Interplay between diet, gut microbiota, and autoimmune mechanisms: a focus on diet and fungal alterations in Multiple Sclerosis

Abstract

The human holobiont consists of the human host and the microbiota—a diverse community of microorganisms that interact with the host either transiently or over the long term. In particular, the human gut harbors the body's most diverse microbial community, the gut microbiota. The gut microbiota comprises organisms from several kingdoms, including bacteria, fungi, and archaea, most of which are stable colonizers. Under homeostatic conditions, the gut microbiota supports host physiology by aiding the digestion of complex dietary components and, in return, producing microbial metabolites, such as short-chain fatty acids, with immunomodulatory functions for the host. The gut microbiota also contributes to immune regulation by shaping adaptive responses and limiting pathogen colonization through competitive interactions within the niches of the gastrointestinal tract. While intrinsic host factors, such as host genetics, may influence microbiota composition, environmental factors, especially diet, play a major role in shaping the gut ecological dynamics. Dietary shifts that vary from a probiotic fiber-rich pattern can alter microbiota composition and function, promoting a transition from communities dominated by beneficial taxa (e.g., Clostridia) to ecosystems enriched in pro-inflammatory pathobionts. Clinical and epidemiological evidence indicates that Western-style diets characterized by high fats and simple sugar composition are associated with increased risk of gut microbiota-derived inflammatory conditions, including autoimmune disorders such as Multiple Sclerosis (MS). Moreover, other anthropogenic environmental factors, such as urbanization and high sanitation, predispose the gut-immune axis to a pro-inflammatory state. In this context, the degree of urbanization of the gut microbiota has been shown to influence its composition and its resilience. In this thesis, high-salt intake (a key component of the Western diet) was shown to induce more pronounced dysbiotic changes in animals with an urbanized, highly sanitized microbiota (conventional laboratory mice, CLM) compared to genetically identical mice harboring a wild-derived microbiota (wildling mice), with effects observed from a dietary salt content of 4%. In CLM, this perturbation was characterized by the depletion of beneficial taxa such as Bifidobacterium and Lactobacillus, an increase in Akkermansia, and an overall reduction in microbial richness. In contrast, the wild-derived microbiota displayed greater resilience, showing minimal changes in overall community composition under the same conditions. Given its central role in immune modulation, the gut microbiota has been increasingly implicated as both a risk factor and a trigger of pathogenic immune responses against self-antigens, a key mechanism underlying autoimmune diseases. While the bacterial component of the microbiota (the gut bacteriota) is the most abundant and functionally characterized, it represents only part of this complex ecosystem. Most research has focused on bacterial communities within the intestinal niche; however, growing evidence highlights a potential role for the gut mycobiota in the etiology and development of autoimmune diseases, including MS. Emerging evidence links the gut mycobiota to MS through: (i) increased antifungal immune responses, including elevated anti-Candida antibodies in serum and CSF; (ii) higher susceptibility to fungal infections in MS patients, particularly under immunosuppressive therapies; (iii) genetic associations such as HLA-DRB1*15 linked to aberrant antifungal immunity; (iv) fungal antigen–driven Th17 responses (e.g., mannoproteins inducing IL-17); and (v) biomarkers of fungal-related immune activation, such as increased chitotriosidase and MBL pathway activity. Based on this rationale, this thesis investigated the impact of diet on microbiota composition and clinical development in MS. In this thesis, the beneficial effects of a flavonoid-enriched Mediterranean diet were shown to promote an eubiotic compositional shift in primary-progressive MS patients, characterized by a progressive worsening, leading to an increase of health-promoting bacteria such as Agathobacter, Barnesiella, Butyricimonas, Enterocloster, and Roseburia. Particularly, the intensity of the overall microbial shift upon flavonoid-enriched Mediterranean diet was directly correlated with increased clinical disability, indicating that PPMS patients with more severe disease were those who benefited the most. Furthermore, this thesis demonstrated that, under a full Western diet (high-fat, high-sugar, and high-salt), antifungal treatment further worsened the gut dysbiosis of wild-derived gut bacteriota. More specifically, depletion of the gut fungal commensal Kazachstania pintolopesii by antifungal treatment led to an expansion of pathogenic bacteria, such as Enterorhabdus, mucin-degrading bacteria, such as Mucispirillum, and sulfate-reducing bacteria, such as Desulfovibrio. Finally, this thesis investigated the direct role of antifungal immunity in modulating autoreactive responses. An improvement in the clinical condition of wildling mice with Experimental Autoimmune Encephalomyelitis (EAE) was observed upon depletion of the commensal fungus Kazachstania pintolopesii. The role of Kazachstania pintolopesii in worsening EAE in a wild-derived gut ecosystem was further validated in CLM mice upon fungal colonization of the gut, with results comparable to those of a food-borne fungus, Maudiozyma humilis, associated with high levels of cytotoxic mucosal-associated immune cells in relapsing-remitting MS patients. In CLM, the administration of these fungi activated the mucosal-associated invariant T cells (MAIT) immune axis to drive inflammatory autoreactive responses in the spinal cords of EAE-induced mice, through pathways dependent on MR1, a non-classical MHC class I-like molecule that regulates MAIT cell activation by presenting small microbial metabolites. Together, the findings of this thesis highlight the interplay between diet, gut microbiota, and antifungal immunity in shaping immune dysregulation in Multiple Sclerosis. Environmental factors, such as a Western-like diets and gut fungi contribution in gut homeostasis, were shown to alter the resilience and immunomodulatory functions of the gut microbiota, promoting pro-inflammatory states. This work showed with high resolution the gut mycobiota profile as a critical contributor to the gut immune axis, showing that dysbiosis of fungal commensals such as Kazachstania pintolopesii promotes dysbiosis and the expansion of pro-inflammatory bacterial taxa. These alterations might be responsible for the modulation of autoreactive immune responses via the MAIT-immune axis, or more generally of mucosal-derived immunity, involving MR1-dependent presentation of microbial metabolites. Overall, these findings provide suggestions of possible mechanistic insight into how host–microbiota interactions contribute to MS clinical condition and suggest that targeting the bacteriota–mycobiota networks could be a promising strategy to improve immune homeostasis. Future investigations should further dissect the molecular mechanisms underlying these interactions, with particular focus on the metabolic networks within the gut ecosystem that involve the gut mycobiota, and how fungal-derived metabolites integrate with bacterial pathways to modulate immune responses.