An EAU model was established using IRBP
1 − 20, PTX, and CFA as previously reported [
6]. After immunization, the EAU mice were daily received oral administration of DMF for 14 days. DMF administration efficiently ameliorated EAU symptoms and reduced the clinical and pathological scores (Fig.
1A and B). Additionally, to test the therapeutic effect of DMF on established EAU, we administered established EAU mice (day 10 after immunization) with DMF for 5 days. We found that the therapeutic effect still existed (Additional file 1: Fig.
S1B). In the following experiments, we started DMF administration on the first day of modeling in the DMF group.
To identify the genomic variations associated with DMF administration, scRNA-seq was performed on isolated CDLN cells derived from normal mice, EAU mice, and EAU mice administered with DMF (Fig.
1C). After initial processing, including quality filtering, integration, and batch effect correction, the cells were divided into 8 distinct clusters. Uniform manifold approximation and projection (UMAP) was used for visualization (Fig.
1D). The cell clusters were annotated via their expression of marker genes (Fig.
1D and E, Additional file 1: Fig.
S1C). 8 classical immune cell types, namely, T cells, B cells, natural killer cells (NKs), conventional dendritic cells (cDCs), monocytes (Monos), plasmacytoid dendritic cells (pDCs), macrophages (Macros), and neutrophils (Neus), were identified. We then explored the proportional alterations of immune cells post-DMF administration. The proportional changes during EAU were partly reversed by DMF treatment in several immune cell types, including T cells, B cells, monocytes, pDCs, and macrophages (Fig.
1F). To explore the overall effect of DMF on the transcriptome of EAU mice, we performed DEG analysis of all immune cells between the DMF and EAU groups as well as between the EAU and normal groups (Fig.
1G and H). DMF reduced the expression of genes related to AP-1 (
Fos, Fosb, and Jun), antigen presentation (
B2m), interferon signaling (
Ifi27l2a and Isg15), lymphocyte differentiation (
Pim1), and cell migration (
Cxcr4) (Fig.
1G). These genes were included in the DEGs which were upregulated during EAU compared to the normal condition (Fig.
1H). Meanwhile, DMF treatment upregulated two genes involved in RNA metabolism,
Rps28 and
Rps29, which were downregulated during EAU (Fig.
1G and H). These results indicated that DMF partially reversed the EAU-induced transcriptional changes. To investigate the specific beneficial mechanisms of DMF on EAU, we further identified “rescue DEGs” to better elucidate the EAU-induced transcriptional alterations that were reversed following DMF treatment (Fig.
1I). The genes upregulated in EAU and downregulated in the DMF group were identified as downregulated rescue DEGs. Similarly, the genes downregulated in EAU and upregulated in the DMF group were identified as upregulated rescue DEGs (Fig.
1I). Subsequently, a GO analysis was performed to characterize the biological significance of these rescue genes. Pathways related to IL-17 signaling, antigen processing and presentation, IL-6 signaling, and Th17 cell differentiation were enriched by the downregulated rescue DEGs (Fig.
1J). Meanwhile, pathways related to RNA metabolism, G protein-coupled receptor signaling, and endocytosis were enriched by the upregulated rescue DEGs (Fig.
1J). These results indicated that DMF partly reversed EAU-induced transcriptional alterations. We also conducted DEG analysis between the DMF group and the normal group (Additional file 1: Fig.
S1D). We observed that the expression of
Fos,
Ifi27l2a,
Isg15, and
Cxcr4 was lower in DMF group compared to normal group (Additional file 1: Fig.
S1D). In addition, the expression of genes related to the function of macrophages and neutrophils (
Ccl5,
Ctsb, and
Grn) was higher in the DMF group compared to the normal group. GO analysis was conducted to annotate these DEGs. The downregulated DEGs in the DMF group compared to the normal group were enriched in pathways related to electron transport chain and ATP process, while the upregulated DEGs were enriched in pathways related to neutrophil degranulation and innate immune response (Additional file 1: Fig.
S1E).
Overall, DMF treatment efficiently ameliorated EAU symptoms and partly reversed EAU-induced transcriptional alterations.