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Autism spectrum disorders (ASD) gather heterogeneous neurodevelopmental disorders, often diagnosed in childhood. ASD are psychiatric disorders described in the Diagnostic and Statistical Manual of Mental Disorders, 5th edition [1]. The set of ASD disorders have some common neurocognitive symptoms as difficulties to have social and affective interactions, repetitive sensory-motor behaviors, and dysfunctions of perception and integration of information [2,3]. These disorders have a worldwide impact, but epidemiological data about ASD people are not very accurate [4]. On the one hand, the rate of ASD patients is increasing in developed countries, reaching up to 5% of the overall population. On the other hand, it is difficult to obtain accurate data in emerging countries. Moreover, there are few diagnoses of ASD in adulthood, which could explain an underestimation of ASD worldwide population [4,5]. Nevertheless, it is known that more men are diagnosed with ASD than women in the world [5]. Environmental factors such as food, prenatal and postnatal lifestyle, genetics, neurobiology, microbiota or gut dysbiosis (GD) could cause ASD [6,7]. GD is particularly interesting to study because it represents a common symptom of ASD. About 70% of children with ASD could suffer of gastro-intestinal symptoms (GIS), such as diarrhea, constipation, or abdominal pain. There seems to be a correlation between severity of GIS and neurological ASD symptoms [6,8,9]. The prevalence of GIS in ASD population is more important than in the general population [10,11]. Moreover GIS is still one of the most common symptom found in ASD population associated to GD [5,8]. The composition of gut microbiota (GM) could be a little different between individuals, but some specific phenotypes linked with ASD are established [12,13]. Bacterial signatures of the GM vary from one subject to another, but some bacterial genus are found and others disappear specifically in ASD [13]. GM could also be modified by many prenatal and postnatal factors as type of birth, breastfeeding, circadian rhythm, antibiotics, stress [7,14]. GM and brain have a bidirectional communication and constitute the microbiota-gut-brain-axis [12]. In fact, healthy GM has several essential roles in the organism, including producing essential nutrients for the brain (short-chain fatty acid or essential vitamins) and also some neurotransmitters as gamma-aminobutyric acid (GABA) or serotonin [7]. The communication between GM and brain is essential and impacts the physiological functions of the brain [15].
Most of what we know about the role of the GM in normal brain development comes from experiments carried out in mice treated with antibiotics, or in pregnant mice, either germ-free (GF mice) or reared in specific pathogen-free environment (SPF mice). Depletion of the maternal microbiome early to mid-gestation altered the expression of 333 genes in fetal brains of embryos at 14.5 days, including many involved in fetal thalamocortical axonogenesis and yields adult offspring with disrupted neurobehavioral responses to forepaw and hindpaw tactile stimuli [16]. Both in vivo and ex vivo magnetic resonance imaging revealed that SPF mice with commensal bacteria demonstrated several larger regions in grey matter and other brain regions than GF mice without commensal bacteria. This suggests that commensal bacteria are necessary for normal morphological development and maturation in the grey and white matter of the brain regions with implications for behavioral outcomes such as locomotion and cognitive functions [17]. Furthermore, a study revealed a role for the maternal microbiome in supporting placental growth and vascular development during pregnancy [18]. Placental vascular deficiencies characteristic of hypertensive disorders of pregnancy, and preeclampsia in particular, are associated with increased risk for myriad diseases in adulthood, including cardiovascular disease, kidney disease, and cognitive impairment [19]. In addition, authors showed that the offspring from viral-infected pregnant mice exhibits behavioral abnormalities including abnormal communication, increased repetitive behaviors, and deficits in sociability, and displays patches of disorganized cortical cytoarchitecture during embryonic development as well as in adulthood [20,21]. Similar cortical patches were observed in prefrontal and temporal cortical tissues of children with autism [22].
In this way, GD could have many impacts on the health and particularly on cerebral or neurological diseases [12]. Somes diseases are linked with modifications of microbiota, such as anxiety, depression, schizophrenia, Alzheimer’s disease (AD) and ASD [12].
