Antibiotic-Induced Dysbiosis of Gut Microbiota Impairs Corneal Nerve Regeneration by Affecting CCR2-Negative Macrophage Distribution.

Although antibiotics are useful, they can also bring negative effects. We found that antibiotic-treated mice exhibit an alteration in the gene expression profile of corneal tissues and a decrease in corneal nerve density. During corneal wound healing, antibiotic treatment was found to impair corneal nerve regeneration, an effect that could be largely reversed by reconstitution of the gut microbiota via fecal transplant. Furthermore, CCR2- corneal macrophages were found to participate in the repair of damaged corneal nerves, and a decrease in CCR2- corneal macrophages in antibiotic-treated mice, which could be reversed by fecal transplant, was observed. Adoptive transfer of CCR2- corneal macrophages promoted corneal nerve regeneration in antibiotic-treated mice. The application of probiotics after administration of antibiotics also restored the proportion of CCR2- corneal macrophages and increased the regeneration of corneal nerve fibers after epithelial abrasion. These results suggest that dysbiosis of the gut microbiota induced by antibiotic treatment impairs corneal nerve regeneration by affecting CCR2- macrophage distribution in the cornea. This study also indicates the potential of probiotics as a therapeutic strategy for promoting the regeneration of damaged corneal nerve fibers when the gut microbiota is in dysbiosis.

Local Group 2 Innate Lymphoid Cells Promote Corneal Regeneration after Epithelial Abrasion.

Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2- corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.

Short-term High Fructose Intake Reprograms the Transcriptional Clock Rhythm of the Murine Extraorbital Lacrimal Gland.

Purpose: To determine whether high fructose intake (HFI) influences the daily transcriptional clock rhythms of murine extraorbital lacrimal glands (ELGs). Methods: Timed ELGs were collected from two groups of C57BL/6J mice subjected to a 12-hour light/12-hour dark (LD) cycle for 10 days; the first group received water-only feeding and the second received water with 15% fructose. Total RNA was extracted and subjected to RNA sequencing. A JTK_CYCLE algorithm and computational software were used to determine the periodicity, rhythmicity, and amplitude of the cycling transcripts. The rhythmic transcripts from different conditions were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Results: HFI feeding caused massive remodeling of the preexisting rhythmic genes in the normal control (NC)-fed ELGs. The induced transcripts in HFI-fed mice resulted in a profound reorganization of the coordinated transcriptional oscillations and KEGG pathways. Moreover, HFI feeding significantly altered the distribution of the KEGG pathways over an LD in the ELGs. Finally, we found that the ELGs have a robust core clock machinery and HFI feeding altered amplitude and the peak phase of clock gene transcriptional oscillation in ELGs. Conclusions: Short-term HFI reprograms the daily transcriptomic oscillation of murine ELGs. This information may deepen our understanding of the outcomes of lacrimal glands altered by nutritional challenge.

Acute tobacco smoke exposure exacerbates the inflammatory response to corneal wounds in mice via the sympathetic nervous system.

Exposure to tobacco smoke is a major public health concern that can also affect ophthalmic health. Based on previous work demonstrating the important role of the sympathetic nervous system (SNS) in corneal wound repair, we postulated that acute tobacco smoke exposure (ATSE) may act through the SNS in the impairment of corneal wound repair. Here we find that ATSE rapidly increases the markers of inflammatory response in normal corneal limbi. After an abrasion injury, ATSE exaggerates inflammation, impairs wound repair, and enhances the expression of nuclear factor-κB (NF-κB) and inflammatory molecules such as interleukin-6 (IL-6) and IL-17. We find that chemical SNS sympathectomy, local adrenergic receptor antagonism, NF-κB1 inactivation, and IL-6/IL-17A neutralization can all independently attenuate ATSE-induced excessive inflammatory responses and alleviate their impairment of the healing process. These findings highlight that the SNS may represent a major molecular sensor and mediator of ATSE-induced inflammation.

Epothilone B Speeds Corneal Nerve Regrowth and Functional Recovery through Microtubule Stabilization and Increased Nerve Beading.

The successful restoration of corneal innervation and function after a corneal injury is a clinically challenging issue. Structural and functional recovery after a nerve injury involves a complex series of steps in which microtubules play a key role. The aim of the current study was to investigate the effects of epothilone B (EpoB), a microtubule-stabilizing agent, on corneal innervation and the functional recovery of the corneal nerve in mice after corneal epithelial abrasion. The pretreatment of mice with EpoB has a remarkable effect on the stabilization of beta-III tubulin, as demonstrated by substantial increases in the visualization of beta-III tubulin, nerve beading, corneal reinnervation, and reaction to stimuli. Furthermore, a pharmacokinetic analysis showed that EpoB remains at a high concentration in the cornea and the trigeminal ganglion for at least 6 days after administration. In addition, the administration of EpoB at 24 hours after corneal abrasion has a marked therapeutic effect on nerve regrowth and functional recovery. In conclusion, EpoB treatment may have therapeutic utility for improving corneal reinnervation and restoring sensitivity following corneal injury.

The mouse autonomic nervous system modulates inflammation and epithelial renewal after corneal abrasion through the activation of distinct local macrophages.

