Abstract
Purpose :
The conserved miR-183/96/182 cluster (miR-183C) are specifically expressed and play important roles in primary sensory neurons and innate immune cells. Cell type-specific inactivation of miR-183C in trigeminal ganglion (TG) sensory neurons results in a decrease of corneal sensory nerve density, however, an increased number of CRMCs, suggesting an intrinsic regulation of miR-183C on corneal nerve density and an extrinsic regulation on CRMCs through sensory innervation. The purpose of the current study is to unravel the molecular mechanism underlying these intrinsic and extrinsic regulations.
Methods :
3’RNA sequencing (Seq) was done in the TG and cornea of miR-183C conventional knockout (KO) and sensory neuron-specific conditional knockout (SNS-CKO) mice and their age- and sex-matched wild-type (WT) controls. Target prediction was conducted using the TargetScan algorithm. Quantitative (q)RT-PCR of Cx3cl1 was performed in TG and corneal RNA; ELISA assays in protein lysate; and immunofluorescence in whole mount and/or cross-sections of TG and cornea. Cx3cr1 knockout allele is bred into the miR-183C KO and SNS-CKO mice to test the effect of different dosages of Cx3cl1-Cx3cr1 signaling on corneal sensory nerve density and number of CRMCs.
Results :
1) 3’RNA seq data revealed that Cx3cl1 was upregulated in the TG and cornea of miR-183C KO and SNS-CKO vs their WT control mice.
2) Target prediction showed that Cx3cl1 was a conserved predicted target gene of miR-183.
3) ELISA assay confirmed that Cx3cl1 was significantly increased in the TG and cornea of miR-183C KO mice, suggesting that inactivation of miR-183C disinhibits Cx3cl1 in the TG sensory neurons and resulted in increased expression of Cx3cl1 in the cornea of SNS-CKO mice.
Conclusions :
Cx3cl1 is a target of miR-183C in TG sensory neurons. Neuron-produced chemokine Cx3cl1 is known to promote chemotactic migration of microglia in the central nervous system, mediate the homing of MHC-II-positive dendritic cells in the cornea and recruitment of macrophages in other tissues, enhance neuron adhesion to extracellular matrix and reduce neuronal migration. Our data suggest that miR-183C regulates corneal sensory nerve density and CRMCs through its regulation of Cx3cl1. The Cx3cl1-Cx3cr1 axis is central to the neuroimmune interaction in the cornea.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.