Hazlett, L. D. | bioRxiv Science

bioRxiv2026

The miR-183/96/182 Cluster Regulates Trigeminal Ganglion Sensory Neurons Response to Pseudomonas aeruginosa Infection

PurposeTo uncover the molecular mechanisms of corneal sensory nerves (CSN)s involvement in the initiation of Pseudomonas aeruginosa (PA) keratitis and the roles of the miR-183/96/182 cluster (miR-183C) in this process. MethodsmiR-183C conventional knockout (KO) or sensory neuron-specific (SNS) conditional (C)KO mice and their age- and sex-matched wild type (WT) controls were used. TG SN were isolated. Neurite growth and branching were analyzed by neurite tracing. Custom-made microfluidic chambers (MFC) were used to separate the neuronal cell bodies in the soma chamber and their neurites/nerve endings in the axon chamber. TG SNs response to lipopolysaccharide (LPS) or PA infection of the neurites/nerve endings was studied by ELISA assays of CX3CL1 and substance P (sP) in the axon chamber. Target luciferase reporter assays were performed to validate key downstream target genes of miR-183C. ResultsThe total neurite length and number of branches per TG SN were decreased in the CKO vs WT mice, and in the male vs female WT mice. PA infection, but not LPS alone, induced the production and secretion of CX3CL1 and sP in WT mice; while TG SN of miR-183C KO mice responded to both LPS and PA and were significantly enhanced when compared to WT mice. Antagonists to TLR4 and/or FPR1 inhibited PA-induced responses. Target luciferase reporter assays confirmed that genes encoding NRP1, TAC1-the precursor gene of sP, CX3CL1 and ADAM10, a metalloproteinase involved in the production of soluble CX3CL1, were direct targets of miR-183C. ConclusionsPA directly activates TG SN and induces chemokine and neuropeptide production/secretion through TLR4 and FPR1 receptors, which may contribute to the initiation of PA keratitis. miR-183C regulates TG SN neurite growth, chemokine and neuropeptide production/secretion and the response to PA infection by targeting a collection of key genes involved in axon guidance/projection-, chemokine and neuropeptide biogenesis- and receptors mediating PA-induced activation.

immunology↗

bioRxiv2025

New Insight Into the Neuroimmune Interplay In Pseudomonas aeruginosa Keratitis

PurposeThe 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 Pseudomonas aeruginosa (PA) keratitis. MethodsDouble-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. ResultsIn 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. ConclusionsThis 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.

immunology↗