to dysbiosis. Higher levels of SCFAs can also alter the intercellular spaces between the cells, resulting in a leaky gut that allows for more metabolites and bacteria to pass through the epithelial barrier, where bacterial endotoxins and other microbial-derived metabolites can gain entry into the bloodstream [17] Furthermore, dysbiosis can affect host immunity and neurobehavioral responses [17,29]. Among SCFAs, butyrate is a promoter of colonic functionality and physical integrity, via cholesterol-rich membrane microdomain, as well as the preferred metabolic substrate for the colonocytes' energy requirements [54].
Essential vitamins such as folate, vitamin K, and vitamin B12, for the host's growth, are synthesized by gut microbiota, which in turn may affect DNA and histone protein methylation [55][56][57]. Certain hormones and vitamins also participate in drug and poison removal [58].
Apart from carbohydrates, GI bacteria also metabolize complex lipids and proteins that are indigestible by the host [59][60][61]. Expression of colipase, a critical protein factor for lipid metabolism, and subsequent stimulation of the release of pancreatic lipases appear to be regulated by Bacteroides thetaiotaomicron [59].
The metabolism of tryptophan by the HIM and/or gut and immune cells follows three alternative pathways: (a) the transformation to ligands of the aryl hydrocarbon receptor (AhR), (b) the kynurenine pathway (via indoleamine 2,3-dioxygenase 1), and (c) the serotonin (5-HT) production pathway. These pathways are performed by HIM, enterocytes/immune cells, and enterochromaffin cells, respectively. The HIM pathway yields several molecules, indole-3-aldehyde, indole-3-acid-acetic, indole-3-propionic acid, indole-3-acetaldehyde, and indoleacrylic acid. AhR signaling is crucial for gut epithelium renewal and barrier integrity and acts over many immune cell types for responsiveness [62].
Microbial metabolism of tryptophan is very important for intestinal AhR activity, since the absence or imbalance of tryptophan-metabolizing organisms generate deficiency of AhR agonists [46]. The production of AhR ligands have been determined among few HIM species, Peptostreptococcus russellii and Lactobacillus ssp. Many GI and neuropsychiatric diseases have been related to dysbiotic impairment of tryptophan metabolism or to accumulation of the end products [62].
Several HIM species not only can synthesize but respond as well to hormones and neurotransmitters of bacterial and human origin, which impact their growth and virulence. Beneficial Lactobacillus spp. are able to synthesize acetylcholine and gammaaminobutyric acid (GABA), while Bifidobacterium spp. produce GABA. Escherichia spp. produce norepinephrine, serotonin, and dopamine; other Firmicutes species Bifidobacterium spp.
Reduced adiposity [52] F. prausnitzii Improved insulin sensitivity [53] I3A, indole-3-aldehyde; IPA, indole-3-propionate; HYA, 10-hydroxy-cis-12-octadecoate (linoleic acid derivative); CLA, conjugated linoleic acid. Modified from [29]. belonging to Streptococcus and Enterococcus produce serotonin, and Bacillus produce norepinephrine and dopamine [17]. Those bacteria-derived neurotransmitters released directly to the intestinal lumen may either induce epithelial cells to in turn release molecules that modulate neural signaling within the enteric nervous system (ENS) or, after passing through the gut wall, gain entry into the portal circulation to exert direct effects on afferent axons [63,64]. Indeed, several reports documented elevated levels of noradrenaline and adrenaline in the plasma of subjects coursing with systemic infections by gram-negative Proteobacteria, like Escherichia coli. (Reviewed in [64]).

Stability of HIM
Bacterial colonization of the human gut likely occurs at the time of birth, when infants born via vaginal delivery are inoculated with a complex mixture of maternal vaginal microorganisms. According to Dominguez-Bello et al. [65], those infants had colonizing Lactobacillus, Prevotella, or Sneathia species in their skin and mucosae, which resembled their own mother's vaginal microbiota. In contrast, infants delivered by cesarean section had predominantly Staphylococcus, Corynebacterium, and Propionibacterium species, akin to their mothers' skin microbiota. Thus, there is concern that babies delivered via cesarean section may receive an insufficient maternal bacterial load [17].
After birth, breastfeeding is the main factor defining the composition of newborn's GI microbiota, since breast milk provides a variety of specific antibodies and immediate immunity molecules that neutralize pathogenic bacteria. Breast milk also contains more than 200 oligosaccharides (prebiotics) that favor the growth of bifidobacteria [66,67], which have been reported to prevent gastrointestinal infections by competitive exclusion of pathogens based on common binding sites on epithelial cells [67]. Therefore, in breastfed children, bifidobacteria reaches up to 90% of GI microbiota, followed by lactobacilli, Bacteroides, coliforms, and clostridia. In contrast, infants fed with infant formula have predominance of Bacteroides, enterococci, coliforms, and clostridia, with much lesser bifidobacteria, resembling the more diverse GI microbiota of adults [66,67].
The initial breastfeeding-driven colonization is essential for induction of adaptive immunity and for early metabolic programming. After the introduction of complementary feeding, the microbiota differences between breastfed children and those fed with formula tend to disappear. It is assumed that the predominant bacteria in the intestinal microbiome of 3-year-olds are similar to those of adults and remain relatively stable lifelong [66,67].
Daily variability of the HIM composition has been assessed in controlled feeding studies, specifically short-term administration of extreme amount of fat and fiber intake, which revealed disturbance of the intestinal microbiome, but this effect was of low-scale and transient that not changed the individual's enterotype designation [66,67].

