Gut Microbiota and Autism Spectrum Disorder: A Neuroinflammatory Mediated Mechanism of Pathogenesis?

The human gut microbiota is a complex biome and variable collection of microorganisms interacting with one another and the human host, including bacteria, fungi, archaea, and viruses. The gut microbiome influences many aspects of host health, including immune system control, gut hormone regulation, and neuronal transmission. It modifies the ingested medications and their metabolism, toxin clearance, and the generation of numerous host-affecting agents [1]. The gut microbial load can directly or indirectly influence the brain via a mutual relationship known as the "gut-brain axis." The gut microbiota can directly influence the brain by producing neuroactive substances such as neurotransmitters, amino acids, and microbial metabolites. These substances can potentially interfere with the host immune system and metabolism, affecting the gastrointestinal (GI), nervous system and vagus nerve. The gut microbiota can also influence the integrity of the gut barrier, which limits the passage of luminal substances into the bloodstream. Accessibility of such bacterial structural components like lipopolysaccharides or by-products of metabolic activities like short-chain fatty acids (SCFAs) may result in an inflammatory cascade that affects the CNS [2].

Autism spectrum disorder (ASD) is a persistent psychological abnormality characterized by impaired social communication and limiting and repetitive behavior patterns, hobbies, or activities [3]. Both genetic and environmental variables have been involved in ASD. Recent studies have shown that inflammation and inflammatory mediators have a role in disease genesis. Inflammatory elements that contribute to ASD include unusual microglia activation and polarization phenotypes, higher systemic levels of pro-inflammatory mediators, and altered patterns of immune cell responsiveness to activation triggers [4].

Numerous types of research in recent years have implicated gut bacteria in the etiology of ASD. However, studies have found that the structure of the gut microbiota is meaningfully changed in ASD; the significance of the gut microbiota as an etiology of ASD is yet unclear. It has been accepted that the microbiome of autistic children differs from healthy individuals [4]. Inflammatory deviations are potential etiology candidates in how gut microbiota can influence the gut-brain axis of ASD patients. Neuroinflammatory factors in ASD result from changes in the regulation of intestinal barriers, activation and function of microglia, and levels of neurotransmitters [5, 6].

ASD is currently diagnosed based on clinical symptoms, which can lead to delays and misinterpretation. Biomarkers based on neuroinflammatory processes associated with gut microbiota may provide a more objective and precise way of detecting ASD. Several examples of these markers include microRNAs that modulate immune signaling; brain-derived neurotrophic factor (BDNF), which promotes brain growth; S100B, which reflects neural immunity; and chemokines that facilitate immunological activation, such as RANTES and eotaxin [7]. This review aims to evaluate and discuss neuroinflammatory biomarkers in the pathogenesis and potential diagnostic trials of ASD in more detail.

Nearly two decades earlier, a potential association between gut microbiota and ASD was proposed [8]. While the exact cause of ASD is still unknown, existing literature has shown that gut dysbiosis, along with a neuroinflammatory condition, is found in individuals with ASD [9]. Studies found inconsistent differences in the gut microbiota composition of ASD cases. Overall, the ASD population shows signs of dysbiosis, with a different abundance of Bacteroidetes/Firmicutes, Prevotella, Clostridium, Lactobacillus, Bifidobacterium, Faecalibacterium, Streptococcus, Enterobacteriaceae, Verrucomicrobia, Fusobacteria, Escherichia coli, Enterococcus, Akkermansia, Phascolarctobacterium, and lots of other microbes compared to healthy controls [10‐12]. Nonetheless, specific microbial patterns associated with ASD remain unidentified.