Inflammation and reepithelialization after corneal abrasion are critical for the rapid restoration of vision and the prevention of microbial infections. However, the endogenous regulatory mechanisms are not completely understood. Here we report that the manipulation of autonomic nervous system (ANS) regulates the inflammation and healing processes. The activation of sympathetic nerves inhibited reepithelialization after corneal abrasion but increased the influx of neutrophils and the release of inflammatory cytokines. Conversely, the activation of parasympathetic nerves promoted reepithelialization and inhibited the influx of neutrophils and the release of inflammatory cytokines. Furthermore, we observed that CD64+CCR2+ macrophages in the cornea preferentially expressed the ß-2 adrenergic receptor (AR), whereas CD64+CCR2- macrophages preferentially expressed the α-7 nicotinic acetylcholine receptor (α7nAChR). After abrasion, the topical administration of a ß2AR agonist further enhanced the expression of the proinflammatory genes in the CD64+CCR2+ cell subset sorted from injured corneas. In contrast, the topical administration of an α7nAChR agonist further enhanced the expression of the anti-inflammatory genes in the CD64+CCR2- subset. Thus crosstalk between the ANS and local macrophage populations is necessary for the progress of corneal wound repair. Manipulation of ANS inputs to the wounded cornea may represent an alternative approach to the treatment of impaired wound healing.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for quantification of SR1001, an inverse agonist of retinoid-related orphan receptors, and its application to pharmacokinetic studies in streptozotocin-induced diabetic mice.

Retinoic acid receptor-related orphan receptors (RORs) play critical roles in the onset and progression of type I diabetes, an autoimmune disease characterized by the destruction of pancreatic ß-cells. SR1001, an ROR inverse agonist, has been proven to be an effective diabetes treatment in the non-obese diabetic (NOD) mouse model. However, optimization of this treatment is challenging because knowledge of SR1001 pharmacokinetic (PK) behaviors in type I diabetic animals is limited. The aim of our study was to develop and validate a specific and sensitive ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to measure the concentrations of SR1001 in plasma and biological samples. Using the developed UPLC-MS/MS method, SR1001 linearity ranges in biological matrices were determined to be 5-1000ng/mL, with correlation coefficients of >0.99. The limit of detection (LOD) and limit of quantification (LOQ) values of SR1001 were 1 and 5ng/mL, respectively. And the intra-day and inter-day variances were less than 10%, and accuracy was within 90%-110%. The extraction recoveries of SR1001 were ≥80%, and no significant matrix effect was observed. Using the validated UPLC-MS/MS method, levels of SR1001 in plasma and six major organs (heart, liver, spleen, lung, kidney, and brain) were determined in streptozotocin (STZ) -induced diabetic mice. The PK parameters of SR1001 were also calculated. The SR1001 drug concentration-time curves for organs and plasma showed similar trends, and the elimination half-lives of SR1001 in diabetic mice were about 12h. SR1001 was highly bound to plasma protein, resulting in a much higher maximum concentration (Cmax=144394ng/mL) and area under the concentration-time curve (AUC0-t=2728258ng/mL*h), but a low tissue/plasma partition coefficient (Kp) value of <0.3.

Modulation of Circadian Rhythms Affects Corneal Epithelium Renewal and Repair in Mice.

Purpose: In mammalian corneal epithelium, mitosis shows a distinct circadian pattern. However, how this circadian pattern is maintained, and how it or its disruption influence renewal and regeneration remain unclear. Methods: C57BL/6 mice were maintained under 12-hour light/12-hour dark (LD), 12-hour light/12-hour light (LL), 12-hour dark/12-hour dark (DD), or reversed LD (DL, 12-hour dark/12-hour light; jet-lag defined as a shift of 12 hours) conditions. Mitotic cells in corneal epithelium were enumerated and analyzed via immunofluorescence at different zeitgeber times (ZTs). Expression of core clock genes (Clock, Bmal1, Period2, Cry1, and Rev-erbα) was qualified via quantitative RT-PCR. The rate and quality of healing at different ZT times and after administration of two small-molecule modifiers of the circadian clock, KL001 and SR8278, was evaluated. Results: In this study, photic cues were found to influence the 24-hour rhythm of corneal clock gene expression and epithelial cell mitosis in mice. Disruption of the circadian clock by exposure to constant light, constant dark, or jet-lag conditions modified the normal 24-hour patterns of corneal epithelial mitosis and corneal clock gene expression. The time of day of wound occurrence affected the rate and quality of corneal healing, with both of these parameters peaking during the more mitotically active hours of the morning. The two small-molecule modifiers of the circadian clock, KL001 and SR8278, had negative and positive effects on corneal wound healing, respectively. Conclusions: Circadian rhythms significantly influence corneal epithelium renewal and repair in mice. Our findings reveal possible opportunities for biological rhythm-based interventional strategies to control corneal healing and restore corneal homeostasis.

Insulin Restores an Altered Corneal Epithelium Circadian Rhythm in Mice with Streptozotocin-induced Type 1 Diabetes.

The mechanisms of corneal epithelial lesions and delayed wound repair, as well as their association with diabetes mellitus, are critical issues for clinical ophthalmologists. To test whether the diabetic condition alters the circadian rhythm in a mouse cornea and whether insulin can synchronise the corneal clock, we studied the effects of streptozotocin-induced diabetes on the mitosis of epithelial cells, the recruitment of leukocytes to the cornea, and the expression of main core clock genes (Clock, Bmal1, Per2, Cry1, and Rev-erbα) in the corneal epithelium. We also assessed the possible effect of insulin on these modifications. Diabetes downregulated Clock, Bmal1, and Per2 expression, upregulated Cry1 and Rev-erbα expression, reduced corneal epithelial mitosis, and increased leukocyte (neutrophils and γδ T-cells) recruitment to the cornea. Early treatments with insulin partially restored the altered rhythmicity in the diabetic cornea. In conclusion, insulin-dependent diabetes altered the normal rhythmicity of the cornea, and insulin administration had a beneficial effect on restoring normal rhythmicity in the diabetic cornea.