Characterising neuroimmune changes in the gut-brain axis in autism: alterations in gut-associated lymphoid tissue and the enteric nervous system
Characterizing neuroimmune changes in the gut-brain axis in Autism: Alterations of the Gut-Associated Lymphoid Tissue (GALT) and the Enteric Nervous System
Gastrointestinal (GI) issues have been well recognized in autistic patients since the disease was first described in the 1940s (1, 15). GI dysfunction is a hallmark symptom for individuals with autistic spectral disorders (ASDs) (1). Children with ASDs are four times more prone to GI dysfunction compared to their healthy counterparts (2, 15). As a result, most patients of ASD tend to limit their diet to certain favored food items (3). The reason for such specificity towards specific food items remain unexplored (3). It is contended that changes in gut morphology and emotive functions could attribute to such phenomenon (15). Although different studies have confirmed the presence of neuronal deficits in patients with ASD, there is inconclusive evidence regarding the involvement of gastrointestinal histopathology with such deficits (2, 17). It is contended that the genes that are implicated for the neuronal deficits in ASD patients might also be responsible for the associated GI histopathology. For example, mutations in the Shank-3 and Neuroligin-3 have been strongly associated with the neuronal deficits observed in ASD patients (4, 5). Various researchers have provided mounting evidence regarding the involvement of Shank-3 gene in the genesis of GI dysfunctions that are presented by patients with ASD (4, 5, 6).
The genes that control neuronal activity are also contended to mediate gastrointestinal functions across concerned stakeholders. Hence, different researchers have proposed the involvement of gut-brain axis in driving the symptoms of ASDs and vice-versa (6, 7, 17). The mechanism of interaction between the central and peripheral nervous systems in regulating the gut-brain axis remains inconclusive (6, 7). Studies suggest that the vagus nerve might play a key role in mediating the bidirectional communication between the gut and the brain (8, 10). However, recent evidence suggests the role of other neurotransmitters in regulating the gut-brain axis (17). In one study, Veenstra-VanderWeele et al. showed that individuals with autistic disorders exhibit reduced serotonergic activity in the gut of ASD patients. These patients exhibit increased serotonin reuptake due to the over activity of the SERT receptor. The authors attributed such phenomenon to mutations in the SERT (serotonin reuptake) gene (17). The SERT gene expresses the serotonin-reuptake protein which is responsible for regulating the turnover of serotonin in the neuronal synapses. Mutations in the SERT gene are related to higher rate of reuptake of serotonin from the neuronal synapses (17). Serotonin is a key neurotransmitter of the central nervous system and the enteric nervous. Veenstra-VanderWeele et al. speculated that specific neurotransmitters can mediate bidirectional communication between the gut and brain in patients with ASD or their healthy counterparts (15). Therefore, a dysfunction in the secretion or expression of such specific neurotransmitters could attribute to the cognitive and GI dysfunctions in patients with ASD.
In one of their earlier studies, the same researchers highlighted that mice with SERT-mutations vocalized less, exhibited repetitive behavior, and avoidance with other mice (18). Therefore, the role of serotonergic mutations in the genesis of symptoms of autism could be established through such findings. The next question was to explore the relation between such symptoms and the genesis of GI histopathology in patients with ASD (22). To address this question, Margolis and Gershon explored the histopathological changes of the gut mucosa in mice experimental models (15). The authors showed that mice induced with ASD exhibited poor gut lining and had fewer neurons than their healthy counterparts. To recall, low serotonin levels inhibit the development of neurons in the GI tract and also reduces GI motility. Reduction in GI motility could manifest as constipation that s common in patients with ASD (19).
Very few studies have explored the histopathological changes in the large bowel (specifically the mid-colon) and its implications in ASD patients. Studies are also scarce in regard to the isolation of specific gene mutations that are simultaneously responsible for GI and neuronal dysfunction in ASD patients (13). Changes in gut micro biota, inappropriate immune responses, and altered intestinal permeability have also been implicated for the genesis of GI dysfunctions in patients with ASD (9, 10, 11, 12). Hence, the present study was conducted to draw a cause-and-effect relationship between the symptoms and histopathological changes in the gut across patients presenting with ASDs. The study further explored the immunological changes that could likely contribute to the genesis of altered GI function across concerned stakeholders.