Moreover, Cuomo et al. [9] recently indicated that gut dysbiosis and inflammation were identified by host fecal DNA-specific methylation in autistic children. They revealed that autistic patients with dysbiosis significantly enriched inflammatory and immune pathways, including the production of interleukin (IL)-2, 6, and 12 and the activation of the toll-like receptor (TLR) 3 signaling pathway. Consistently, several studies corroborated earlier findings of the neuroinflammation caused by dysbiosis in various neurodegenerative and neuropsychiatric conditions [12, 13]. Disruptions in immune signaling pathways like the NLRP3 inflammasome, type 1 interferon, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathways are among the possible dysbiotic consequences. Alterations to the T-helper 17 cell/T-reg proportion and imbalances in macrophage polarization, tumor necrosis factor (TNF)-α, IL-1β, 18, and 6 are also possible [14, 15].

On the other hand, the involvement of inflammation and immunological dysregulation has been indicated in the development and/or severity of ASD [16, 17]. Prior investigations on autistic cases have shown elevated levels of inflammatory markers such as TNF-α, interferon-γ, IL-2,4,5,6,8,17, and 10 [12, 18, 19]. A study also reported that autistic children with an innate proinflammatory response or impaired T cell activation indicate more severe behavioral issues compared to those with noninflamed or non-T cell-activated immunological profiles [20]. Given the lack of research on gut microbial composition and metabolites in autistic patients concerning inflammatory conditions (Table 1), the precise relationship between these three factors remains unclear and requires further investigation. Overall, gut microbiota seems to play a crucial role in ASD through inflammation.

Protein and peptide-based biomarkers have been the subject of some research for early diagnosis of ASD [7]. The S100 calcium-binding protein beta subunit (S100B) is expressed in astrocytes and other extra-neural cells, including enteric glial cells (EGCs). It affects neurons depending on the concentration, which can be trophic up to a few nanomolar doses and toxic at micromolar levels. Extracellular protein S100B contributes considerably to neuroinflammation by acting synergistically with pro-inflammatory cytokines and, at higher concentrations, acting as a cytokine [32]. Despite the existing conflict [33‐35], multiple studies have found a significant elevation of S100B in autistic individuals compared to healthy cases, supporting the possible role of this factor in the etiology and development of ASD [36‐40]. The source of this elevated S100B concentration in the periphery can be injured neurons or EGCs. In a recent study, the correlation between plasma S100B levels and fecal concentrations of calprotectin (an objective marker of GI inflammation status) revealed that not only brain astrocytes but also EGCs might be involved in the pathophysiology of autism [41]. One hypothesis says that the alternation of enteric glial-derived S100B expression in autistic patients can result from changed microbiota, disruption of the intestinal barrier, and even pathogenic bacteria, altogether inducing intestinal inflammation and converting EGCs to reactive EGCs [38]. Another in vivo study in mice showed that gut microbiota biodiversity increases with S100B levels or oral administration. Firmicutes phylum, including Lactobacillus and Bacteroidetes, including Barnesiella and Butyricimonas spp, are affected by S100B levels [42]. However, higher levels of Bacteroidetes and lower levels of Firmicutes were observed in a group of children with autism [43]. No study clarified the correlation between gut microbial alternation and the effect of probiotic use and S100B levels in autistic patients. Studies can be directed to know the effects of probiotic administration as a manipulative factor of gut microbiota on the levels of S100B in ASD patients. Furthermore, it is suggested that S100B can be investigated as a potential biomarker both in the diagnosis and treatment of autism.