In the context of neurodevelopmental disorder as ASD, GD have an impact on the brain development that could lead to behavioral alterations. It was demonstrated that the transfer of GM into germ-free mice could lead to extensive alternative splicing of gene involve in axonal growth, neuronal migration and synaptic communication in the offspring [23]. Another study demonstrated that transfer of GM from ASD children could induce an alteration in tryptophan and serotonin metabolism in germ-free mice [24]. These studies suggest that GM could lead to an alteration in brain development and functions in ASD context. The GD in neuropsychiatric disorders could be modified and partially reversible in different ways based on the microbiome, as fecal microbiota transplantation (FMT) [25], administration of prebiotics or probiotics. There is a link between reversibility of dysbiosis and decreasing of symptoms [26]. In AD patients, studies have shown a beneficial effect of FMT in cognitive impairments [27,28]. In ASD, there are human trials about prebiotics and probiotics, and about FMT [26]. Prebiotics and probiotics induce a reduction of GIS and cognitive impairments [26]. FMT has been administered to children in different trials, and the results look promising [6,29].
Firstly, the different gut bacterial signatures associated to ASD are described. Secondly, the microbiota mechanisms involved in the ASD are developed. The third part gathers the different therapeutic strategies based on the modulation of GM.
The GM begins to develop before birth and is modulated by many perinatal factors such as type of birth, diet or maternal GM. For example, an infection of the mother by filamentous bacteria during pregnancy can stimulate the mother’s immune system, and increase the production of interleukin 17 (IL-17). This cytokine can go through the placenta and impact the brain of the fetus (Fig. 1) [30]. Another study showed that maternal GM of mice could produce some metabolites such as imidazole propionate or 3-indoxyl sulfate, which could promote thalamic axogenesis in fetuses. Models of SPF mice or intestinal microbiota-depleted mice by antibiotic treatment have shown reduced thalamic axogenesis in their fetuses [16]. Moreover, some bacteria can cross the placenta, and colonize the fetal gut during pregnancy [31]. The intestinal microbiota is considered mature in human at 3 to 5-year-old [32]. Nevertheless, early modifications in GM can have an impact on the child’s future mental health. Using quantitative polymerase chain reaction (qPCR) analysis of bacterial genus found in the fecal microbiota of 1-month-old babies, a study showed significant differences in bacterial genera under the different conditions studied. Bacterial Actinobacteria and Bacteroides phyla were found in greater quantities in vaginally-born infants than in those born by caesarean section [32-34]. Firmicute phylum is more common in babies born by caesarean section [33]. Breastfeeding favors the presence of Firmicutes phylum and Bacteroides genus [32,33]. Exclusive breastfeeding helps to reduce the number of Enterococcus genus at 1 month. The decrease of Enterococcus is significant in babies born vaginally and with breastfeeding, compared to babies born vaginally and with mixed feeding (Fig. 1) [32]. Another study, which examined 16S rRNA in the meconium of newborn infants and in the stools of 1-month-old infants showed that the quantity of bacteria differed according to the conditions, but that the bacterial phyla found in both conditions remained identical [33]. An analysis compared the microbiota found at birth and after 1 month in different conditions, and showed significative changes such as a decrease in Proteobacteria phylum at 1 month, and an increase in the presence of Firmicutes [33]. The microbiota develops and evolves throughout life, but GD often appears in toddlers and elderly people [14]. Factors such as the use of antibiotics in children can reduce microbiota diversity and delay maturation [34,35]. A child taking azithromycin for 3 days reduces the diversity of the intestinal microbiota, notably impacting Proteobacteria. Macrolides administered for 6 months also have an impact on the microbiota, notably with a decrease in the Actinobacteria phylum [36]. Penicillin and other antibiotic classes also have an impact on the microbiota, sometimes even long term [35]. Other factors can modulate the microbiota, such as diet or circadian rhythm which can increase stress (Fig. 1) [7,37]. Indeed, stress can induce a decrease in rodent’s Lactobacillus population [37].