Our study explored the role of Shank3 KO mutation on the morphology and histopathological features of the mouse colon. The histopathology studies (using H&E staining) reflected that the intensity of stain was clearly distinguishable within the inner longitudinal and outer circular muscles, mucosal and sub mucosal layers of the mid-colon sections of both Wild-type (n=14) and Shank 3 KO mutated mice (n=23). Mutations in the Shank3 KO gene are common across patients presenting with ASD. The study involved both types of Shank3 mutations; Shank3 KO Heterozygous (n=12) and Shank3 KO Homozygous (n=11). The homozygous variant (Shank3 KO HOM) was double recessive for the proposed mutations within the Shank3 gene. The morphological features that were explored in this study include villus height, villus width and crypt depth in the TS (transverse section) of the mid-colon.
Changes in the morphology of the mid-colon or the large intestine, in general, are responsible for the gastrointestinal symptoms witnessed across ASD patients. One study reflected that changes in the lumen diameter of the mid-colon could delay transit time within the large intestine and cause constipation across concerned stakeholders. Likewise, another study reflected that changes in colon morphology were responsible for the classical malabsorption syndrome. Margolis and Gershon highlighted that constipation and malabsorption are also the hallmark GI dysfunctions witnessed across ASD patients (15).
In fact, the changes in GI morphology across ASD patients are attributed to the alterations in the turnover of serotonin and other growth factors (18, 20). The same study further highlighted that serotonin is a key mediator of the gut-brain axis that accounts for the neurological and GI dysfunctions in ASD patients (14, 15). Our study reflected that the height of the villus in Shank3 KO HOM and Shank3 KO HOM mice significantly larger compared to their WT counterparts (p<0.05). However, there was no significant variation in the villus width and crypt depth between the three experimental genotypes (WT, Shank3 KO HOM, and Shank3 KO HOM) (p>0.05).
Our study further showed that the three experimental genotypes of the mid-colon sections (WT, Shank3 HET, and Shank3 HOM) differed in immune response and neuronal distribution. The primary antibodies Hu and Tuj1 were used to label the neurons while Iba1 was used to label the inflammatory cells such as monocytes and macrophages in the colonic isolates. Quantitative analyses revealed higher density of Iba-1 marker in Shank3 KO (HOM) sections in comparison to WT (163.4 ± 7 versus 120 ± 12cells per µm2, p=0.008). However, the Tuj 1 immunoreactivity was found to be non-specific. The Tuj-1 immunoreactivity was used to inspect neuronal cells processes and microtubules in the musculature. The Hu-immunoreactivity was isolated from the surface of cell bodies and the neuronal processes of the mid-colon.
Iba1 antibody is a rabbit polyclonal antibody that is known to react specifically with brain microglia and macrophages. Our study showed that the positive cells of Iba-1 were attached to the blood vessels and there were elongated cell bodies which were speculated to be perivascular macrophages. These findings suggest that individuals with ASD exhibit higher inflammatory characteristics in their gut mucosa compared to their healthy counterparts. Such speculation is not surprising considering the alterations in bowel functions across patients with ASD. Studies suggest that accumulation of inflammatory mediators narrow the lumen and motility of the gut (18, 19). As a result, the respective individual is at the risk of developing constipation or diarrhea. Such symptoms are the hallmark features of GI dysfunction in patients with ASD. The findings of this study complemented the findings of Veenstra-VanderWeele et al. and Margolis and Gershon. In our study, the neuronal cell populations as evident from Hu and Tuj-1 tagging was found to be higher in the mid-colon sections of WT mice compared to their Shank3 KO mutated counterparts. The observations were based on secondary antibody tagged imaging.