BDNF is a protein member of the nerve growth factor family (neurotrophins). BDNF has a key role in both the pre-synaptic site (modulates neurotransmitter release) and post-synaptic site (augments the function of ion channels), so it generally contributes to affect neuroplasticity and, thereby, behavior-related conditions [44]. Abnormal levels of BDNF were seen in a wide range of neurological diseases, including schizophrenia, depression, and even autism [45]. According to recent studies, altered BDNF levels were observed in ASD patients compared to the controls, revealing that BDNF might play a role in autism pathophysiology [46‐50]. A relatively higher level of BDNF was seen in mild phenotypes compared to severe autism, emphasizing the probable protective function of this factor [51]. Downregulation of the BDNF in the antiapoptotic signaling pathway in the brains of autistic individuals is one of the possible underlying mechanisms in the pathophysiology of autism [52]. The reduction of BDNF expression as a neuroprotective agent can be caused by raised inflammatory factors, including IL-1β and TNF; therefore, it may have a negative regulatory role in neuroinflammation [53, 54]. The dysbiotic gut microbiota in autistic patients may contribute to this inflammatory condition through immune dysregulation and the release of inflammatory factors such as IL-1β, which crosses the BBB [55]. Animal studies showed that BDNF has been lower in germ-free mice's cortex and hippocampus [56]. Probiotic administration in these sterile mice also resulted in partial and complete normalization of behavior and BDNF levels, respectively. It has also been suggested that probiotics, specifically a combination of the Lactobacillus and Bifidobacterium genera, may be effective in increasing BDNF levels and improving mental health parameters in patients with depression and neurological disorders [57, 58]. Balance of fecal Clostridium spp. and normal BDNF expression were both achieved through fecal microbiota transplantation or Bifidobacterium treatment in an animal model of autism [21]. In another rat model study, Lactobacillus supplementation could increase BDNF levels and attenuate behavioral anomalies [59]. Regarding these relations, further studies are needed to know if the induction and modification of microbial alteration in the gut of autistic patients can be monitored and controlled by BDNF levels.

Regulated upon Activation, Normal T Cell Expressed and Secreted, RANTES (CCL5), and eotaxin (CCL11) are pro-inflammatory chemokines released by a variety of cells, including blood cells, fibroblasts, endothelium, epithelium, neurons, and glial cells [60, 61]. RANTES [60, 62‐66] and eotaxin [65‐68] plasma levels are considerably higher in autistic children. Since RANTES and eotaxin act as pro-inflammatory mediators, their rise implies that both play a neuroinflammatory role in ASD [60, 61, 69, 70]. Although Shen et al. [63] reported no significant correlations between RANTES or eotaxin and behavioral patterns of ASD, Han et al. [64, 65] and Hu et al. [67] found RANTES and eotaxin related to ASD, respectively. Besides, other studies demonstrated that the rise of both factors is ASD related [66]. Moreover, gut microbiota seems to induce RANTES-mediated inflammation [71‐73]. Earlier studies uncovered the NOD‐like receptor family pyrin domain containing 6–gut microbiota axis and subsequent IL-6 and TNF release as one possible connection of gut microbiota dysbiosis with RANTES-mediated immune dysregulation [74, 75]. Concerning the expression of gene encoding, it has been found that gut microbiota can manipulate eotaxin expression levels [76]. On this matter, antibiotic-treated mice had an altered microbiome with elevated eotaxin and different structures in their microglia [77]. Also, it has been found that mice's eotaxin levels changed after fecal microbiota transfer [78]. Regarding gut microbiota and ASD relation, modified anxiety-like and repetitive behaviors were observed while the levels of RANTES and eotaxin were improved through gut microbiota transplant in ASD mice. These results showed that RANTES and eotaxin play important roles in CNS synaptic transmission and development, and their levels are associated with the structure of microbiota in mice [22]. Clostridiaceae, Erysipelotrichaceae Prevotella families, Candidatus Arthromitus, and Proteus genus were found to be inversely associated with the level of RANTES and eotaxin [22]. In-vivo topical and oral probiotic administrations have reported a connection of RANTES with strains Lactobacillus paracasei SGL 04, Lactobacillus plantarum SGL 07, Lactobacillus fermentum SGL 10, and Lactobacillus brevis SGL 12 lysates, and Lactobacillus rhamnosus GG [79, 80]. Similarly, Probiotics containing Lactobacillus acidophilus, Lactobacillus rhamnosus GG, and Bifidobacterium also changed eotaxin gene expression in an animal [81]. Overall, the important findings implied from these studies suggest a potential mechanism of gut microbiota in ASD pathogenesis and severity through inflammatory factors of RANTES and eotaxin.

The cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) drives many aspects of myeloid hemopoietic cell biology, including survival, proliferation, differentiation, and functional activity. It also affects the immune system through dendritic and T-cell functions [82, 83]. GM-CSF triggers chronic inflammation in the CNS and acts as a neuronal growth factor to stimulate neuronal and glial differentiation [82‐84].

Although some earlier studies presented a low GM-CSF level in autistic patients [85, 86], higher levels of GM-CSF were found consequently in the brains of ASD patients [70, 82‐84]. Perroud et al. reported higher levels of GM-CSF- IL-1α, TNF-α, and interferon-α among ASD children experiencing co-morbid GI symptoms [87]. The changes in GM-CSF levels in ASD can indicate that an inflammatory process may be involved in developmental and neuroimmune impairment [83]. Results of co-culture experiments by Takada et al. are the first to show that GM-CSF-induced macrophages inhibit the dendritic outgrowth of neurons in autistic individuals. This phenomenon is mediated through the secretion of pro-inflammatory cytokines, IL-1α and TNF-α, and may lead to more severe behavioral effects [88].

Interestingly, GM-CSF levels vary with alterations in gut microbiota [89‐92] and mostly with IL-17a, a cytokine that correlated with the severity of behavioral symptoms in individuals with ASD [89, 90]. Different species of gut bacteria have been linked to GM-CSF, including Parabacteroide, Prevotella, Streptococcus, Clostridium, Lactobacillus reuteri, Lactobacillus crispatus Enterococcus faecalis, Blautia, Butyricimonass, Roseburia, Anaerotruncus, and Blautia [89, 92]. An important finding showed that gut microbiota-derived metabolites like SCFAs may alter GM-CSF levels [90]. Within a study, GM-CSF as a neuroimmune factor was increased with the administration of probiotics containing Bifidobacterium longum, Lactobacillus delbrueckii bulgaricus, and Streptococcus thermophilus [93]. Altogether, the change of GM-CSF neuroinflammatory factors by gut microbiota alteration provides insight into the mechanism of pathogenesis in this way in ASD patients.

The high mobility group box 1 protein (HMGB-1) is one of the most abundant members of the HMGB protein family and has many potential roles [94]. It has a key role in DNA regulatory activities as a nuclear protein [95]. As an extracellular factor, it is actively released when immune cells respond to an inflammatory condition [96] and also passively released by necrotic or damaged cells [95]. HMGB1 has numerous membrane receptors called pathogen recognition receptors, TLR4, TLR9, and receptors for advanced glycation end products (RAGE) are the dominant ones. Through its interactions with these receptors, HMGB1 promotes inflammation in cells [97]. HMGB1 can cross the blood–brain barrier, promote neurite outgrowth and cell migration, or mediate neuroinflammation after injury [98].

It has been understood that plasma levels of HMGB-1 can elevate in ASD patients [99] and positively correlated with the severity of autism [100]. Another effective inflammatory molecule, the epidermal growth factor receptor, was considered to be related to symptom severity in children with autism, and the HMGB1 level seems to correlate with that [101, 102]. Interestingly, higher HMGB1 levels are found to be associated with higher GI dysfunctions in individuals with autism, which can imply an intestinal concept of pathogenesis [23, 103]. It is similarly studied that fecal levels of HMGB1 were correlated with GI sign severity in ASD children, which regards ASD-related dysbiosis [23]. Microbiome dysbiosis accompanied by intestinal inflammation can lead to the activation of monocytes, upregulating HMGB1 excretion for a pro-inflammatory feedback loop [104].

