Gupta Naman, LoGrasso Giovanni, Hazlett Linda D, Xu Shunbin
Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine/Kresge Eye Institute, Detroit, Michigan.
bioRxiv. 2025 Mar 11:2025.03.06.641908. doi: 10.1101/2025.03.06.641908.
The miR-183/96/182 cluster (miR-183C) is required for normal functions of sensory neurons (SN) and various immune cells, including myeloid cells (MC). This research aims to reveal the roles of miR-183C of SN in the interplay of corneal sensory nerves (CSN) and MC during (PA) keratitis.
Double-tracing mice with SN-specific (SNS) conditional knockout of miR-183C (CKO) and age- and sex-matched wild type (WT) controls were used. Their CSN are labeled with Red Fluorescent Protein (RFP); MC with Enhanced Green (EG)FP. The left corneas were scarified and infected with ATCC19660 PA. Corneal flatmounts were prepared at 3, 6, and 12 hours post-infection (hpi) and 1, 3, and 5 days (d)pi for confocal microscopy. Myeloperoxidase (MPO) assay and plate count were performed at 1 dpi.
In WT mice, CSN began to degenerate as early as 3 hpi, starting from the fine terminal CSN in the epithelial/subepithelial layers, most prominently in the central region. By 1 dpi, CSN in the epithelium/subepithelial layer were nearly completely destroyed, while stromal nerves persisted. From 3 dpi, CSN were obliterated in both layers. In CKO vs WT mice, CNS followed a slightly slower pace of degeneration. CSN density was decreased at 3 and 6 hpi. However, at 3 dpi, residual large-diameter stromal CSN were better preserved.MC were decreased in the central cornea at 3 and 6 hpi, but increased in the periphery. Both changes were more prominent in CKO vs WT mice. At 12 hpi, densely packed MC formed a ring-shaped band circling a "dark" zone nearly devoid of MC, colocalizing with CSN most degenerated zone in the central cornea. In CKO vs WT, the ring center was larger with fewer MC. At 1 dpi, the entire cornea was filled with MC; however, MC density was lower in CKO mice. An MPO assay showed decreased neutrophils in PA-infected cornea of CKO mice. This led to a decreased severity of PA keratitis at 3 dpi.
This double-tracing model reveals the interplay between CSN and MC during PA keratitis with greater clarity, providing new insights into PA keratitis. CSN-imposed modulation on innate immunity is most impressive within 24 hours after infection. Functionally, the miR-183C in CSN modulates CSN density and the dynamics of MC fluxes- a neuroimmune interaction in display.
miR-183/96/182簇（miR-183C）是感觉神经元（SN）和包括髓样细胞（MC）在内的各种免疫细胞正常功能所必需的。本研究旨在揭示SN中的miR-183C在铜绿假单胞菌（PA）角膜炎期间角膜感觉神经（CSN）与MC相互作用中的作用。
使用miR-183C特异性（SNS）条件性敲除（CKO）的双标记小鼠以及年龄和性别匹配的野生型（WT）对照。它们的CSN用红色荧光蛋白（RFP）标记；MC用增强型绿色（EG）FP标记。刮伤左眼角膜并感染ATCC19660 PA。在感染后3、6和12小时（hpi）以及感染后1、3和5天（d）pi制备角膜平铺片用于共聚焦显微镜检查。在感染后1天（dpi）进行髓过氧化物酶（MPO）测定和平板计数。
在WT小鼠中，CSN早在感染后3小时就开始退化，从上皮/上皮下层的细小终末CSN开始，最明显的是在中央区域。到感染后1天，上皮/上皮下层的CSN几乎完全被破坏，而基质神经仍然存在。从感染后3天起，两层中的CSN都消失了。在CKO小鼠与WT小鼠中，CNS的退化速度稍慢。在感染后3和6小时，CSN密度降低。然而，在感染后3天，残留的大直径基质CSN保存得更好。在感染后3和6小时，中央角膜中的MC减少，但周边增加。在CKO小鼠与WT小鼠中，这两种变化都更明显。在感染后12小时，密集排列的MC形成一个环形带，围绕着一个几乎没有MC的“暗”区，与中央角膜中CSN退化最严重的区域共定位。在CKO小鼠与WT小鼠中，环形中心更大，MC更少。在感染后1天，整个角膜充满了MC；然而，CKO小鼠中的MC密度较低。MPO测定显示CKO小鼠PA感染角膜中的中性粒细胞减少。这导致感染后3天PA角膜炎的严重程度降低。
这种双标记模型更清晰地揭示了PA角膜炎期间CSN与MC之间的相互作用，为PA角膜炎提供了新的见解。CSN对先天免疫的调节在感染后24小时内最为显著。在功能上，CSN中的miR-183C调节CSN密度和MC通量的动态变化——一种神经免疫相互作用。
