The mast cells - Cytokines axis in Autism Spectrum Disorder

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Introduction
Autism spectrum disorder (ASD) is a neurodevelopmental disturbance, diagnosed in early childhood when the children loose acquired skills or the acquisition of new skills is delayed.It is associated with varying degrees of dysfunctional communication and social skills, repetitive and stereotypic behaviors (Theoharides et al., 2008).Over the last 30 years, there has been an impressive rise in ASD cases.According to the Centers for Disease Control and Prevention, Autism and Developmental Disabilities Monitoring Network, approximately 1 in 36 children is identified with ASD (https://www.cdc.gov/ncbddd/autism/data.html).The pathogenesis of the disease is still unclear, and there is a lack of targeted treatment (Manoli and State, 2021).However, a number of papers have suggested that ASD may be related to immune dysfunction of the patient (Ashwood et al., 2006) or of the mother during gestation (Atladottir et al., 2009).Many patients with ASD also have food allergies (Jyonouchi, 2010) and allergic-like symptoms (Angelidou et al., 2011), often in the absence of any positive objective test results, suggesting MC activation by nonallergic triggers (Theoharides et al., 2012b;Kempuraj et al., 2010).
MCs arise from bone marrow progenitors and mature in tissues depending on microenvironmental conditions.They participate not only in the development of allergic reactions, but are also implicated in immunity (Kalesnikoff and Galli, 2008) and inflammation (Theoharides et al., 2012a).MCs produce mediators, both pro-and anti-inflammatory, and may have immuno-modulatory functions (Kalesnikoff and Galli, 2008;Galli et al., 2008).They are equipped with a number of receptor systems that allow recognition of a wide spectrum of stimuli, which leads to MC activation and release of a broad set of molecules (Moon et al., 2010).
As MCs produce a number of biologically active substances and their function is under the control of a variety of soluble factors, we performed a study of protein-protein interactions (PPI) to elucidate the complex interplay between these molecules.
The aim of the present research is to enrich the current knowledge regarding the relationship between activation of MCs and MC-derived molecules and mediators in the pathogenesis of ASD.In addition, we provide data based on PPI network analysis that reveal key molecules and immune pathways associated with MCs and may provide new insights to discover drug targets for modeling the inflammatory processes observed in ASD.
The significance of the present study is in revealing the interactions between key molecules and immune pathways, underlining the role of MCs in the pathogenesis of ASD through PPI networks.It provides a direct evidence for involvement of cytokines, markers for MCs degranulation (serotonin, leukotrienes, prostaglandins, histamine), tryptase alpha/beta-1 (TPSAB1) and various receptors in ASD patients.We believe that understanding the relationship between ASD and the activation of MCs, as well as the involved molecules and interactions between them, may help the development of effective future treatments for ASD patients by providing novel therapeutic targets and strategies.

Materials and methods
In order to examine the role of MCs in the pathogenesis of ASD and the associations between key molecules related to these cells, we firstly searched PubMed, Scopus, Google Scholar and Science Direct for papers dealing with the contribution of MCs in the development of ASD.Secondly, we delineated PPI networks in which these cells and molecules are involved with the purpose to reveal novel drug targets for modulating the aforementioned molecules, immune pathways and neurotoxicity.
We constructed six types of expanded networks: 1) PPI network based on receptors for serotonin, leukotrienes, prostaglandins and histamine with 10 interactions in the first shell and none in the second (with a minimum required interaction score of 0.400 and set organism: homo sapiens), 2) PPI network with TPSAB1 with 10 interactions in the first shell and none in the second shell (with a minimum required interaction score of 0.400 and set organism: homo sapiens), 3) PPI network with TPSAB1 with 10 interactions in the first shell and none in the second shell (with a minimum required interaction score of 0.700 and set organism: homo sapiens), 4) PPI network with TPSAB1 with 10 interactions in the first shell and none in the second shell (with a minimum required interaction score of 0.900 and set organism: homo sapiens), 5) PPI network with TPSAB1 with 50 interactions in the first shell and none in the second shell (with a minimum required interaction score of 0.400 and set organism: homo sapiens), 6) PPI network based on serotonin and histamine receptors and cytokines (with a minimum required interaction score of 0.700 and set organism: homo sapiens).
The networks were constructed using STRING version 12.0 (htt ps://string-db.org).STRING and Gene Ontology (GO) net were used to visualize the physical interactions between the proteins/genes for TPSAB1.