Higher levels of HMGB1 and TLR4 have also been reported to be associated with autistic-like behaviors in mice, possibly through activation of the HMGB1/TLR4 signaling cascade [105]. Serum levels of TLR4 were elevated in ASD children and positively associated with their hyperactivity scores [106]. Activation of the HMGB1/RAGE/TLR4 axis leads to leukocyte infiltration into nerve cells and results in persistent CNS inflammation. It is suggested that neuroinflammation is strongly related to ASD occurrence [107] through activating the inflammasome system as a mechanism [108]. In addition, it is described that HMGB1 can bind to endogenous secretory RAGE, resulting in a decline in plasma RAGE levels. This may contribute to the pathophysiology of autism by interfering with neuropeptide oxytocin transport from the periphery to the brain [109].

The effect of probiotics and gut microbiota alteration on HMGB1 levels in ASD patients can strengthen the idea and can be further studied. HMGB1 might play a key role in ASD pathogenesis through neuroinflammation and can conduct treatment strategies. However, it is a highly potential factor in the pathophysiology of autism, not precisely clarified, and more research is needed.

Osteopontin (OPN) is both a soluble proinflammatory cytokine with a well-established role in autoimmune neuroinflammatory diseases and a component of the non-collagenous bone matrix that controls biomineralization in bone tissue [110]. Depending on its location and context, OPN is involved in local inflammation, cell adhesion, immune response, chemotaxis, and protection from apoptosis [111]. Heilmann et al. hypnotized that OPN can activate the immune system, reduce tissue damage, and stimulate mucosal repair during acute inflammation while promoting the Th1 response and strengthening inflammation under chronic circumstances [112].

OPN has been related to the pathogenesis of neuropsychological disorders like multiple sclerosis and Alzheimer's disease [110, 113]. Expression of secreted phosphoprotein 1 and its encoded protein OPN by CD11c + cells were associated with cognitive impairment and common neuropathologies in Alzheimer’s disease [114]. Studies on OPN levels in autistic patients are limited. However, Al-ayadhi and Mostafa [111] found an association between elevated serum levels of OPN and disease severity, indicating the role of OPN in neuroinflammation and the development of brain-specific auto-antibodies. Their findings can support the idea of OPN as an important neuroinflammation factor in the mechanism of ASD.

The possible interaction of OPN with gut microbiota has been discussed in metabolic disorders [115]. However, the role of OPN is not yet studied in association with gut microbiota in neurological disorders, especially in ASD patients, and can be a potential target for future studies. The finding of alterations in specific strains of gut microbiota connected to OPN and symptoms of ASD may help to improve diet, treatment methods, and probiotic supplements.

Calprotectin is a protein that binds to calcium and is mainly found in neutrophils, which are white blood cells that increase when inflammation and cell damage occur. Calprotectin in stool can indicate intestinal inflammation and serve as a biomarker [116]. Considering the possible role of gut inflammation in the development of ASD, a number of research have studied the association of calprotectin levels in ASD patients, but their results were inconsistent. Some reports show that ASD patients and their relatives may have higher calprotectin levels than control groups [41, 117]. Interestingly, Babinská et al. found that calprotectin levels of ASD individuals were significantly related to all domains of autism diagnostic interview-revised, which measures social interaction, communication, and restricted and repetitive behaviors [41].

Similarly, Iovene et al. reported a significant correlation between autism severity, calprotectin level, and Clostridium spp—abundance [24]. Contrarily, Azouz et al. found no relation between calprotectin and disease severity, though they revealed a moderate correlation between calprotectin and GI symptoms [118]. Tomova et al. also revealed a positive correlation between Costridiacae bacteria, the severity of GI manifestations, and behavioral symptoms of ASD children. Calprotectin levels were also moderately correlated with higher expression of macrophage inflammatory protein 1β, which was associated with communication subscale and total score of autism diagnostic observation schedule, indicating that it may play a role in microbial-neuronal cross-talk [25]. Unlikely, some investigations found no statistically significant difference in calprotectin levels between ASD patients and controls [119‐122] and, consequently, no appreciable variation in calprotectin levels of ASD patients with and without GI symptoms.

