Psychobiotics and fecal microbial transplantation for autism and attention-deficit/hyperactivity disorder: microbiome modulation and therapeutic mechanisms

One study found reduced D-arabinitol levels, a metabolite of Candida species, in the urine of children with ASD after probiotic supplementation (Shaw et al., 1995). This result suggests that probiotics may prevent gastrointestinal colonization by Candida species. In another study, the levels of Bifidobacterium (known as beneficial bacteria, such as Lactobacillus species) were significantly lower in the stool of children with ASD (Shaaban et al., 2018). After probiotic supplementation, there was a significant increase in the colony counts of Bifidobacterium and Lactobacillus with significant improvement in the severity of ASD and gastrointestinal symptoms. A study in 2015 reported the effect of mixed probiotic administration on gut microbiota composition in children with ASD (Tomova et al., 2015). The abundance of Clostridia and Desulfovibrio and Bacteroidetes/Firmicutes ratio were related to the severity of ASD and gastrointestinal symptoms. After probiotics treatment, the amount of Firmicutes significantly decreased, which resulted in an increase in the Bacteroidetes/Firmicutes ratio to a level similar to that observed in healthy children, Bifidobacterium increased, and Desulfovibrio decreased significantly. Moreover, a study of a mixture of Lactobacillus spp. and Bifidobacterium spp. in two different rodent ASD models indicated that a probiotic mixture could improve social behavioral symptoms by modulating the gut microbial population (Mintál et al., 2022).

Psychobiotics have a potential to not only reconstruct the gut barrier function by resisting harmful bacteria, but also exert an immunomodulatory effect by reducing circulating hormones and pro-inflammatory cytokines in serum. The gut microbiota has been demonstrated to serve as a regulator of intestinal, systemic, and CNS resident immune cell function (Zheng et al., 2020). Gut microbiota can communicate with the CNS by regulating intestinal and peripheral immune cells and peripheral immune responses via circulating cytokines (Arrieta and Finlay, 2012).

Any peripheral inflammatory event induces VN to cause the suppression of the release of proinflammatory cytokines from intestinal macrophages (Daliri et al., 2016). Probiotics reduce gut inflammation through various mechanisms, such as reducing inflammatory cytokines and other immunomodulatory effects. For example, anti-inflammatory cytokines (IL-4 and IL-10) and proinflammatory cytokines (TNF-a, IL-1b, IL-2, IL-6, IL-8, IL-12, IL-17, and IL-18) are significantly changed by Lactobacillus rhamnosus GG (LGG) (Miyazawa et al., 2015; Cicenia et al., 2016; Fong et al., 2016; Wang et al., 2016; Aoki-Yoshida et al., 2017; Clarke, 2018; Cai et al., 2019).

In clinical studies, Tomova et al. found that TNF-α levels were strongly correlated with GI symptoms and showed a trend toward correlation with ASD severity (Tomova et al., 2015). Probiotic supplementation significantly decreased TNF-α levels in the feces of children with ASD. Similar to this study, Sanctuary demonstrated that psychobiotic supplementation could reduce the intracellular expression of certain cytokines in CD4+ T cells (Sanctuary et al., 2019). The frequency of CD4+/IL-13+ T cells was significantly lower after the treatment.

Adıgüzel et al. demonstrated that dietary treatment with multispecies probiotics formulations (Sptreptococcus thermophilus, Bifidobacterium breve, B. animalis, Lactobacillus helveticus, L. plantarum, L. acidophilus, and L. paracacei) attenuated the inflammatory responses in a VPA-induced rodent ASD model. In particular, this study also showed that psychobiotic treatment decreased serum pro-inflammatory cytokine, IL-6 levels, and increased anti-inflammatory cytokine, IL-10 levels, with the improved status of diverse behavior tests including social interaction, anxiety, and repetitive behaviors (Adıgüzel et al., 2022). Alonazi also demonstrated that dietary psychobiotic supplementation could reduce levels of various serum inflammatory cytokines, such as IL-1β, IL-8, IL-10, and IFN-γ, in an ASD rat model induced by a neurotoxic dose of propionic acid (Alonazi et al., 2022).

The gut microbiota influences the brain directly through neural pathways, including the VN and ENS. The VN connects the ENS and CNS and it could be activated by cytokines, which could be modulated by bacteria, and byproducts from bacteria including endotoxins and peptides. In particular, the neuropeptide could be sensed by receptors associated with dendritic cells in the gut, which could transfer the signals to the brain (Perez-Burgos et al., 2013). Psychobiotics modulate CNS-related behaviors through the VN pathway and the physiological response of various metabolites, including SCFAs, enteroendocrine hormones, cytokines, and neurotransmitters (Bravo et al., 2011; Dinan et al., 2014; Sgritta et al., 2019).