Therefore, it could be possible that specific neuronal populations (such as serotonergic neurons) might be less developed in Shank3 KO mutated counterparts compared to WT mice. A reduction in neurotransmitter secretion could have accounted for the increased inflammatory response and morphological changes in the mid-colon section that was reflected in this study. In this study, Shank3 KO mutated mice showed marked reduction in neuronal components. Hence, a deficit of certain population or sub-population of neurons or neurotransmitters might have been responsible for the GI and cognitive dysfunctions that are specific for the Shank3 KO-mutated model of ASD. The study further implicated that heightened inflammatory or immune response may account for luminal thinning (8, 18, 20). Our study showed that increase in length of the villi and narrowing of the intestinal lumen due to the accumulation of inflammatory mediators might reduce bolus formation. Such issues indicate a possible correlation between autism and reduced gastric motility that manifests as constipation and malabsorption in concerned individuals.
The present study exhibited certain strength and limitations. The study provided conclusive evidence regarding the cause-and-effect relationship between the morphological changes in mid-colon sections in the presence of autistic spectral disorders. The sample size for the immunohistochemical studies was quite small. Hence, the chances of experimental bias cannot be ruled out in this study. Future studies should be conducted with large sample sizes to provide conclusive evidence regarding the genesis of GI dysfunctions in ASD patients. Studies should be also conducted to identify the neuronal and neurotransmitter populations that are reduced in patients affected with ASD. Such findings could help to correlate the neuronal deficits and its impact on GI architecture across affected individuals. The present study provided the roadmap for investigating the neuronal correlates of GI dysfunction in ASD patients. These findings might be helpful for managing the symptoms and pathology of autistic disorders. Hence, the present study also pointed that cross-talk mechanisms might be involved in regulating the gut-brain axis in patients with ASD (21, 22, 23).
Although the role of neuronal activity in mediating different immune or inflammatory responses remains undisputed, the detailed mechanism through which neurotransmitters modulate such pathways remain inconclusive. However, studies suggest that neurotransmitters might play a role in modulating cytokine signaling and binding of antigens to their receptors. The present study attempted to elucidate the neuroimmune changes in the gut-brain axis that led to alterations in the Gut-Associated Lymphoid Tissue (GALT) and the enteric nervous system. The study showed that autistic mice models exhibit histopathological changes in their gut morphology. The villi height was significantly increased in the mid-colon of such models compared to their healthy counterparts. However, the experimental isolates exhibited comparable villi width and crypt depth as their healthy (WT) counterparts.
Our study further showed that the neuronal population in mice autistic models within the gut mucosa was less distributed compared to their WT counterparts. On the contrary, mid-colon sections in autistic mice exhibited higher inflammatory response compared to their healthy controls. The findings of the present study and past studies reflect that serotonin could play a key role in integrating the functional correlates of GI morphology with alterations in the morphology of the ENS. Serotonin is a key neurotransmitter in the brain and is primarily responsible for mood elevation. Studies suggest that serotonin is pro-inflammatory in the CNS, while it is anti-inflammatory outside the CNS. It is contended that serotonin acts on the subtype-2B receptors (5-HT2B) to produce its anti-inflammatory properties. Recent evidence suggests that serotonin subtype-2B receptors (5-HT2B) are widely distributed in the urinary bladder and other smooth muscles of the body. Since the gastric mucosa is lined by smooth muscles, the involvement of serotonergic pathways in influencing the GI pathophysiology of autistic mice models cannot be ruled out as per our study.
Discussion on Cigarette Smoke Part
The study further showed that mice exposed to cigarette smoke exhibit thinner mucosal lining in their mid-colon section compared to their non-smoking (Sham) counterparts (p<0.05). Such findings further confirmed the involvement of the gut-brain axis in the genesis of GI symptoms across ASD patients. Loss of mucosal lining or thinning of the gastric mucosa could account for the reduced GI motility that is witnessed across autistic patients.
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