The Role and Mechanism of Anti-ICOS mAb in Experimental Autoimmune Encephalomyelitis

Abstract

Targeting costimulatory signalling pathways, especially inducible T-cell costimulatory (ICOS)-ICOS ligand(ICOSL) co-stimulatory signal, has been widely used as a therapeutic target in autoimmune diseases. Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating and neurodegenerative disease in the central nervous system. At present, few studies are addressing the role of the ICOS-ICOSL co-stimulatory pathway in MS. We aimed to explore the role of anti-ICOS mAb in the immune response of EAE and further reveal the regulatory mechanism. C57BL/6 female mice (6-8 weeks old, 18-20 g) were randomly divided into EAE group, T1 group, T9 group and T30 group. The EAE model was established in the three groups by MOG active immunisation. Symptom scores and weights of mice were recorded every day after modelling. Mice in the T1 and the T9 groups were given intraperitoneal injections of anti-ICOS mAb on Day 1 or 9 after active immunisation, respectively. Anti-ICOS mAb were injected three times total, each injection separated by 72 h. On the 19th day after modelling, the mice were sacrificed and the spinal cord tissues of each group were collected. Mice in the T30 group were given an intraperitoneal injection of anti-ICOS mAb on Day 9 after active immunisation. These anti-ICOS mAb were injected continuously until Day 30. On Day 30 after the establishment of the model, the mice were sacrificed and spinal cord tissues were collected. HE staining was used to observe the inflammatory infiltration of the spinal cord in each group of mice. Transcriptome sequencing was used to detect gene transcription in spinal cord tissues of mice and to explore the signalling pathways that might be involved. Finally, the signalling pathway was verified by Western blot. (1) Compared with the EAE group, there are significantly lower behavioural scores, heavier weight, delayed onset time and fewer inflammatory infiltrations of the spinal cord in the T9 group and T30 group. However, there were no significant differences in behavioural scores, weight, onset time and inflammatory infiltration between the EAE group and the T1 group. (2) The R² value of the spinal cord sample in each group was greater than 0.8. There were 6569 differentially expressed genes (DEGs) between T9 group and EAE group, including 2579 up-regulated genes and 3990 down-regulated genes. There were 487 DEGs between T1 and EAE groups, including 131 up-regulated genes and 356 down-regulated genes. There were 7116 DEGs between T9 and T1 groups, including 2921 up-regulated genes and 4195 down-regulated genes. KEGG enrichment showed that DEGs between T9 group and EAE group were mainly enriched in Epstein-Barr virus infection, NF-κB signalling pathway, FcγR-mediated phagocytosis, natural killer cell-mediated cytotoxicity, as well as Th17, Th1 and Th2 cell differentiation pathways. In the most significantly enriched EB pathway, we found that the fold change of key genes SYK, PI3K and AKT was -4.3457, -2.0985 and -0.69373, respectively (p < 0.001). However, in KEGG enrichment analysis between T1 group and EAE group, DEGs were mainly enriched in retrograde endocannabinoid signalling, Parkinson's disease, morphine addiction, Alzheimer's disease, endocrine disorders, Huntington's disease, cholinergic synapses, oxidative phosphorylation and other pathways. In addition, in KEGG enrichment analysis between T9 group and T1 group, DEGs were mainly in Epstein-Barr virus infection, Th17, Th1 and Th2 cell differentiation, NF-κB signalling pathway, B/T cell receptor signalling pathway, TNF signalling pathway, FcγR-mediated phagocytosis and other pathways. (3) Compared with the control group, the relative expression of SYK, AKT and PI3K protein in the spinal cord of the EAE group was significantly increased. The relative expression of SYK, AKT and PI3K protein in the T9 group and T30 group was significantly decreased compared with the EAE group, while there was no significant difference between the EAE group and the T1 group. Anti-ICOS mAb treatment showed a therapeutic effect in EAE mice during the immune response period (Days 9 and 30 after active immunisation), but not in the immune activation period (Day 1 after active immunisation). The therapeutic effect of anti-ICOS mAb through blocking ICOS-ICOSL signalling in the immune response of EAE mice may be mediated by inhibiting the SYK/PI3K/AKT pathway.

Keywords: AKT; PI3K; SYK; anti‐ICOS mAb; experimental autoimmune encephalomyelitis; immune response.