The levels of oxytocin and DHEA-S, which have been considered to be etiologies of ASD, were significantly lower in the plasma of children with ASD in a clinical study, and there was a trend towards a correlation between decreased DHEA-S levels and a lower Bacteroidetes/Firmicutes ratio which increased after probiotic implementation (Tomova et al., 2015). According to Grimaldi, increases in butyrate production, potentially positively affecting ASD, were detected in children with ASD following exclusion diets (Nankova et al., 2014; Grimaldi et al., 2018). Additionally, lower levels of amino acids (isoleucine, leucine, valine, alanine, and glutamine) and lactate were detected in the B-GOS group. The presence of amino acids in feces is associated with problems in gut barrier function (Marchesi et al., 2007).

A study of psychobiotics in a rodent ASD model, which was induced by oral propionic acid ingestion, proposed that Lactobacillus bulgaricus and Bifidobacterium infantis could ameliorate glutamate excitotoxicity, a major autistic feature in this model. The therapeutic effect of these psychobiotics might be due to the reduction of oxidative stress, restoration of the depleted GABA signaling pathway, and upregulation of the GABA receptor’s gene expression (Bin-Khattaf et al., 2022). Ingestion of Lactobacillus rhamnosus could connect bidirectional communication of the gut-brain axis, and it could regulate emotional behaviors by controlling the GABA receptor expression in the VN (Bravo et al., 2011). In 2018, the specific bacterial species of ASD were identified in Shank3 knock-out mice, and this study suggested that oral Lactobacillus reuteri ingestion could decrease repetitive behaviors by up-regulation of the γ-aminobutyric acid (GABA)-related metabolism (Tabouy et al., 2018).

Bacteria can synthesize and respond to hormones and neurotransmitters. Lactobacillus species produce acetylcholine and GABA, Bifidobacterium species produce GABA, Escherichia produces norepinephrine, serotonin, and dopamine, Streptococcus and Enterococcus produce serotonin, and Bacillus species produce norepinephrine and dopamine (Galland, 2014). Other bacterial strains (Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Lactobacillus plantarum, Streptococcus thermophilus, Escherichia coli, Morganella morganii, Klebsiella pneumoniae, and Hafnia alvei) produce serotonin (O’Mahony et al., 2015).

Various studies have suggested the dopamine hypothesis, which indicates that the enteric neurotransmitter release by nutrient intake could affect brain health and responses. And Bacillus, one of the representative bacteria to modulate the dopaminergic system, is known for its ability to produce dopamine and noradrenaline directly in the GI tract (Satti et al., 2023). Dysregulated dopaminergic and noradrenergic neurotransmission has been widely implicated in the pathophysiology of ADHD, and dopamine and norepinephrine play essential roles in behavioral, cognitive, and affective functions (Del Campo et al., 2011). In a study using patients with ADHD, Bifidobacterium was increased in patients with ADHD, which was linked with the enzyme involved in the dopamine precursor (phenylalanine) synthesis (Aarts et al., 2017)

Serotonin also plays a role in ADHD pathogenesis, however, it affects brain function not directly, but via the nervous system (Banerjee and Nandagopal, 2015; Hou et al., 2018). On the other hand, gut microbiota directly act a biological role on the brain by modulation tryptophan’s peripheral availability because tryptophan can cross the BBB and affect serotonin synthesis in CNS (Richard et al., 2009; Schwarcz and Stone, 2017).

A recent experimental study has demonstrated that Lactobacillus rhamnosus regulates, via the VN, emotional behavior and the central GABAergic system, which is also associated with neuropsychiatric disorders (Enticott et al., 2010). According to Pärtty, the early supplementation of Lactobacillus rhamnosus GG decreases the risk of developing ADHD, and Liang-Jen Wang suggested that oral probiotic Bifidobacterium bifidum (Bf-688) improves the clinical symptoms of ADHD. In addition, food supplement treatments containing Lactobacillus acidophilus and Bifidobacterium improve the self-control and attention of children with ADHD (Pärtty et al., 2015; Wang et al., 2022). These results are thought to be due to the role of Lactobacillus and Bifidobacterium as producers of GABA, which is known to decrease in patients with ADHD (Yunes et al., 2016).