Studies on probiotic effects on calprotectin levels and autism are limited in the literature. Laghi et al. showed that greater calprotectin levels were associated with more Prevotella and fewer Akkermansia bacteria in the gut, indicating these bacteria may have inflammatory or protective effects, respectively [26]. However, Santocchi et al. found probiotic therapy, including eight strains of Streptococcus, Bifidobacterium, and Lactobacillus, to have a favorable impact on adaptive functioning in ASD patients but no discernible impact on calprotectin levels with or without GI symptoms [123]. This indicates that the probiotic effect on autistic patients is more complex than the reduction of gut inflammation, and the role of calprotectin as a probable neuroinflammatory mediator should be more studied.

Overall, the heterogeneities of calprotectin studies could be due to the diversity of trialed individuals, the accuracy of the used methods, and insufficient simultaneous studies of microbiota alterations and calprotectin. However, it is still possible to understand that host-microbiota dysbiosis and inflammation-induced calprotectin trigger neuroinflammatory mechanisms that cause autistic aspects.

Many gut microbiota-derived metabolites are highlighted in ASD, such as complex polysaccharides or metabolic amino acids, which can be neurotransmitters [124]. Several of them have been recently discussed as early diagnostic biomarkers of ASD [7]. One significant group of metabolites through which gut microbiota regulates the host physiology is short-chain fatty acids, which primarily constitute acetate (AA), butyrate (BTA), and propionate (PPA).

The genera Prevotella, Bifidobacterium, and Ruminococcus are the primary producers of acetate [125], the most prevalent SCFA, which is reported to be decreased in ASD [29, 126]. BTA is mainly produced by the Firmicutes phylum, more precisely by Lachnospiraceae and Ruminococcaceae families [127], and PPA is synthesized by the Bacteroidetes phylum (including Bacteroides and Prevotella) and Firmicutes phylum (including Roseburia, Blautia and Coprococcus) [128]. However, alongside Bacteroides, the elevated level of PPA is associated with increased Clostridium and Desulfovibrio species in autistic individuals [129]. Also, a study on autistic children revealed lower Bifidobacterium and higher PPA levels, both of which attenuated at older ages [27].

Unlike some studies [28, 29, 126], others reported higher levels of AA, PPA, and BTA in autistic patients compared to control groups [27, 30, 31, 130, 131]. These gut microbiota-related SCFAs exhibit conflicting pro-inflammatory and anti-inflammatory effects in the host's inflammatory response, possibly due to the differences in binding receptors and local concentrations [132]. Some animal studies revealed that supplementation with the microbial metabolites AA and BTA could reverse the social behavioral phenotypes [133‐136]. In contrast, intracerebroventricular injection of PPA in rat brains has induced ASD-like symptoms, including reactive gliosis [137]. It has been understood that PPA can lead to gliosis, disturbed neuro-circuitry, and neuroinflammatory response through modulation of the PTEN/AKT pathway in ASD [138]. As the finding data regarding SCFA levels in autistic patients are inconsistent and yet to be studied [7, 28, 124, 139], additional research is required to verify the potential role of SCFAs in the pathophysiology of ASD. They might be considered as neuroinflammatory biomarkers and indicators of gut microbiota modification in autism patients.

Over 60% of human genes are controlled by microRNAs (miRNAs), small, non-coding RNAs of around 18–24 nucleotides that function as epigenetic regulators. MiRNAs modify brain plasticity and neuronal development, and their dysregulation causes a broad spectrum of neurological impairments, including ASD [140‐144]. The importance of miRNAs as regulators of numerous cellular and physiological processes, including hematopoiesis, immune reactions, and inflammation, is well-established [145]. Additionally, miRNAs are affected by host-microbiota interactions and play a key role in dysbiosis and induced inflammations [146‐149]. An intensive study found over-expressed miRNAs in ASD and their possible role in impaired neurodevelopment through dysregulated inflammatory genes [150]. Besides, several studies have identified that miRNAs directly and indirectly activate inflammasomes through their interaction with 3'-UTR genes that modulate inflammasome expression [151].