PPI between receptors for key molecules, markers of MC degranulation
The PPI analysis revealed significant relationship between receptors of key MC-derived molecules, such as serotonin, leukotrienes, prostaglandins and histamine.These interconnections might be important for ASD patients, as their MCs are often activated, followed by degranulation and release of mediators.
Fig. 1 shows a PPI network constructed using STRING with receptors of key molecules (serotonin, leukotrienes, prostaglandins and histamine), markers of MC degranulation.The analysis is performed using minimum required interaction score>0.400-medium confidence.This network consists of 34 nodes, and the number of edges (n = 161) exceeds the expected number of edges (n = 2), with p-enrichment value of 1.0e-16, average node degree = 9.47 and average local clustering coefficient = 0.729.

Serotonin
Serotonin (5-hydroxytryptamine, 5-HT) acts as a mono amine neurotransmitter in the central (CNS) and peripheral (PNS) nervous systems.The quantity of 5-HT present in the CNS is relatively low (5%) compared to PNS (95%) (Abela et al., 2020).In the PNS, 5-HT is synthesized by both gut neurons and enterochromaffin cells (ECCs), located in the gastrointestinal system.It plays several roles as a hormone, autocrine, or paracrine factor.Because 5-HT cannot cross the blood-brain barrier (BBB), these two central and peripheral 5-HT systems are entirely independent (Sahu et al., 2018).5-HT effects are determined by interacting with seven subtypes of transmembrane receptors in the CNS and PNS, and a number of non-neuronal cells in the gastrointestinal, blood, and endocrine systems.All 5-HT receptors are heteroreceptors and postsynaptically expressed on non-serotonergic neurons.Because of the key role of 5-HTRs in the brain, there is a strong rationale to associate their ligands with the treatment of various neuropsychiatric disorders (Pourhamzeh et al., 2022).

Cysteinyl leukotrienes
The family of cysteinyl leukotrienes (CysLT), including leukotriene C4, leukotriene D4, and leukotriene E4, promotes neutrophil and eosinophil rolling and recruitment and regulates vascular permeability and smooth muscle contraction (Butterfield et al., 2018).CysLTs are released by MCs, basophils, myeloid dendritic cells, eosinophils, and macrophages.After MC activation, leukotriene C4 is converted into leukotriene E4, which is secreted and contributes to symptom development in systemic mastocytosis patients.Urinary LTE4 can be also elevated in systemic mastocytosis compared to healthy controls and correlates with serum tryptase and histamine metabolite levels (Butterfield, 2010).

Prostaglandin D2
Prostaglandin D2 (PGD2) is synthesized from prostaglandin H2 by PGD2 synthase after MC activation and contributes to increased vascular permeability, leukocyte recruitment and neuronal activation (Butterfield et al., 2018;Butterfield, 2020;Butterfield and Weiler, 2020).Levels of PGD2 metabolites are elevated in patients with anaphylaxis (Nassiri et al., 2016).Prostaglandin metabolites are also elevated in patients with MC-related disorders.For example, 11-β-prostaglandin F2α (11β-PGF2α) is increased in most patients with MC activation syndrome (MCAS) and correlates with symptoms, especially with flushing (Butterfield and Weiler, 2020).Therefore, patients with mastocytosis or MCAS and elevated 11β-PGF2α can benefit from treatment with aspirin because of its irreversible inhibition of both cyclooxygenase (COX) 1 and 2 activities preventing the conversion of arachidonic acid to prostaglandins.Indeed, aspirin treatment might decrease or normalize 11β-PGF2α levels in patients with mastocytosis and mediator related symptoms (Butterfield and Weiler, 2008;Ravi et al., 2014).

