Biodegradable Electrospun PLGA Nanofibers-Encapsulated Trichinella Spiralis Antigens Protect from Relapsing Experimental Autoimmune Encephalomyelitis and Related Gut Microbiota Dysbiosis

Ljiljana Sabljić,1 NataÅ¡a Radulović,2 Jelena Đokić,3 Dusica B Stojanovic,4 DuÅ¡an Radojević,3 Sofija Glamočlija,1 Miroslav Dinić,3 NataÅ¡a Golić,3 SaÅ¡a Vasilev,1 Petar Uskoković,4 Ljiljana Sofronić-Milosavljević,1 Alisa Gruden-Movsesijan,1,* Sergej Tomić1,* 1Institute for the Application of Nuclear Energy, University of Belgrade, Belgrade, Serbia; 2Institute for Biological Research “SiniÅ¡a Stanković”, University of Belgrade, Belgrade, Serbia; 3Institute for Molecular Genetics and Genetical Engineering, University of Belgrade, Belgrade, Serbia; 4Faculty of Metallurgy and Technology, University of Belgrade, Belgrade, Serbia*These authors contributed equally to this workCorrespondence: Sergej Tomić, Principal Research Fellow, Institute for the Application of Nuclear Energy, University of Belgrade, Banatska 31b, Zemun, Belgrade, 11080, Serbia, Tel +381 11 2610 126, Fax +381 11 2618 724, Email [email protected]: Trichinella spiralis has evolved complex immunomodulatory mechanisms mediated by excretory-secretory products (ESL1) that enable its survival in the host. Consequently, ESL1 antigens display excellent potential for treating autoimmune diseases such as multiple sclerosis (MS). However, whether timely controlled delivery of ESL1 antigens in vivo, as in natural infections, could enhance its therapeutic potential for MS is still unknown.Methods: To test this, we encapsulated ESL1 antigens into biodegradable poly (lactide-co-glycolic) acid (PLGA) nanofibers by emulsion electrospinning as a delivery system and assessed their release dynamics in vitro, and in an animal MS model, experimental autoimmune encephalomyelitis (EAE), induced 7 days after PLGA/ESL1 subcutaneous implantation. PLGA/ESL1 effects on EAE symptoms were monitored along with multiple immune cell subsets in target organs at the peak and recovery of EAE. Gut barrier function and microbiota composition were analyzed using qPCR, 16S rRNA sequencing, and metabolomic analyses.Results: ESL1 antigens, released from PLGA and drained via myeloid antigen-presenting cells through lymph nodes, protected the animals from developing EAE symptoms. These effects correlated with reduced activation of myeloid cells, increased IL-10 expression, and reduced accumulation of proinflammatory natural killer (NK) cells, T helper (Th)1 and Th17 cells in the spleen and central nervous system (CNS). Additionally, CD4+CD25hiFoxP3+ regulatory T cells and IL-10-producing B cells were expanded in PLGA/ESL1-treated animals, compared to control animals. The migration of ESL1 to the guts correlated with locally reduced inflammation and gut barrier damage. Additionally, PLGA/ESL1-treated animals displayed an unaltered microbiota characterized only by a more pronounced protective mevalonate pathway and expanded short-chain fatty acid-producing bacteria, which are known to suppress inflammation.Conclusion: The delivery of T. spiralis ESL1 antigens via biodegradable electrospun PLGA nanofiber implants efficiently protected the animals from developing EAE by inducing a beneficial immune response in the spleen, gut, and CNS. This platform provides excellent grounds for further development of novel MS therapies. Keywords: electrospinning, PLGA nanofibers, drug delivery, tolerogenic cells, immune modulation, gut microbiota