In detail, animal studies suggest that an increase or decrease of miR-146a can be a potential cause of ASD [152]. A clinical study of the postnatal period compared miRNAs of ASD and healthy controls and confirmed miR-146a as the most dysregulated miRNA in ASD [152]. Using in vitro models and postmortem human brain tissues, another study also found that miR-146a overexpression in the brains of ASD patients is detectable as early as childhood [153]. The changes in Gut microbiota-host interaction could induce miR-146a and consequently promote neuroinflammatory pathways [154]. It is highlighted that miR-146a-induced nuclear factor kappa-B augments the inflammation signaling pathway in the gut-brain axis. It has been shown that Bacteroides fragilis, Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus delbrueckii Bulgaricus, and Escherichia coli Nissle 1917 were linked to miR-146a expression [149, 154, 155]. Another research indicates that miR-146a is essential for certain inflammatory cytokine expression and that its absence in the brain leads to an overall compensatory upregulation of miR-155. Enhanced protein carbonylation and decreased cysteine thiol levels were additional indicators of this elevated neuroinflammatory flux due to an upsurge in oxidative stress mediators [156].

Several studies have identified miR-146a and miR-155 to various pathologic conditions indicated by chronic inflammation [157]. A possible explanation is that gut-derived toxins, such as LPS, capable of traversing the blood–brain barrier and are in systemic circulation, can potentially activate the NF-kB-miRNA-146a-miRNA-155 signaling pathway. This pathway would then transmit pathogenic signals originating from the microbiome to the brain, which might disturb the innate immune reactions and lead to neuroinflammatory conditions [158]. MiR-155 could also be altered by gut microbiota dysbiosis [159]. One study added evidence of increased miR-155 expression in the amygdala, frontal cortex, and cerebellum of children with ASD [62]. miRNA-155 is involved in TLR activation by bacterial lipopolysaccharides, activation of tumor necrosis factor-alpha and IL-6, and regulation of suppressor of cytokine signaling 1 on dendritic cells. These activities, alongside the variation with microbiota dysbiosis, can give a candidate role to miRNA-155 in the neuroinflammatory mechanism of the gut-brain axis and ASD [152, 159]. Earlier studies identified probiotics of Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus delbrueckii, Bifidobacterium bifidum, and E coli Nissle 1917 could change the level of miR-155 [149, 155, 159, 160].

Moreover, studies found upregulated miR-181 in ASD patients, expected to impact the ASD-related neurexin 1 gene [152, 161, 162]. Neuroinflammation and immunological dysregulation are two of the many physiological processes linked to the miR-181 family [163‐165]. On the other hand, some studies show that gut microbiota could regulate miR-181 in mice [148, 166, 167]. It has also been revealed that Lactobacillus rhamnosus and Lactobacillus delbrueckii probiotics affect the miR-181a expression in inflammatory diseases [160]. Additionally, metabolites derived from gut microbiota could affect miR-181 expression in different states [148]. Altogether, these pieces of evidence strengthen the argument about the possible miR-mediated role of gut microbiota through the neuroinflammatory process in ASD.

Living microorganisms known as probiotics can influence host health through various mechanisms. According to recent research, they can be used as a therapeutic tool to treat ASD by restoring a healthy balance in the gut microbiota, adjusting the levels of neurotransmitters in the tissues, and reducing inflammation in the gut [168, 169].