Histamine
Histamine is the major preformed MC mediator and it is stored in large quantities within secretory granules.Histamine predominantly binds to H1 and H2 receptors expressed on endothelial bronchial smooth muscle and gastric parietal cells, respectively.It causes skin wheal and flare reactions, bronchoconstriction, hypotension, vasodilation, increased capillary permeability and gastrointestinal hyperactivity (Butterfield et al., 2018;Butterfield, 2020;Leru et al., 2020;Lieberman, 2011;Lieberman et al., 2015).

PPI between receptors for MC-derived molecules, cytokines and neurotrophic factors
Further PPI analysis on the receptors of MC-derived molecules showed an interconnection between them and MC-released cytokines and neurotrophic factors, in the center of which is BDNF.As many studies have shown high BDNF levels in ASD (Zheng et al., 2016), this relationship may reveal new therapeutic targets for these patients due to the protective role of this neurotrophic factor.
Fig. 2 shows a PPI network constructed using STRING with serotonin and histamine receptors, and cytokines.The analysis is performed using minimum required interaction score>0.700-high confidence.This network consists of 39 nodes, and the number of edges (n = 118) exceeds the expected number of edges (n = 9), with p-enrichment value of 1.0e-16, average node degree = 6.05 and average local clustering coefficient = 0.697.
IL-6, a cytokine with pleiotropic effects on different tissues/organs, plays a role in hematopoiesis, inflammation, bone metabolism, and immune responses (Zheng et al., 2016).IL-6 is produced by MCs, and patients with mastocytosis have increased IL-6 plasma levels probably related to the constitutional activation of the KIT signaling pathway (Zheng et al., 2016;Theoharides et al., 2002).Therefore, IL-6, although not specific for MCs, can be considered a good diagnostic and prognostic biomarker of the severity of mastocytosis (Parente et al., 2023).
MCs can secrete IL-31, a well-known pruritogenic mediator.Its circulating levels are increased in mastocytosis and correlate with disease severity, especially with the intensity of pruritus, tryptase levels, osteosclerosis, and the extent of bone marrow infiltration by MCs (Hartmann et al., 2013).IL-31 levels are influenced by sex and body mass index and are elevated also in other diseases, such as atopic dermatitis, cutaneous lymphomas, and psoriasis (Parente et al., 2023).
IL-1β is a pro-inflammatory cytokine crucial for defence responses to infections and injuries (Dinarello, 1996).It is produced and secreted by a variety of cell types, although most research has focussed on its production within cells of innate immunity, such as monocytes and E. Kovacheva et al. macrophages (Takeuchi and Akira, 2010).
IL-6 and IL-8 (CXCL8) are widely used as a diagnostic and prognostic markers for infectious and other inflammatory conditions (Livaditi et al., 2006;Kraft et al., 2015).The main function of IL-8 is induction of chemotaxis.In addition, IL-8 causes a release of lysosomal enzymes, an upregulation of adhesion molecules, an increase of intracellular calcium, and priming of the oxidative burst (Harada et al., 1994;Huber et al., 1991;Detmers et al., 1990;Brechard et al., 2005;Henkels et al., 2011).
IL-10 is a potent anti-inflammatory cytokine that plays a crucial, and often essential, role in preventing inflammatory and autoimmune pathologies (Sabat et al., 2010;Kuhn et al., 1993).Deficiency or aberrant expression of IL-10 can enhance inflammatory response to microbial challenge but also leads to development of inflammatory bowel disease and a number of autoimmune diseases (Sellon et al., 1998;O'Garra et al., 2008;Gazzinelli et al., 1996).Thus impaired IL-10 expression or signaling can enhance the clearance of pathogens during an acute infection, but also exaggerates the inflammatory response, resulting in exacerbated immunopathology and tissue damage (Li et al., 1999;Siewe et al., 2006;Sun et al., 2009;Ejrnaes et al., 2006).
The IL-12 family is comprised of 4 members, IL-12, IL-23, IL-27 and IL-35.IL-12, IL-23 and IL-27 are secreted by activated antigen presenting cells (APC) during antigen presentation to naïve T cells, while IL-35 is a product of regulatory T and B cells (Collison et al., 2007;Wang et al., 2014;Shen et al., 2014).They provide the bridge between innate and adaptive immune response by priming naïve CD4 + T cells to differentiate into cytokine-producing T-helper subsets and memory T cells (Steinman, 2006).In addition to their influence on cell fate decisions of differentiating lymphocytes, IL-12 cytokines regulate cellular pathways required for proper functioning of the immune system.Some IL-12 members activate pro-inflammatory responses that confer protection against infection while others restrain unbridled immune responses that cause autoimmune diseases (Collison et al., 2007;Hunter, 2005;Trinchieri et al., 2003).
IL-38 (IL-1F10) is the 10th member of the IL-1 family.IL-38 is an IL-36 antagonist and functions as a typical receptor antagonist (Ra) similar to IL-1Ra and IL-36Ra (van de Veerdonk et al., 2012).IL-38 reduces inflammation by preventing the binding of agonist receptor ligands to IL-36R, a specific receptor of IL-38 (Yuan et al., 2015).
Interferons (IFNs) are crucial modulators of the immune response against various viruses as well as cancer cells.IFNs are subdivided into three types: I (α, β, ϵ, k, ω), II (γ), and III (λ), based on the IFN receptors through which they signal.In humans, IFN-α can be further categorized into 13 different IFN-α subtypes, which all signal through a shared type I IFN heterodimeric receptor complex comprising two IFN-α receptor subunits (IFNAR1 and IFNAR2).These IFNAR receptor subunits are present on nearly all nucleated somatic cells (Hoffmann et al., 2015).
TNF-α has pleiotropic effects on a large number of cell types.The cytokine is reported to be an important regulator of inflammatory responses and it is involved in the pathogenesis of several inflammatory and autoimmune diseases (Bradley, 2008).Furthermore, TNF-α is known to trigger other inflammatory molecules, such as cytokines and chemokines (Jiang et al., 2017).
Nerve growth factor (NGF) is a neurotrophin enhancing the growth and survival of peripheral sensory and sympathetic mammalian nerve cells (Levi-Montalcini, 1987).It is reported that peripherally innervated tissues produce and release NGF which is retrogradely transported by specific receptors, to finally provide protective and functional neuronal integrity (Reichardt, 2006).

