STAT3 Knockout Cell Lysate

Aims To identify transcriptome signatures underlying epileptogenesis in temporal lobe epilepsy (TLE). Methods Robust rank aggregation analysis was used to integrate multiple microarrays in rodent models of TLE and determine differentially expressed genes (DEGs) in acute, latent, and chronic stages. Functional annotation and protein-protein interaction analysis were performed to explore the potential functions of the DEGs and identify hub genes with the highest intramodular connectivity. The association between hub genes and hippocampal sclerosis/seizure frequency was analyzed using publicly available RNA-sequencing datasets from TLE patients. We subsequently established a pilocarpine-induced status epilepticus (SE) model in rats and validated mRNA expression of hub genes by quantitative reverse transcription PCR (qRT-PCR). Results The DEGs in the acute, latent, and chronic phases of TLE in animal models were prominently enriched in inflammatory response. Hub genes identified in the acute phase mainly participated in biological processes including inflammation, blood-brain barrier damage, and cell adhesion. The hub genes in the latent phase were related to microglia/macrophage activation (Emr1 and Aif1) and phagocytosis (Cd68, Tyrobp, and Lyz). In the chronic phase, the hub genes were associated with activation of complements and microglia/macrophages. We further found that some hub genes identified in human TLE, such as Tlr2, Lgals3, and Stat3, were positively correlated with seizure frequency. Other hub genes, including Lgals3 and Serpine1, were associated with hippocampus sclerosis. qRT-PCR analysis confirmed that the mRNA levels of hub genes in rat hippocampus were significantly up-regulated after SE induction. Conclusions Our integrated analysis identified hub genes in different stages of epilepsy. The functional annotations suggest that the activation and phagocytic activities of microglia/macrophages may play critical roles in epileptogenesis of TLE.

Interleukin-10 (IL-10) is a dimeric cytokine with both immunosuppressive and immunostimulatory activities; however, IL-10-based therapies have shown only marginal clinical benefits. Here, we explored whether the stability of the IL-10 receptor complex contributes to the immunomodulatory potency of IL-10. We generated an IL-10 mutant with enhanced affinity for its IL-10R. receptor using yeast surface display. Compared to the wild-type cytokine, the affinity-enhanced IL-10 variants recruited IL-10R. more efficiently into active cell surface signaling complexes and triggered greater STAT1 and STAT3 activation in human monocytes and CD8(+) T cells. These effects, in turn, led to more robust induction of IL-10-mediated gene expression programs at low ligand concentrations in both human cell subsets. IL-10-regulated genes are involved in monocyte energy homeostasis, migration, and trafficking and in CD8(+) T cell exhaustion. At nonsaturating doses, IL-10 did not induce key components of its gene expression program, which may explain its lack of efficacy in clinical settings. Our engineered IL-10 variant showed a more robust bioactivity profile than that of wild-type IL-10 at low doses in monocytes and CD8(+) T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with wild-type IL-10. Our study provides insights into how IL-10 receptor complex stability fine-tunes IL-10 biology and opens new opportunities to revitalize failed IL-10 therapies.