The bacterial signatures of the GM in autistic children aged 2 to 4 years compared with non-autistic children appeared significantly different, in a study identifying bacterial phenotypes from stool. Moreover, 16S rRNA gene analysis identified 91 different operational taxonomic units (OTUs) in all children [38]. Other studies showed that some OTUs are found only in ASD children compared to control children [39]. In another study, 65 OTUs were found to be more abundant in ASD children, while 29 were found to be less abundant in ASD children than in control children [38]. However, the results of bacterial phylum and genus identification remain heterogeneous even in ASD children [38,40]. The Actinobacteria phylum appears to be less abundant in ASD children, particularly the Bifidobacterium genus [41,42]. The Coriobacteriaceae family is less abundant in ASD children too [41]. These two bacterial types normally constitute an important part of the child’s microbiota [39,41]. Bifidobacterium genus could play a protective psychobiotic role, help production of neurotransmitters that could have a positive influence on the gut-brain-axis. For example, it produces the amino acid tryptophan, which is a precursor of the neurotransmitter serotonin. Serotonin could promote functioning of serotoninergic neurons and help reduce anxiety and depression [38,43]. Bifidobacterium, and more specifically Bifidobacterium longum, could play a role to reduce the level of an inflammatory cytokine such as IL-6 in fetal enterocytes in vitro [40]. There is also an increase in Firmicutes and Bacteroidetes phyla in ASD children [38,39]. Firmicutes is normally the most abundant phylum in healthy subjects, but the increase of the phylum Bacteroidetes in ASD children causes an inversion of the Firmicutes/Bacteroidetes ratio (Fig. 1). All genera of the phylum Firmicutes are increased in ASD children, except Streptococci, that is found in lesser quantities than in control children [38]. The Proteobacteria phylum is also more abundant in ASD children, particularly the Enterobacteriaceae family. This bacterial family and other Gram-negative bacteria could be linked with higher level of lipopolysaccharides (LPS) in the serum of ASD patient, that could stimulate the production of inflammatory cytokine and a decrease in the social behavior score in ASD children [38]. Some genera are more common in ASD children than in control children, such as Streptococcus, Lactobacillus, Ruminococcus, Ruminoclostridium and Lactococcus [39]. Inversely, other genera, such as Flavonifractor and Acinetobacter, or some families such as Gemellaceae or Streptococcaceae were scarce in ASD group as in healthy children [38,39]. These differences in microbiota composition and abundance between children could have an impact on communication via the gut-brain-axis [39], which will be detailed below.
Other studies have shown that ASD children were colonized by low level of Faecalibacterium [44] and more specifically Faecalibacterium prausnitzii. This bacterial type could be involved in the production of anti-inflammatory cytokines such as IL-10 and IL-12. Moreover, F. prausnitzii could produce butyrate which could increase mucin production and reduce the permeability of epithelial gut barrier [45,46]. A correlation seems to exist between the relative abundance of some bacterial genera, as Flavinofractor, Acinetobacter or Ruminococcus, and the presence or absence of some ASD symptoms in childhood [39].
There is a bidirectional communication between the GM and the brain, notably via the vagus nerve, which enables the transmission of information via neurotransmitter molecules such as serotonin. There is another pathway named hypothalamic-pituitary-adrenal (HPA) system, which communicates via the bloodstream with entero-endocrine molecules. The third known pathway is the immune system with pro- or anti-inflammatory cytokines that could impact brain functions [47,48]. The microbiota also produces certain fatty acids or amino acids with neuroprotective properties (Fig. 1). When GD is present, these pathways may be impacted, notably by amount variations of factors involved in these pathways.
One mechanism which is particularly suspected in ASD is neuroinflammation [49]. This mechanism could lead to synaptic dysfunction, failure of microglia maturation or some other negative impacts on the brain functions [50]. One hypothesis is that neurological symptoms similar to those found in ASD children could elicit increased intestinal permeability and neuroinflammation via cytokines. The increased intestinal permeability could improve the passage of some inflammatory cytokines, and have an impact on a cerebral inflammatory reaction [47,51].
Significant levels of inflammatory cytokines were found in the blood and brain of ASD children [51]. The bacterial phyla most frequently associated with higher inflammatory cytokine levels in ASD children are Prevotella, Bacteroidetes and Bifidobacterium [40]. Some studies suggest that the inflammatory cytokine IL-6, whose production can be induced by GM, could increase inflam-mation and brain dysregulation, modify the integrity of the intestinal barrier and reduce the social behavior score in ASD [48]. In ASD children, there seems to be a lack of B. longum, which could help to reduce and regulate the production of IL-6. Moreover, a study has shown that LPS could increase pro-inflammatory cytokines interferon gamma, IL-6 and tumor necrosis factor alpha (TNFα) [52].