Gut-to-brain axis
The basis of the GBA cross-communication includes an array of multichannel sensing and trafficking pathways (neural, endocrine, immune, and metabolic) to transfer the enteric signals to the brain (Figure 1), which ultimate results in keeping proper maintenance of GI homeostasis, although its multiple effects likely impacts on brain performance and higher cognitive functions [1][2][3]68].
The GBA comprises highly interconnected body systems. Those systems are the CNS, the autonomic nervous system (vagal and spinal nerves), and the ENS Interplay between Human Intestinal Microbiota and Gut-to-Brain Axis: Relationship… DOI: http://dx.doi.org /10.5772/intechopen.89998 (the arrangement of neurons and supporting cells throughout and embedded within GI tract, from the esophagus to the anus). Other critical components of GBA include the hypothalamic pituitary adrenal axis (HPA; release of gut hormones), the immune system (release of multiple cytokines), and bacteria-derived metabolites (SCFAs and free amino acids). In fact, gut microbes have evolved alongside their host, through complex relationships, so influencing their own genotypic and phenotypic features [1][2][3]. However, failures in the GBA cross talk may lead to a number of health disorders, from inflammatory to metabolic and neurodevelopmental conditions, including ASD [1].
The following pathways may explain the influence of the gut microbiota on neurologic disorders through GBA: (a) production of neurotransmitters, (b) triggering release of gut hormones from entero-endocrine cells, (c) stimulation of the ENS and signaling to the brain via ascending neural pathways, and (d) activation of the immune system via cytokine release by the mucosa-associated immune cells.
At physiological conditions, GBA modulates the digestive processes like motility and secretion, immune function, and perception and emotional response to visceral stimuli [17]. The high comorbidity of stress-related neurologic disorders with GI disorders proves the impact of altered function of GBA [3].

Autism spectrum disorders
ASD is a group of neurodevelopmental abnormalities whose clinical manifestations begin in early childhood (although their diagnosis may delay months to years later in life). Clinically ASDs show complex and heterogeneous features but generally are defined by a core symptomatology including impaired social communication (oral and nonverbal languages, eye contact), behavioral problems (fixated interests in the daily routine, engagement in repetitive manners, exacerbated responses to external stimuli), and self-isolation, with or without impairment of cognitive abilities and competences [9,69].
According to the latest American Psychiatric Association's diagnostic criteria [69], ASDs include conditions known as autism disorder (AD), Asperger's syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS).
Noteworthy ASD clinical features show extensive heterogeneity among affected subjects, according to the developmental stage, to chronological age, and to specific disorder within the spectrum (and even within the same disorder) [9,69].
Until of April 2018, ASD were estimated to affect, in average, 1 in every 160 children worldwide, with a yearly rising incidence, and an estimated boy to girl ratio of 5:1 [70]. Data from the USA reveal that prevalence of ASDs has dramatically increased from 4.5 in 10,000 children in 1966 to 1 in 68 in 2010 and finally to 1 in 59 children in 2014 [71].
This recent outburst in frequency may be partly attributed to increased public awareness and or to better diagnosis; however, the occurrence of other factors, such as exposure to environmental chemicals, diet alterations, metabolic status, and changes in microbiota composition, cannot be excluded [17].
Despite the alarming rise trend in frequency of diagnosed cases in developed countries, the etiopathogenesis of ASD is still unknown; thus, there are no consensus in medical, neurologic, or psychiatric treatments [10]. Moreover, a diversity of comorbidities also affect ASD individuals, including one or more of the following: anxiety, intellectual disability, epilepsy/seizures, attention deficit and hyperactivity disorder, GI disorders, sleep disorders, obesity, depression, bipolar disorder, and Tourette's syndrome, among others (Figure 2) [6,7,9].

