Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Acknowledgments We thank Professor Lai Wei (Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China) and Feng Jiang (University of Rochester School of Medicine and Dentistry, New York, USA) for their help with the bioinformatics analysis. Supplementary Material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2022.846106/full#supplementary-material Click here for additional data file.(115K, pdf). dominantly expressed in orbital fibroblasts (OFs). RNA sequencing of paired unstimulated and transforming growth factor (TGF)-1-stimulated samples demonstrated that upregulation of IL-11 expression defined the dominant transcriptional response. IL-11 signaling was also confirmed to be downstream of TGF-1 and IL-1. Therefore, we deduced that IL-11 protein is secreted in an autocrine loop in TAO. We also indicated that IL-11 mediated the profibrotic phenotype switch by inducing the expression of myofibroblast differentiation markers, including -smooth muscle actin and collagen type I 1, which Vegfb could be abrogated by an anti-IL-11 neutralizing antibody. Furthermore, we revealed that extracellular regulated protein kinase may be a crucial factor in the pro-fibrotic, translationally specific signaling activity of IL-11. P005091 These data demonstrate that IL-11 plays a crucial role in orbital fibroblast phenotype switching and may be a potential therapeutic target candidate for the treatment of TAO. studies. Materials and Methods Study Participants and Sample Collection Peripheral blood samples were obtained from TAO patients (n = 40) and healthy volunteers (n = 18), who were recruited from the Zhongshan Ophthalmic Center, Sun-Yat Sen University. Clinical and demographic descriptions of the participating subjects are provided in Table?1 . Table?1 Demographic Data of Patients with TAO and Controls. (v1.3.3b) (29). Differential expression testing was performed using the DESeq2 R P005091 package (1.20.0). Genes with a P-value 0.05 as identified by DESeq2 were considered differentially expressed. Single Cell RNA Sequencing Single-cell suspensions P005091 from orbital connective tissues were derived from 1 TAO patient and 1 healthy control as previously described (30). Single cells were isolated and lysed with collagenase buffer composed of 5 mg/mL collagenase IV and 10 U/mL DNase I at 37C for 50 minutes (all from Sigma-Aldrich, St. Louis, USA). Subsequently, filtration and red blood cell removal P005091 were performed using the MACS Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). RNA was then reverse-transcribed into cDNA libraries using a 10x Genomics Chromium machine following the manufacturers instructions. DNA sequencing was performed on a NovaSeq 6000 Sequencing System (Illumina, CA, USA) using a paired-end 150 sequencing mode. The Illumina output was processed using Cell Ranger 3.0.2 (10Genomics, CA, USA). Cells of sufficient complexity were clustered and t-distributed stochastic neighbor embedding (t-SNE) plots were generated for visualization using the Seurat R package (31). RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction Following the manufacturers instructions, the total RNA was extracted from cultured cells using the RNA Purification Kit, and reverse transcription was performed using an Evo M-MLV RT kit (Accurate Biology, Hunan, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using SYBR Premix Pro Taq HS (Accurate Biology, Hunan, China) on a Light Cycler 480 Real-Time System (Roche, Basel, Switzerland). GAPDH was selected as the internal control and relative fold change was calculated using the 2 2?CT method. The primers used in this experiment are listed in Table?3 . Table?3 Primer Sequences of qRT-PCR. gene expression in single cells (P adjust = 4.1310-71). (D) The concentration of IL-11 in the OFs supernatant after a 48-h incubation without stimulus were detected by enzyme-linked immunosorbent assay (TAO, n = 8; control, n = 8). The data are expressed as the mean standard deviation (SD). *P 0.05. assessed by two-tailed students t-test. To investigate IL-11 expression, ELISA was performed on the culture supernatants of OFs from patients with TAO or from healthy controls. We found that IL-11 expression levels of TAO cells were greater than P005091 those control fibroblasts (P = 0.022) ( Figure?3D ). Taken together, these data provided evidence that OFs are both a source and a target of IL-11, indicating that IL-11 protein is perhaps secreted in an autocrine loop. IL-11 Was Secreted by OFs in Response to TGF-1 or IL-1 Transforming growth factor-beta1 (TGF-1) represents the most prominent profibrotic cytokine and plays a pivotal role in TAO (32). To elucidate the involvement of IL-11 signaling in the profibrotic TGF-1 signaling, RNA sequencing (RNA-seq) was performed on paired unstimulated and TGF-1-stimulated samples. Genes were ranked based on the significance of the differences in expression levels. Among the most-upregulated genes, we detected several typical genes involved.