Short-chain fatty acids (SCFA) can be produced by GM. Certain types of anaerobic bacteria, such as Bifidobacterium, Prevotella and Lactobacillus, are particularly involved in SCFA synthesis via dietary fibers fermentation [53]. The majority of SCFAs are produced from precursors such as propionate, acetate or butyrate [49]. This last one is produced specifically by Faecalibacterium which is found in GM [43]. Because of GD in ASD children, SCFA and butyrate production by Faecalibacterium could be deficient in these children [43,47]. As they could cross the blood-brain barrier, SCFA could have a neuroprotective role and anti-inflammatory properties. Thus, they could reduce neuroinflammation [49]. GM metabolizes some essential amino acids such as tryptophan, which is the precursor of serotonin (5-hydroxytryptamine [5-HT]), a neurotransmitter that plays a role in mood. Serotonin depletion could be at the root of sad or depressed moods, or of cognitive disorders [54]. These symptoms are common in ASD children. Tryptophan is also a precursor for the synthesis of other metabolites, such as 5-methoxytryptophol, which may play a protective role against the genesis of ASD in utero [55]. In addition, certain bacterial strains as Lactobacillus or Bifidobacterium could modulate the production of other neurotransmitters such as GABA [7,56], which is the main excitatory neurotransmitter in fetuses, whereas it becomes inhibitory in adults [57]. An imbalance between neuronal inhibition and excitation via the neurotransmitters GABA or glutamate could be a significant element in the mechanisms of ASD [58]. This imbalance could lead to abnormalities in cerebral information processing and behavioral alterations, and thus different perception of environment [57,58]. Several studies suggest that GABA may be deficient in the brains of ASD children, although others show equivalent levels of this neurotransmitter in all children [58,59].
It appears that HPA hyperactivity is correlated with increased corticotropic hormone levels and stress, as well as a tendency to depression in mice with sterile microbiota. However, Bifidobacterium infantis may reduce this hyperactivity in SPF mice. To date, many mechanisms remain unclear [44]. In ASD children, the HPA hyperactivity with high level of cortisol in blood is also modulated by adrenocorticotropic hormone in hypothalamus [60].
Prebiotics are non-digestible components of the human body that benefit the health of the host by selectively stimulating the growth and activity of intestinal bacteria [61]. By contrast, probiotics are living microorganisms which, when ingested or applied topically in sufficient quantities, can correct dysbiosis. Strains of Bifidobacterium and Lactobacillus, as well as Saccharomyces boulardii, are commonly used as probiotics [61].
A double-blind study has shown for the first time the concomitant benefit of an exclusion diet combined with 6-week treatment with the prebiotic galacto-oligosaccharide immuno B-GOSⓇ (Clasado Biosciences) (Table 1) [62]. The latter could stimulate butyrate production and modulate intestinal bacterial populations. The exclusion diets used were casein-free or gluten-free. Administration of B-GOSⓇ for 6 weeks in combination with an exclusion diet significantly increased GM diversity, with an increase in Bifidobacterium spp. The study showed a trend towards a reduction in gastrointestinal disorders, although the results were not significant under the same conditions. There was also a significant improvement in the anti-sociality scores used to assess neurological criteria in ASD [62]. The study also showed an increase in Bifidobacterium spp., Ruminococcus spp., and the Lachnospiraceae when B-GOSⓇ was administered for 6 weeks without a restrictive diet. GM diversity was also increased, with no significant difference compared to no prebiotic administration [62].
Among the prebiotics, omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been suggested as a potential treatment for several neurodevelopmental disorders [63]. A meta-analysis showed that n-3 PUFAs, compared with placebo, improved social interaction and repetitive and restricted interests and behaviors [64]. However, the findings cannot be generalized to all children with ASD because the included children were of different age groups (< 8 years old to 28 years old), displaying different symptom severity or high hyperactivity level and predominantly comprised males [64].
A study which included 35 ASD and 25 control patients, 3 to 25 years old, investigated the impact of the association of probiotic (Lactobacillus plantarum) and oxytocin. With association of probiotic and oxytocin, there was behavioral improvements and remodulation of GM with increasing of Ruminococcacaeae and Lachnospiraceae. On the contrary, these results were not significant on all studied aspects when oxytocin or the probiotic were administered alone (Table 1) [65].
Other randomized controlled trials have shown that administration of VISBIOMEⓇ (ExeGi Pharma) probiotics, based on Lactobacillus and Bifidobacterium, improves gastrointestinal disorders associated with ASD (Table 1) [66,67]. Another study showed that concomitant administration of Bifidobacterium infantis with bovine colostrum product improved gastrointestinal disorders and certain behavioral aspects, notably with an improvement in the Aberrant Behavior Checklist (ABC)-lethargy score, whereas other ABC-scores about behavior improvement were not significant (Table 1) [68].
Pre- and pro-biotics nevertheless have a number of limitations in the treatment of ASD. Studies produced limited efficacy evidence, due to small sample sizes, short intervention duration and different drugs used [69,70]. In addition, there was no standardized probiotic regimen, with multiple different strains and concentrations of probiotics [71].
