Single cell analysis of short-term dry eye induced changes in cornea immune cell populations.

Background: Dry eye causes corneal inflammation, epitheliopathy and sensorineural changes. This study evaluates the hypothesis that dry eye alters the percentages and transcriptional profiles of immune cell populations in the cornea. Methods: Desiccating stress (DS) induced dry eye was created by pharmacologic suppression of tear secretion and exposure to drafty low humidity environment. Expression profiling of corneal immune cells was performed by single-cell RNA sequencing (scRNA-seq). Cell differentiation trajectories and cell fate were modeled through RNA velocity analysis. Confocal microscopy was used to immunodetect corneal immune cells. Irritation response to topical neurostimulants was assessed. Results: Twelve corneal immune cell populations based on their transcriptional profiles were identified at baseline and consist of monocytes, resident (rMP) and MMP12/13 high macrophages, dendritic cells (cDC2), neutrophils, mast cells, pre T/B cells, and innate (γDT, ILC2, NK) and conventional T and B lymphocytes. T cells and resident macrophages (rMP) were the largest populations in the normal cornea comprising 18.6 and 18.2 percent, respectively. rMP increased to 55.2% of cells after 5 days of DS. Significant changes in expression of 1,365 genes (adj p < 0.0001) were noted in rMP with increases in cytokines and chemokines (Tnf, Cxcl1, Ccl12, Il1rn), inflammatory markers (Vcam, Adam17, Junb), the TAM receptor (Mertk), and decreases in complement and MHCII genes. A differentiation trajectory from monocytes to terminal state rMP was found. Phagocytosis, C-type lectin receptor signaling, NF-kappa B signaling and Toll-like receptor signaling were among the pathways with enhanced activity in these cells. The percentage of MRC1+ rMPs increased in the cornea and they were observed in the basal epithelium adjacent to epithelial nerve plexus. Concentration of the chemokine CXCL1 increased in the cornea and it heightened irritation/pain responses to topically applied hypertonic saline. Conclusion: These findings indicate that DS recruits monocytes that differentiate to macrophages with increased expression of inflammation associated genes. The proximity of these macrophages to cornea nerves and their expression of neurosensitizers suggests they contribute to the corneal sensorineural changes in dry eye.

Characterization of the 3'UTR of the BTD gene and identification of regulatory elements and microRNAs.

Reduced biotinidase activity is associated with a spectrum of deficiency ranging from total deficiency to heterozygous levels, a finding that is not always explained by the pathogenic variants observed in the BTD gene. The investigation of miRNAs, regulatory elements and variants in the 3'UTR region may present relevance in understanding the genotype-phenotype association. The aims of the study were to characterize the regulatory elements of the 3'UTR of the BTD gene and identify variants and miRNAs which may explain the discrepancies observed between genotype and biochemical phenotype. We evaluated 92 individuals with reduced biotinidase activity (level of heterozygotes = 33, borderline = 35, partial DB = 20 or total DB= 4) with previously determined BTD genotype. The 3'UTR of the BTD gene was Sanger sequenced. In silico analysis was performed to identify miRNAs and regulatory elements. No variants were found in the 3'UTR. We found 97 possible miRNAs associated with the BTD gene, 49 predicted miRNAs involved in the alanine, biotin, citrate and pyruvate metabolic pathways and 5 genes involved in biotin metabolism. Six AU-rich elements were found. Our data suggest variants in the 3'UTR of BTD do not explain the genotype-phenotype discrepancies found in Brazilian individuals with reduced biotinidase.

Hearing Impairment in Mucopolysaccharidosis: A Systems Biology Approach

Abstract Mucopolysaccharidoses (MPS) are lysosomal diseases caused by deficiencies in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Sensorineural hearing impairment is a common feature in MPS patients, but there is no consensus on its etiology. For this reason, we aimed to identify genes and pathways related to hearing loss and to correlate them with gene expression data in MPS. We used HPO and Disgenet to identify candidate genes. We constructed the network with string and Cytoscape, and hub genes were identified in Cytohubba. Expression data were obtained from the MPSBase website. We found the NDUFA gene family as the major hub genes and 114 enriched pathways related to hearing loss. These genes and biological pathways may serve as potential candidates for clinical studies to better understand hearing impairment mechanisms in lysosomal storage diseases like mucopolysaccharidosis.