PPI with TPSAB1 as a seed protein
Tryptase is usually stored within MCs and is released upon stimulation, thereby serving as a biomarker of MC activation.This fact prompted us to use it as a seed protein in the PPI analysis and to search for its relationship with other molecules.
Tryptase is a neutral serine-proteinase with trypsin-like characteristics and a molecular weight of 134 kDa.It is the most abundant protease in MC secretory granules.Four non-covalently bound subunits constitute the enzyme.With regard to the amino acid sequences, human MC tryptases are categorized into four major types α, β, γ, and δ, with tryptase α having two subtypes (α1, α2) and tryptase β having three subtypes (β1, β2, β3) (Kolmar et al., 2012;Payne and Kam, 2004;Pereira et al., 1998;Presnell and Taft, 2003).The first two types are soluble proteinases and can proteolytically form active tetramers packed in the granules in complex with proteoglycans, largely heparin proteoglycans (Hanna et al., 1993;Schwartz, 1990).Based on an Asp246/Gly246 mutation in its catalytic domain, tryptase α displays a slight enzymatic activity.Moreover, it is not stored in MC granules and appears to act as a pro-enzyme (Schwartz et al., 1995).On the contrary, tryptase β is the chief enzymatically active type, and, by being a granule component, it is released from MCs upon activation and degranulation (Lindstedt et al., 1998).In the extracellular fluid, tryptase can diffuse away from the secretory granules (Payne and Kam, 2004).
Human tryptase genes are clustered on the short arm of chromosome 16, encoding an immature tryptase (protryptase) that, following proteolytic activation, assembles into an active tetramer presenting as a soluble serine proteinase (Fukuoka and Schwartz, 2007;Kido and Nakai, 2002;Maffitt, 2001).The activation process consists of two proteolytic steps (Ni et al., 2017).An autocatalytic intermolecular cleavage comes in the first step, followed by proteolytic processing by cathepsin C when exposed to heparin or dextran sulphate under acidic pH and optimal conditions in MCs.The second step is to remove the remaining precursor dipeptide via dipeptidyl peptidase I converting spontaneously the mature peptide into a mature tetramer.There has been some evidence that processing of human tryptase involves activation by cathepsin L or B. Though more likely to be involved in a minor portion of protryptase processing, cathepsin C is not critical for mature tryptase synthesis (Le et al., 2011a;Le et al., 2011b).
Fig. 3 A shows a PPI network constructed using STRING with TPSAB1 as a key mediator released during MC degranulation.The analysis is performed using 10 interactions in the first shell (none in the second shell; minimum required interaction score>0.400-medium confidence).This network consists of 11 nodes, and the number of edges (n = 33) exceeds the expected number of edges (n = 10), with p-enrichment value of 1.06e-08, average node degree = 6 and average local clustering coefficient = 0.802.Fig. 3. A: PPI network constructed using STRING with TPSAB1 as a seed protein with 10 interactions in the first shell (none in the second shell; minimum required interaction score>0.400);B: PPI network constructed using STRING with TPSAB1 as seed protein with 10 interactions in the first shell (none in the second shell; minimum required interaction score>0.700);C: PPI network constructed using STRING with TPSAB1 as seed protein with 10 interactions in the first shell (none in the second shell; minimum required interaction score>0.900).Nodes indicate proteins and edges indicate PPIs.