Prior to clinical trials, preclinical FMT trials were carried out, in particular in fragile X messenger ribonucleoprotein 1 knock-out (FMR1 KO) mice displaying autistic-like symptoms. FMT from healthy mice to FMR1 KO mice improved their behavioral symptoms and restored social preference. Furthermore, the levels of inflammatory factors (TNFα, Ionized calcium-binding adapter molecule 1) were significantly reduced in the FMR1 KO mouse brains after FMT [61]. A study in mice also showed that a critical neurodevelopmental window existed for GM remodeling, and that this remodeling contributed to social behavior improvement [72]. FMT of healthy humans in an ASD mouse model could improve autistic-like symptoms such as anxiety [61]. Open-label studies on children treated with FMT from a stool-controlled donor were published (Table 2) [29,73]. After vancomycin treatment to eliminate some of the GM already present [73], FMT was administered for 4 or 8 weeks. FMT treatment showed an improvement in gastrointestinal disorders and an increase in GM diversity thanks to the graft, notably with an increase in Prevotella and Bifidobacterium, which multiplied to reach levels equivalent to neurotypical children 2 years post graft. Results also showed the improvement of behavioral symptoms, through the evaluation of the Parent Global Impressions-III score. These results remained stable for at least 8 weeks after the end of FMT treatment, and most improvements in GI symptoms were maintained after two years (Fig. 1) [73,74]. Moreover, significantly different levels of 5-HT and GABA were found in the blood of children after 4 weeks of FMT treatment, and they remained stable thereafter throughout follow-up up to 8 weeks [29]. Furthermore, in a study of 48 children separated into 2 equivalent groups, one receiving 2 FMTs every 2 months, and the other receiving rehabilitation training, behavioral symptoms studied using the Childhood Autism Rating Scale test were significantly improved compared with children who did not receive FMT. GIS improved after the first FMT, and microbiota changes were similar to those of non-ASD patients (Table 2 and Fig. 1) [75]. A case report about a 7 year-old female child with severe ASD and GIS has shown that after 5 FMT/10 weeks that GM was modified, capacity to produce SCFA by GM was improved and intestinal inflammation was improved too [76]. In addition, a retrospective study of 49 children with ASD who received a washed fecal microbiota (WMT) between June 2019 and July 2021 showed that WMT can relieve constipation and improve sleep disorders in these children without serious side effects [77].
These results look promising, but despite some randomized, controlled, double-blind trials, these studies have some limitations. Interventional placebo controlled FMT studies include a limited number of patients. A larger number of patients would be needed to confirm the results and ensure a genuine link between FMT and symptom improvement. Similarly, the patients recruited come from the same geographical region. Diversifying patients’ ethnic origins and standardizing on their age could strengthen the results obtained and erase potential confounding factors linked to an age-dependent microbiota [61]. Moreover, there are different ways to transfer microbiota as capsules or by liquid colonoscopy, and theses ways are not comparable because quantity and quality of live bacteria and localization of instillation are not similar [78,79].
Fecal transplantation is a recent treatment of patients with ASD through clinical trials. It is important to emphasize that several parameters need to be taken into account for future studies, and in particular to define a transplantation procedure (location of the transplant, better characterization of the transplant’s bacterial signature) with inclusion and exclusion criteria validated by a scientific and medical consortium, and to take into account the one-health concept, since the environment and lifestyle habits can modify the intestinal microbiota (Table 2).
The GM is impacted by a wide range of factors throughout life. Inversion of the Firmicutes/Bactoroidetes ratio, or lack of Bifidobacterium are retained elements of GD in ASD. The neuroinflammation by cytokines, decrease of SCFA, reduced 5-HT production, or imbalance between GABA and glutamate in fetuses were involved in the GD of ASD. These mechanisms could affect behavioral responses in ASD. However, GD could be modulated by administration of pre- or probiotics, such as Bifidobacterium spp. and Lactobacillus, or by FMT, which seems to improve GIS and some behavioral impairment features. Further studies with larger cohorts and standardized treatments will be needed to confirm these promising results about FMT.
No potential conflict of interest relevant to this article was reported.
Conceptualization: Lisa Poupard, Zahyra Kaouah. Data acquisition: Lisa Poupard, Zahyra Kaouah. Formal analysis: Lisa Poupard, Zahyra Kaouah. Supervision: Zahyra Kaouah, Guylène Page. Writing—original draft: Lisa Poupard. Writing—review & editing: Zahyra Kaouah, Guylène Page, Vincent Thoreau.
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