Nebulized bone marrow-derived stem cell supernatant induces tolerogenic dendritic cells via upregulation of FOXO3 for EAE treatment

Abstract

Background: Bone marrow mesenchymal stem cells (BMSCs) exert potent paracrine effects that can reshape the immune microenvironment. Nebulized inhalation enables non-invasive, targeted delivery to the lung, a pivotal immune interface capable of modulating systemic immunity. This study introduces a novel therapeutic strategy using nebulized BMSC supernatant to activate the FOXO3 signaling pathway in pulmonary dendritic cells (DCs), reprogramming them toward a tolerogenic phenotype. This approach suppresses autoreactive T cell infiltration and alleviates central nervous system (CNS) inflammation in the experimental autoimmune encephalomyelitis (EAE) model, offering a potential acellular therapy for multiple sclerosis (MS) and other autoimmune diseases. Methods: EAE mice were treated with nebulized BMSC supernatant or adoptive transfer of pretreated DCs. Disease progression was assessed by body weight and clinical scores. Hematoxylin and eosin (HE) staining and myelin immunofluorescence staining were used to evaluate CNS inflammation and demyelination. Flow cytometry measured T cell differentiation in spleen and lymph nodes, as well as DC antigen presentation and cytokine secretion in lung tissue. Quantitative Polymerase Chain Reaction (QPCR) assessed inflammatory cytokines, and immunofluorescence determined FOXO3 expression in DCs. FOXO3 involvement was validated using inhibitor-pretreated DCs in adoptive transfer experiments. Results: Nebulized BMSC supernatant reduced clinical severity and weight loss in EAE mice, and decreased CNS inflammatory infiltration and demyelination. Treatment suppressed peripheral Th1 and Th17 differentiation while increasing Treg frequency. Lung DCs exhibited reduced antigen presentation and pro-inflammatory cytokine expression, increased IL-10 secretion, and elevated FOXO3 expression. Blocking FOXO3 in DCs reversed these effects, aggravating EAE symptoms and promoting Th1/Th17 differentiation. Conclusions: Nebulized BMSC supernatant effectively treats EAE by inducing pulmonary tolerogenic dendritic cells (tolDC) via FOXO3 activation, reshaping peripheral T cell responses, and reducing CNS inflammation. This strategy highlights the therapeutic potential of targeting the pulmonary immune interface as a novel acellular approach for MS and related autoimmune disorders.