Table 1
Descriptions and functions of proteins belonging to the PPI network (10 interactions in the first shell; none in the second shell; minimum required interaction score>0.400;0.700; 0.900) constructed around TPSAB1.Fig. 3 B demonstrates a PPI network constructed using STRING with TPSAB1 as a key mediator released during MC degranulation.The analysis is performed using 10 interactions in the first shell (none in the second shell; minimum required interaction score>0.700-high confidence).This network consists of 8 nodes, and the number of edges (n = 13) exceeds the expected number of edges (n = 7), with p-enrichment value of 0.0273, average node degree = 3.25 and average local clustering coefficient = 0.786.Fig. 3 C presents a PPI network constructed using STRING with TPSAB1 as a key mediator released during MC degranulation.The analysis is performed using 10 interactions in the first shell (none in the second shell; minimum required interaction score>0.900-the highest confidence).This network consists of 5 nodes, and the number of edges (n = 5) exceeds the expected number of edges (n = 4), with p-enrichment value of 0.37, average node degree = 2 and average local clustering coefficient = 0.833.Shows the descriptions and functions of the involved proteins in this PPI network.
As TPSAB1 is the basic mediator, we tried to summarise the descriptions and functions of the involved proteins in Fig. 3 PPI networks according to their interaction score with TPSAB1 (Table 1).
Further analysis of TPSAB1 as a seed protein revealed a significant number of interconnections with other molecules, such as MAPK 11, MAPK 12 and MAPK 13, which are altered in ASD.
Fig. 4 shows a PPI network constructed using STRING with TPSAB1 as a key mediator released during MC degranulation.The analysis is performed using 50 interactions in the first shell (none in the second shell; minimum required interaction score>0.400-medium confidence).This network consists of 36 nodes, and the number of edges (n = 156) exceeds the expected number of edges (n = 46), with p-enrichment value of 1.0e-16, average node degree = 8.67 and average local clustering coefficient = 0.738.
In order to explain the performed analysis, we display the top twenty Go-enriched biological processes around TPSAB1 in the PPI network.
Table 2 shows the top twenty biological GO-enriched terms in the PPI network (50 interactions in the first shell; none in the second shell; minimum required interaction score>0.400)constructed around TPSAB1, indicating that the five most important over-represented terms are the positive regulation of MC proliferation, melanocyte migration, positive regulation of fibrinolysis and angiotensin maturation.
Observed gene count indicates how many proteins in the network are annotated with a particular term.Strength describes how large the enrichment effect is.It is the ratio between the number of proteins in the network that are annotated with the term and the number of proteins that are expected to be annotated with this term in a random network of the same size.False discovery rate describes how significant the enrichment is.P-values corrected for multiple testing within each category using the Benjamini-Hochberg procedure are shown.