The VN is the longest cranial nerve in the body, and it delivers electronic signals from the body (lungs, liver, heart, GI tract) to the brain through sensory fibers. And this connection administers the GI tract’s function via the metabolites from intestinal microorganisms. The VN is involved in functions such as mood control, immune response, and GI tract function via intestinal permeability and enteric reflex and influences the hypothalamic-pituitary-adrenal axis. Vagal afferent fibers sense microbiota signals indirectly through the diffusion of bacterial compounds, metabolites, or other cells located in the epithelium that relay luminal signals (Del Toro-Barbosa et al., 2020). The gut microbiome has the capacity to modulate the host’s emotional and behavioral responses by acting on vagal afferents.

Various host physiological metabolism could be regulated by SCFAs, in particular, gut barrier integrity, immune defense system, and lipid metabolism could be the major target of the SCFA. (Dalile et al., 2019). Moreover, SCFAs might directly influence neural function by reinforcing BBB integrity, modulating neurotransmission, influencing the levels of neurotrophic factors, and promoting memory consolidation. Increased evidence suggests a potential key role for SCFAs in gut-brain axis signaling (Silva et al., 2020). The ADHD group showed significantly lower concentrations of fecal acetate and butyrate than the control group, and various bacterial strains (Bifidobacteria, L. salivarius, L. agilis, L. acidophilus, LGG, B. longum, B bifdum, and L. gasseri) are known to increase SCFAs production (LeBlanc et al., 2017; Jung et al., 2022).

Lactobacillus rhamnosus GG is known to strengthen the gut permeability barrier by fortifying intestinal tight junctions, mucin layer thickness, and antigen-specific immunoglobulin A production (Asano et al., 2012). In particular, L. rhamnosus GG administrated participants showed a significant decrease in the serum levels of the pro-inflammatory cytokines (IL-6, IL-12 p70, and TNF-α). (Kumperscak et al., 2020).

FMT could serve as a protective treatment for reconstructing the gut microbiota at both the phylum and genus levels and has a therapeutic effect on ASD symptoms and gastrointestinal disorders (Li et al., 2021). A modified FMT protocol for children with ASD, termed microbiota transfer therapy, appears to be a promising approach to alter the gut microbiome and improve GI and behavioral symptoms of ASD (Kang et al., 2017; Kang et al., 2019). This protocol improved GI and ASD symptoms, and the microbiome persisted for two years after treatment, suggesting a long-term impact. Important changes in the gut microbiota at the end of treatment were observed during follow-ups, including significant increases in bacterial diversity and relative abundance of Bifidobacteria and Prevotella.

Li et al. showed that the gut microbial population of ASD children was altered by FMT with donor microbiota toward that of the healthy group. Especially, the FMT response significantly reduces the abundance of Eubacterium coprostanoligenes (Li et al., 2021). These data also indicated that decrement in the population of Eubacterium coprostanoligenes by FMT might be a curative technique for ASD symptoms and behaviors.

Unlike probiotics, FMT refers to the transfer of the full spectrum of gut microbial communities containing more than 1,000 bacterial strains, and it might be more effective than psychobiotics in aspects of physiological regulation in the nervous system, endocrine system, and host behavior (Chen et al., 2022). In recent studies, FMT could exhibit a recovery effect on the serum levels of serotonin, GABA, and DA in the ASD cohort, which means that FMT might be an effective technique in regulating neurotransmitters via the MGB axis (Li et al., 2021). Moreover, FMT in the ASD cohort could decrease GABA and serotonin in serum, but the dopamine level was increased by FMT. It could be assumed that FMT may be an efficient approach to modulate neurotransmitter secretion for regulation of the central nerve via the MGB axis.

Alterations in the gut microbiota composition after FMT could significantly improve behavioral impairments and regulate immune responses in ASD. Chen and colleagues demonstrated that treatment using FMT with in vitro cultured healthy donor’s intestinal microbiota had a positive effect on ASD symptoms in mouse ASD model (Chen et al., 2020). They observed amelioration of anxiety actions and repetitive performance with lower serum levels of metabolites, such as GRO-α and MIP-1α, and a conversely higher level of MCP-3, RANTES, and Eotaxin. Additionally, family or genus levels of S24-7, Clostridiaceae, Prevotella, and Candidatus Arthromitus were key microbial taxa in FMT treatment, and serum levels of chemokines were related to the relative abundance of these taxa.

In this study, both original donor microbiota transplantation and cultured microbiota transplantation improved behavioral abnormalities and chemokine disorders in an ASD mouse model and were effective in the modification of several key differential taxa in the gut microbial composition (Chen et al., 2020). These results of cultured microbiota transplantation suggest the possibility of using “donor-free FMT” and regulating the donor gut microbiota structure before transplantation during in vitro culture. The batch methods are fast, easy, and repeatable culturing techniques.