Interplay between HIM and GBA in the context of ASD
About 40-60% of ASD children suffer from gastrointestinal comorbidities [8], although due to their social and communicative impairments, the real prevalence of gastrointestinal issues among ASD patients may be higher. Such intestinal dysfunction in this group of patients may be caused by disturbances in the pathways underlying the GBA, with a central role of the HIM and including an immune component.
Several studies have demonstrated HIM dysbiosis in ASD subjects; however, little or null correlation between studies has been obtained, mainly due to variations in study groups, control groups, and the use of diverse methods for microbiota/ microbiome determinations and analysis ( Table 2) [67,72]. In short, 13 of the 15 studies showed some degree of dysbiosis among ASD patients as compared with controls (total combined sample of 585 individuals, 339 ASD, 61 control siblings, and 185 unrelated neurotypical controls), whereas 2 of the 15 studies found no significant differences among ASD subjects as compared with siblings controls (no neurotypical controls were included).
Dysbiosis in ASD is also associated with increased permeability of the GI tract, the leaky gut, which leads to the entry of endotoxins, and other bacterial products into the bloodstream [92]. Bacterial lipopolysaccharide (LPS) can alter neuronal as well as microglial activity in brain regions involved in emotional control [93][94][95]. In fact, serum levels of LPS were significantly higher among ASD subjects compared to healthy individuals and correlated with impaired social behavioral scores [96].
Serotonin synthesis in the gut and the brain depends on the availability of dietary tryptophan. High levels of blood serotonin were found in children with ASD [97][98][99], which contrasts with finding of decreased brain serotonin synthesis in ASD subjects [100]. A significant correlation between whole-blood serotonin levels and low-grade intestinal inflammation in ASD was demonstrated [101]. Regarding these findings, a likely explanation was proposed by de Theije et al. (2011) [91]: After GI inflammation, the intestinal serotonin release provokes changes in motility, secretion, vasodilation, and permeability, leading to functional intestinal dysmotility, stool inconsistency, and abdominal pain. Since the majority of dietary tryptophan is transformed in serotonin by HIM during inflammation, less tryptophan (and serotonin) will be available for the brain resulting in mood and cognitive dysfunction in ASD and increased autistic behavior [102].
Propionic acid, a major SCFA produced by clostridia, Bacteroides, and Desulfovibrio, has been associated with ASD, since it can induce ASD-like behavioral deficits in rats [103,104]. Detrimental effects of propionic acid are suggested to be through mitochondrial and epigenetic modulation of ASD-associated genes. In fact, elevated levels of SCFAs are described in the stool of ASD children [82,105]. ↑ Ruminococcus torques (only in ASD-GI+). ↑Ammonia and SCFA (acetic, butyric, isobutyric, valeric, isovaleric acids), likely microbial-derived.
No differences in phenol and p-cresol levels [81][82][83] Interplay between Human Intestinal Microbiota and Gut-to-  Scientific literature supports the notion that the HIM plays a crucial role in the pathogenesis of ASD, so scientists are now targeting gut microbiome as a therapeutic approach for such disorder (reviewed in [106]). First, modification of high lipid and sugar diet for a fiber-and protein-containing one showed improved skills while ameliorated ASD behavioral deficits. Second, supplementation with prebiotics (inulin, fructo-oligosaccharides, galacto-oligosaccharides, and lactulose) allows specific changes, both in the composition and/or activity of the gut microflora, mainly inducing the growth of indigenous lactobacilli and bifidobacteria. Third, probiotics administration, either Bacteroides fragilis or Lactobacillus reuteri, there were improvements in ASD-associated behaviors, counteract effect of harmful infections and stimulation of the host's immune system. Fourth, fecal microbiota transplant, usually applied for treating recurrent Clostridium difficile infection and other GI disorders, consists of a sample containing about a thousand indigenous bacterial species of the GI from a neurotypical donor, treatment showed sustained improvement of both GI-and ASD related symptoms (up to 8 weeks posttreatment).

Conclusion
After the complete sequencing of the human genome was achieved, the scientific community began, in the second half of the past decade, the task of mapping the human microbiota, mainly the intestinal microbiota. In parallel, the notion that the ENS interplay with the intestinal microbiota, generating responses in the CNS, through the GBA and HPA axis, has opened an avenue for the study of gastrointestinal, metabolic, and/or neuropsychiatric disorders.
In this landscape, an increasing body of evidence suggests that HIM has a key role in gut and brain development and functionality but also in pathogenesis of mental disorders, including ASD. Studies on ASD have showed that HIM dysbiosis, with altered Bacteroidetes/Firmicutes ratio, presence of detrimental key species, and dysregulation of bacterial metabolite release, appears to correlate with severity of ASD symptoms. In this regard, intervention measures to restore HIM homeostasis are likely promising.
However, the part concerning the microbiota is only one more piece of the puzzle that are ASDs, mainly because the etiology of such disorders remains elusive.