Discussion
PPI data can be used in a larger scale to map networks of interactions depending on their physical or functional association (Barabasi and Oltvai, 2004;Grindrod and Kibble, 2004).Protein interaction networks are practical means to abstract basic knowledge and to improve biological and biomedical applications.Although protein interaction networks are incomplete (Yu et al., 2008), systematic studies of them have been confirmed to be especially important for deciphering the relationships between network structure and function (Yook et al., 2004), discovering novel protein function (Sharan et al., 2007), identifying functionally coherent modules (Dittrich et al., 2008;Spirin and Mirny, 2003), and conserved molecular interaction patterns (Jaeger et al., 2010;Sharan et al., 2005).
Our study presents novel data on the complex interplay between molecules, which participate and are elevated in both, ASD and MC  activation.We believe that such connections, and especially these between pro-and anti-inflammatory mediators, may improve the treatment of neuroinflammation in ASD patients.Our previous reports on ASD revealed that redox mechanisms regulate and modulate many different immune functions, including but not limited to macrophage and Th cell polarization, phagocytosis, production of pro-and antiinflammatory cytokines, metabolic reprogramming of immune cells, immune training and tolerance, chemotaxis, pathogen sensing, antiviral and antibacterial effects, TLR activity, and endotoxin tolerance (Morris et al., 2022).In a previous study we showed that peripheral activation of immune-inflammatory pathways, most likely induced by viral infections, may result in CNS neuroinflammation and mitochondrial dysfunction.These processes are leading to abnormalities in transsynaptic transmission, and brain neurodevelopment in ASD (Gevezova et al., 2023).Furthermore, our results are supported by reports from other researchers who found imbalance in the number and ratio of immune cells involved in both, the innate and adaptive immune responses, in ASD (Nadeem et al. 2019(Nadeem et al. , 2022)).Abnormalities in these immune cells lead to autoimmune and neuroimmune dysregulation.Moreover, IL-17 signaling in immune cells is found to play an important role in bidirectional brain-peripheral inflammatory communication (Nadeem et al., 2020).IL-17 receptor signaling in monocytes may potentiate the effects of IL-17A released by other immune cells and may aggravate neuroinflammation in ASD (Nadeem et al., 2018).This evidence reveals that neuroinflammation plays a key role in the pathogenesis of ASD.
IL-33 is reported to synergize with inflammatory neuropeptides to stimulate MCs and results in increased vascular permeability (Theoharides et al., 2010).IL-33 has been considered as an alarmin, acting through MCs to alert the innate immune system (Moussion et al., 2008;Enoksson et al., 2011), and has been linked to brain inflammation (Zhao et al., 2012;Pichery et al., 2012;Chakraborty et al., 2010).A number of inflammatory molecules, such as , are found to be increased in the brain and cerebrospinal fluid of many patients with ASD (Zimmerman et al., 2005;Li et al., 2009;Tsilioni et al., 2015).These molecules may derive from the activation of microglia (Rodriguez and Kern, 2011;Gupta et al., 2014;Koyama and Ikegaya, 2015;Takano, 2015), responsible for innate immunity of the brain (Ransohoff and Brown, 2012;Aguzzi et al., 2013).
In addition, a meta-analysis demonstrated significantly lower blood concentrations of IL-10 in patients with ASD compared to controls (Xie et al., 2017;Tostes et al., 2012;Tobiasova et al., 2011;Suzuki et al., 2011;Pecorelli et al., 2016;Pardo et al., 2017;Napolioni et al., 2013;Manzardo et al., 2012;Inga Jacome et al., 2016;Guloksuz et al., 2017;Emanuele et al., 2010;El-Ansary et al., 2016;Bryn et al., 2017;Ashwood et al., 2011).This is not surprising as IL-10 is an anti-inflammatory cytokine, probably resulting in neuroinflammation.IL-10 is a key immunoregulatory cytokine associated with the inhibition of inflammatory response induced by MCs and macrophages.IL-10 is reported to induce apoptosis in immune cells, which occurs with caspase-3 activation and reduced mitochondrial membrane potential.By inhibiting the ability of cells to survive, IL-10 can maintain immune homeostasis and prevent the onset of chronic inflammation (Bailey et al., 2006).Although there is not much evidence for the involvement of IL-31 in neuroinflammation, Franzoi et al. (2018) found a possible link between the cytokine and multiple sclerosis patients.In addition, IL-31 is involved in skin allergic reactions (Hartmann et al., 2013;Ferretti et al., 2017) and MC-associated inflammatory cascade by Th2 lymphocytes (Rabenhorst and Hartmann, 2014;Stott et al., 2013).Therefore, elucidating the roles of mediators will be useful for new immunotherapeutic strategies.Despite the paucity of data on IL-31 in ASD, Ahmad et al. (2020) reported increased levels of IL-31 mRNA and protein compared to a control sample of healthy children.Researchers found that increased production of inflammatory cytokines and transcription factors in CXCR1+ cells lead to immunological imbalance in ASD (Ahmad et al., 2020).Another report stated increased serum levels of IL-38 in about 30% of children with ASD, as compared to controls.Some authors speculate that children with higher serum IL-38 have fewer ASD-related symptoms.In case such cooccurrence is true, it may be assumed that peripheral IL-38 could cross the BBB and partially correct any IL-38 deficiency in the amygdala (Tsilioni et al., 2020).Zhao et al. (2021) found that the levels of peripheral IL6, IL-1β, IL-12p70, MIF, eotaxin-1, MCP-1, IL-8, IL-7, IL-2, IL12, TNF-α, IL-17, and IL-4 were significantly changed in ASD patients compared with controls.
