For the first time, it is suggested that intestinal and hepatic galectin-3+ cells interfere with autistic-like behaviors. Total (Lgals3−/− mice) or partial (Lgals3+/+ mice supplemented with cow´s milk) absence of galectin-3 in the gut-liver axis amplified stereotypies, restrict interest and social retraction in BALB/c mice. These behavioral phenotypes were related with disturbed niches of intestinal epithelial cells, hepatocytes and Kupffer cells in the liver, and shank-3+, Iba-1+, NOS2+ and drebrin+ cells in the CNS.
BALB/c mice showed significant low sociability, self-aggressive behaviors, and stereotypies when compared with neurotypical C57BL/6 mice 5. Our data revealed that both total absence (Lgals3−/− BALB/c mice) or reduction of galectin-3 in the gut-liver axis induced by milk supplementation increased atypical behaviors. The role of galectin-3 as immunomodulatory factor in the gut and liver has been attracted the attention of specialists 15, 20.
Galectin-3 colocalizes with desmoplakin-1 and desmoglein-2 contributing to selective permeability in enterocytes 21. In Lgals3−/− mice, enterocytes have ultrastructural defects in cytoskeleton and basolateral membrane domains resulting in aberrant traffic of luminal molecules 14. In the liver, galectin-3 plays hepatoprotective functions in distinct conditions 12, 22, 23. Lgals3−/− mice develop severe liver steatosis in diet models 24, NASH in aging 25, low regenerative capacity 15, and abnormal organization of collagen fibers in experimental fibrosis 26, 27. Maybe, the reduction or total absence of galectin-3 could modify the intestinal permeability allowing the circulation of luminal components with potential to affect CNS functions.
The strategy to reduce galectin-3 in the gut-liver axis of wild type mice seemed efficient, given that mice supplemented with cow’s milk reduced the epithelial levels of galectin-3 in the intestinal villus and hepatic Kupffer cells. Cow’s milk carries galectin-3 inhibitors, including lactose 28. Intriguingly, milk consumption caused a significant imbalance in intestinal KI67+ and Dll-4+ cell niches, indicating an abnormal turnover of epithelial cells. In corroboration, KI67+ cell niches are disorganized during intervention with milk oligosaccharides and epithelial barrier dysfunctions that permit the abnormal flux of microbiota compounds 29–32. Dll-4 normally synthesized by intestinal epithelial cells is significantly increased during mucosa inflammation 33, 34. In addition, Dll-4−/− mice showed reduced niches of KI67+ cells in the gut 35.
In the liver, milk supplementation was linked to abnormal glycogen accumulation in hepatocytes. Although glycogen naturally accumulates after milk consumption within 18 h of last supplementation 36, long-time glycogen deposit and hydropic degeneration are compatible with subacute liver injury 37, 38. Regarding to increased number of NOS2+ cells, this phenotype has been associated with oxidative stress induced by chemical hepatotoxic processes 39–41.
The atypical pattern of behavior in the absence or partial reduction of galectin-3 in the gut-liver axis led us to investigate the distribution of shank-3+ and Iba-1+ cells, possible oxidative stress sites and the organization of some synaptic proteins in the CNS. Shank-3 stabilizes glutamatergic receptors in synapses 42 and shank-3−/− mice exhibited neurodevelopmental disturbances in neurons of prefrontal cortex 43, GABAergic interneuron dysfunctions 44, and social deficits followed by repetitive stereotyped behaviors 45, 46. In our experimental conditions, reduction or lack of galectin-3 was concomitant with decreased numbers of shank-3+ cell in the cerebral cortex, suggesting that gut-liver axis disorders and atypical behaviors can be correlated by, at least in part, cellular signaling pathways involving galectin-3 and shank-3.
The cerebral cortex of mice marked by partial inhibition of galectin-3 in the gut-liver axis was also characterized by increased numbers of IBA-1+ microglial cells. The same pattern was observed in the cerebellum of these milk-supplemented mice. It is possible that extracerebral inflammation, including inflammatory bowel diseases, activates microglial cells in the CNS 47–49. Particularly in ASD, gliosis and neuroinflammation events have been described in patients and murine models 50, 51. The cerebellum of BTBR T + tf/J (BTBR) mice, an relevant experimental model to study ASD 52, 53, has increased numbers of Iba-1+ cells and NOS2 expression correlated with stereotypies 54, 55. Our data corroborated with these authors since the cerebellum of milk-supplemented mice was also characterized by increased numbers of Iba-1 + microglial cells and NOS2 + Purkinje cells. These data incite a hypothesis that early damages outside of the CNS can be a trigger to initiate behavioral changes.
Pre and postsynaptic proteins were also investigated in mice supplemented with cow’s milk. Drebrin+ cells were significantly reduced in the cerebral cortex of mice receiving milk. Drebrin is an actin-binding protein present in postsynaptic glutamatergic neurons whose concentration in dendritic spines organizes cytoskeleton and morphology of the cells. Dysfunctions were associated with Alzheimer's disease, schizophrenia and ASD related to post synaptic accumulation of glutamatergic receptors 56, 57. The reduction of shank-3+ and drebrin+ cells in the CNS can be directly associated with behavioral changes observed after milk consumption. The distribution of galectin-3+ cells in the gut-liver axis and cortical shank-3+ and drebrin+ cells could be monitored in other ASD experimental models. Perhaps, some genetic predisposition is required to establish this phenotype and more detailed studies are necessary.
In our scenario, we suggest that galectin-3 may be related to the onset of ASD symptoms as co-factor together environmental and genetic causes still for the most part unknown. Coherently with our observation in this study, we can hypothesize that the role of galectin-3 could be crucial at different levels. At early stages, during neurodevelopment and subsequently regulating the synaptic plasticity network pruning and maintenance not directly but through several mediators as BNDF, GABA-AR, NGF, Neurtrophin-3, Synaphtophysin A and Drebrin 58, 59. At peripheral level, this study confirms the key role of galectin-3 as cross-link factor between innate immunity, visceral sensitivity and gut tissues development and homeostasis. The delicate balance between these functions and the influence of dietary habits should be accurately investigated in order to define every molecular pathway involved in the onset of behavioral, cognitive and neurological disturbances associated with autism.
In conclusion, Lgals3−/− mice and Lgals3+/+ mice supplemented with cow’s milk showed atypical behaviors compatible with autistic-like symptoms. Considering that BALB/c Lgals3+/+ mice have been used as experimental model of ASD, for the first time, it is suggested that galectin-3 interferes with gastrointestinal comorbidities and autistic behavior. Data revealed that absence or reduction of galectin-3 in the gut-liver axis increased stereotyped movements, affected sociability indexes and amplified restrict interest for objects. However, it is important to reinforce that cow’s milk did not cause ASD. Although galectin-3+ cells were not detected in CNS of both mice groups, it has been suggested that galectin-3 in the gut-liver axis interferes with brain functions associated with ASD symptoms in genetically predisposed mice. These findings pointed to mechanisms outside of the CNS can be studied as therapeutic target to alleviate symptoms of gastrointestinal comorbidities associated with autism.