Animal models revealed that probiotic supply considerably modified the social and emotional behaviors of the rats as well as blood levels of cytokines like IL-6, IL-17a, and IL-10 [59, 170, 171]. On the other hand, only a few trials assessed the impact of probiotics on ASD with the aspect of inflammatory modulation and immune system regulation (Table 2). Sanctuary et al. evaluated the use of Bifidobacterium infantis in combination with a bovine colostrum product in autistic children. Some patients revealed lower frequency of GI symptoms and aberrant behavior, possibly due to a reduction in TNF-α and IL-13 [172]. Tomova et al. also showed a strong correlation between fecal levels of TNF-α and the severity of autism, indicating the possible involvement of GI inflammation and permeability in ASD through inflammatory pathways. They could significantly decrease the TNF-α levels in the feces of autistic children through probiotic supplementation involving strains of Lactobacillus, Bifidobacteria, and Streptococcus [173]. However, Santocchi et al. found the plasma levels of plasma inflammatory biomarkers, including TNF-α, IL-6, leptin, and plasminogen activator inhibitor 1, and fecal calprotectin contrarily unaffected by the probiotic treatment, involving the same genera as Tomova et al.… Nevertheless, there is a greater improvement in some GI symptoms, adaptive functioning, and sensory profiles in the group treated with probiotics compared to placebo in the subgroup of autistic children with GI problems [123]. Similarly, using strains of Bifidobacterium and Lactobacillus alongside an oligosaccharide could improve disease severity and GI problems in autistic children [174].

Limosilactobacillus genus can also lead to improvement in adaptive symptoms of ASD [175, 177]. However, Schmitt et al. did not see any relevant changes in the plasma TNF-α and HS-CRP, fecal calprotectin, and lactoferrin with the use of this probiotic [175]. Synergic use of Lactiplantibacillus plantarum and oxytocin was also revealed to have an anti-inflammatory effect through the reduction of IL-1β [176]. The probiotic mixture containing five strains of Bifidobacterium longum with anti-inflammatory and high homeostatic intestinal activity, along with Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Ligilactobacillus salivarius, showed significantly alternation the diversity of gut microbiota. The species that are consistent with this formulation of probiotics were found in the feces of autistic children, including Streptococcus thermophilus, Bifidobacterium longum, Limosilactobacillus fermentum, and Ligilactobacillus salivarius [177].

There are no medicines indicated for the core deficits of ASD. Therefore, there is a substantial requirement for the creation of novel pharmacological approaches for patients with ASD. Overall, these findings support that probiotics may serve as a promising therapy due to their beneficial impact on symptoms of ASD. Considering the existing association between immune system dysfunction and behavioral abnormalities [178] and the possible impact of gut microbiota on ASD through inflammatory mediators, it is suggested that neuroinflammatory variables be examined during probiotic administration and the most effective formulation to alter them be determined.

Given the complexity and lack of clarity surrounding the pathophysiology of ASD, research into the role of inflammatory mechanisms and immunological dysregulation has been raised in recent years. Dysregulation pathways in ASD may also be etiologically traced back to gut microbial alterations and host-microbiota dysbiosis. These changes have been associated with ASD symptoms and severities probably through the released metabolites, neural signaling pathway by BDNF, and neuroinflammatory biomarkers, including S100B, HMGB-1, OPN, miRNAs, RANTES, eotaxin, and GM-CSF. In this review, the role of mediators as a triggering mechanism and bridging cause between gut microbiota dysbiosis-induced inflammation from one side, and neuroinflammatory processes of CNS in autism from the other side is emphasized. Probiotics as an applicable therapeutic option to recover microbiota in ASD suggest the relevance of gut microbiota and potential beneficial impacts. However, further studies are essential to evaluate the efficacy of different probiotic formulations considering microbiota alteration types, coincidence neuroinflammatory mediators, intervention length, and autistic age and symptoms. In fact, many ideas have been proposed to explain ASD pathogenesis, but there is currently a lack of intensive immunological, neurochemical, and microbiota studies in the field. This approach can clinically explain the trajectory through microbiota alteration, related metabolites, neurological inflammatory mediators, and the CNS process of ASD. This constructed dogma can be used to create etiologic, diagnostic, prognostic, or therapeutic targets for ASD.