Cumulative evidence supports the role of a dysregulated immune system in neurodevelopmental abnormalities such as ASD, attention deficit hyperactivity disorder and intellectual disability disorder (Sreenivas et al., 2024).Neurodevelopmental conditions are associated with depletion of the compensatory immunoregulatory system (CIRS) (IL-4, IL-10, sIL-1RA and sIL-2R), M1/M2 polarization of macrophages, increased neurogenesis, IL-4 levels.Also enhanced IL-1 signaling associated with increased IL-1α and decreased IL-1-receptor antagonist levels are reported.These immune abnormalities have functional implications for neurogenesis, neurotoxicity and neurodevelopment (Sreenivas et al., 2024).
Microglia and MCs are found to be in close proximity and actively communicating with each other in the CNS.MCs use adhesion molecules, surface receptors and mediators to participate in a complex crosstalk with other brain-resident cells.Recently, proofs suggesting that the initiation and propagation of neuroinflammation are related to the interactions between these cell types are reported (Mukai et al., 2018).MCs also express surface molecules with costimulatory and inhibitory functions, allowing them to communicate with immunocompetent cells, such as T-cells and B-cells.Therefore, they serve as a main bridge between innate and adaptive immunity (Galli et al., 2008).Furthermore, activated MCs in the brain release histamine which causes activation and phenotypic changes of microglial cells (Frick et al., 2016).Exposure of primary microglia to MC-derived tryptase stimulates microglia to secrete TNF-α, IL-6 and RNOS.These effects are mediated by protease-activated receptor-2 (PAR-2) signaling via activation of mitogen-activated protein (MAP) kinase (Erk and p38) and NF-kappa B (NF-kB) pathways (Zhang et al., 2016b).Furthermore, PAR-2 activation induces the expression of ATP-sensitive ionotropic P2X4 receptors on microglia.Correspondingly, the exposure to ATP leads to secretion of BDNF, a potent trophic factor (Yuan et al., 2010).The presence of functional P2X4 receptors are also reported on human MCs lines E. Kovacheva et al. (Wareham et al., 2009).MC-derived pro-inflammatory cytokines such as CCL2, TNF-α and IL1β can also influence microglia activation.In addition, MC degranulation has been shown to activate microglia.Stereotaxic injection of a MC degranulation compound 48/80 (C48/80) and activator of the mas-related G protein-coupled receptor (MRGPR) (McNeil et al., 2015) in the hypothalamus of rats induces MC degranulation, production of pro-inflammatory cytokines and microglia activation (Dong et al., 2014).These effects are mediated by the activation of MAP kinase and AKT pathways followed by increased protein expression of H1R, H4R, PAR-2 and TLR4 on microglial cells.Treatment with a MC stabilizer disodium cromoglycate (cromolyn), inhibits microglial activation and downstream signaling, suggesting MC involvement (Sandhu and Kulka, 2021).
The present study is the first to report PPI network analysis of MCrelated molecules participating in the pathogenesis of ASD.Our results reveal that histamine and serotonin receptors are connected to cytokines via BDNF.Besides this, a meta-analysis of serum and plasma BDNF levels identified 14 studies involving 2707 participants (1131 ASD patients), and noted significantly elevated peripheral BDNF levels in children with ASD (Zheng et al., 2016).In a small post mortem study (n = 7) of mainly male adult ASD patients, BDNF-expression in the basal forebrain was threefold higher than in the control group (Perry et al., 2001).The possibility that BDNF influences brain development is indicated by a MRI study which found that a single nucleotide polymorphism in the BDNF gene affects cortical volume (Raznahan et al., 2009).There is limited literature on neurotrophin and serotonin (5-HT) levels in adults with ASD and very few studies reported original data.Carpita et al. (2024) registered significantly lower platelet-poor plasma 5-HT levels compared to broad autism phenotype and control groups, but no significant difference in BDNF levels.In addition, 5-HT levels have been reported to be associated with some autistic symptoms and support the hypothesis that the biochemical characteristics of ASD in adults are different from those in children.
In children with ASD, MCs are involved in both allergic and inflammatory responses, and have been shown to be 10 times more activated if mastocytosis is present (Theoharides, 2009).Pharmacological studies revealed that some natural substances may block MC activation.That is why progressively more children with autism in the United States are administered supplements containing the natural flavonoids luteolin and quercetin (Theoharides et al., 2012c).Luteolin has antioxidant, anti-inflammatory, MC blocking and neuroprotective effects (Taliou et al., 2013).The results after 4 months of treatment with supplements containing lutein led to improvement in eye contact, attention and social interaction (Theoharides et al., 2012c).Taliou et al. (2013) also reported an improvement in adaptive functioning and behavior.

Limitations of the study
Challenges and limitations of the current data are associated with the fact that PPI analyzes reveal biological relationships between proteins which are enriched in networks, but only future studies can investigate the specific functions of MCs in the different stages of ASD development.This will enable the delineation of various immune clusters that occur in neurodevelopmental disorders.Based on our PPI results, further investigations could determine the molecular mechanisms that link MC dysfunctions with different subtypes of immune disorders in ASD.In addition, future research should focus on MC functions in association with the plethora of immune affects that MCs can produce.Furthermore, potential selection bias should not be ignored in this study.

Conclusion
The incidence of ASD cases continues to rise and there is yet no clinically effective drug for its core symptoms.Unfortunately, the lack of clear understanding of its pathogenesis and the deficit of unquestionable biomarkers makes the development of effective treatments difficult.Identifying ways to inhibit inflammation and the activation of MCs may constitute a novel therapeutic approach for ASD.In the present paper, we found interconnections between MC-derived and MC-related molecules and immune-inflammatory pathways involved in ASD.The built PPI network showed significant interactions between them and their role in MC proliferation and migration, signaling pathways and immune response regulation.Therefore, understanding the exact mechanisms and the reciprocal interactions of all the factors that may have a role in the development of ASD and cognitive alterations is the clue to effective therapeutic strategies.

Fig. 1 .
Fig. 1.STRING protein-protein network analysis performed on receptors of key molecules (serotonin, leukotrienes, prostaglandins and histamine), markers of MC degranulation.Nodes indicate proteins and edges indicate PPIs.

Fig. 2 .
Fig. 2. PPI network constructed using STRING with serotonin and histamine receptors and cytokines with minimum required interaction score>0.700.Nodes indicate proteins and edges indicate PPIs.

Fig. 4 .
Fig. 4. PPI network constructed using STRING with TPSAB1 as a seed protein and 50 interactions in the first shell (none in the second shell; minimum required interaction score>0.400).Nodes indicate proteins and edges indicate PPIs.

Table 2
The top twenty biological GO-enriched terms (biological processes) in the PPI network (50 interactions in the first shell; none in the second shell; minimum required interaction score>0.400)constructed around TPSAB1.