Sprague-Dawley rats (8-week-old male) were obtained from Charles River Laboratories (Wilmington, MA, USA) and kept at the Animal Care of the Neuroscience Center of Excellence, Louisiana State University Health (LSUH; New Orleans, LA, USA). All animals were handled in compliance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at LSUH.
The rats were anesthetized by intraperitoneal injection of Ketamine (50-100 mg/kg) plus xylazine (5-10 mg/kg). A 4 mm diameter filter paper soaked in 1 N NaOH was placed on the central cornea of the right eye for 45 seconds, and then the eye was thoroughly washed with 10 mL of saline. After injury, the rats were randomly divided into five treatment groups: vehicle; lipoxin A4 (LXA4) from Cayman Chemical (Ann Arbor, MI, USA); R,R Resolvin D6 isomer (RvD6i), R,R neuroprotection D (NPD1), and elovanoid (ELV)-N32 synthesized by Dr. R. Nshimiyimana and Prof. N. Petasis. All lipid solutions were prepared at the final concentration of 10 μM using PBS with the minimal contamination of ethanol by evaporating the ethanol and immediately dissolve the lipids in PBS, then vortex well for 2 min. Topical administration (20 μl) was done 3x/day for 14 days. The experiments were double-blinded with the lipid mediators coded during the whole experiments. At the end of the study, when all data was collected, the code was opened.
Injured corneas (n = 5/condition) were harvested and homogenized with TRIzol (Thermo Fisher Scientific) on ice with a glass Dounce homogenizer. RNA sequencing was performed as described14. Briefly, after mRNA extraction and determination of purity, 8 ng of total RNA was reverse transcribed, and total cDNAs were amplified using ISPCR primer, and the library was made with the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). The libraries were pooled with the same molarity and sequenced using the NextSeq 500/550 High Output Kit v2 (75 cycles, Illumina). After demultiplexing, RNA-seq data were aligned to the Rattus Norvegicus reference genome (ftp://ftp.ensembl.org/pub/release-98/fasta/rattus_norvegicus/dna/) using the Subread package v2.0.1 alignment function39. The BAM files for sequencing data alignment were counted using featureCounts function of Subread tool40 using the macOS Catalina. The raw count data were subjected to differential gene expression analysis using DESeq2 package for R41 with the vehicle group as reference. The adjusted p-values were named as the false discover rate (FDR). Significantly changed genes (FDR < 0.05) between each treatment vs. vehicle were subjected to the enrichment analysis using EnrichR42 and NetworkAnalyst 3.043, and pathway analysis using the IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).
Preparation of Alexa 594-conjugated RBD fragment of S protein
RBD fragment of the Spike protein belonging to SARS-CoV-2 (Raybiotech, Peachtree Corners GA. Cat. 230-30162-1000) was labeled using Alexa Fluor™ 594 Protein Labeling Kit (ThermoFisher, Waltham, MA. Cat. A10239) following the manufacturer’s directions. Briefly, 1 mg of protein was dissolved in 0.1 M bicarbonate and then incubated with the Alexa Fluor 594 dye for one hour. The dye was washed using an Amicon-Ultra centrifugal filter cutoff 10KDa (Merck, Millipore Carrigtwohill, CO. Cat. UFC201024). To assess the efficiency of the label, the protein was measured at 280 nm and 590 nm absorbance using NanoDrop One (Thermo Scientific). There was a ratio of 0.4 moles of dye/mole of protein and a recovery of about 80%.
Human corneal epithelial cells (HCEC) culture
All experiments with human corneal epithelial cells were approved by the Institutional Review Board of LSUHNO and conducted in accordance with NIH guidelines. HCEC were kept frozen in the laboratory at passage 2544. Cells were maintained in keratinocyte growth (KGM) medium containing the keratinocyte basal medium (KBM) (Lonza: CC-3101) supplemented with bovine pituitary extract (BPE), hEGF, Insulin, Hydrocortisone and Gentamicin Sulfate-Amphotericin (GA-1000) (Lonza, Cat. CC-4131). For all experiments, cells were seeded at 30,000 cells/cm2.
For screening the stimulation of receptor ACE2 by cytokines, the HCEC were cultured with KGM until 50-60% confluence. Then, changed to KBM containing IL-1β, -2, -6 and 8, IFN-α, -ε, and -γ or TNFα at 1, 10 or 100 ng/ml. The cells were harvested after 6 hours and analyze for the gene expression of Ace2. In other experiments, HCEC were stimulated with IFNγ, and thereafter, lipid mediators were added. For the Alexa 594-conjugated RBD binding, IFNγ was used as a cytokine trigger. At 12 hours after cytokine exposure and lipid mediator treatments, 0.5 μg of labeled RBD was added to the medium. The evaluation of RBD binding was conducted 24 hours after.
Corneal tissue was fixed in Zamboni fixative (MasterTech Scientific, Lodi, CA USA) for 2 hours immediately after euthanasia. After thoroughly washing with PBS, the corneas were embedded in optimal cutting temperature compound, and serial 10-μm cryostat sections were obtained, dried at room temperature for 2 hours, and stored at -20 °C until use. For immunofluorescence, the sections were incubated with primary antibodies at the concentration described in Table 1 in a wet chamber at 4 °C overnight. The sections were washed 3 x/5 min with PBS following by incubation for 1 hour at RT with Alexa Fluor-conjugated secondary antibodies (1:1000 dilution). All sections were counterstained with DAPI (ThermoFisher Scientific, Cat. D1306), and images of rat corneal samples were acquired with an Olympus IX71 fluorescent microscope.
Unbiased imaging-based evaluation of RBD binding
Twenty-four hours after Alexa 594-RBD was added to the HCEC, the cells were washed with PBS (3x/5min) and fixed with 4% paraformaldehyde for 30 minutes at RT. The HCEC were washed 2x with PBS and stained with Hoechst 33342 Solution (ThermoFisher Scientific, Cat. 62249) for 30 minutes at RT. Next, the HCEC were washed 2x with PBS before imaging. For unbiased data collection, 7 designated areas were defined in each well (Supplementary Fig. S7) and captured with an Olympus FV3000 confocal laser scanning microscopy under “Multi Area Time Lapse” (MATL) mode. All images were acquired with the same parameters and Z-section range, converted and inputted in the Imaris software version 9.5.1. The threshold for the control images was defined by the HCEC without Alexa 594-conjugated RBD of S protein and using it as a threshold filter for the Imaris batch image processing function. The sum of total intensity for each image was used to evaluate the binding efficiency. The whole process was summarized in the Supplementary Fig. S7b,c.
Droplet Digital PCR (dd-PCR)
Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Germany), and 1 µg of total RNA was reverse transcribed using an iScript cDNA Synthesis Kit (Bio-Rad, Cat. 170–8841). For ddPCR, 10 ng of cDNA was multiplexed with Ace2 and phosphoglycerate kinase 1 (Pgk1) probes (Bio-Rad, Cat. qHSACEP005-1563 and dHSACPE503-3809) using dd-PCR Supermix for Probes No dUTP (Bio-Rad, Cat. 1863024). Then, 20 µL of the reaction was mixed with 70 µL of Droplet Generation Oil (Bio-Rad Cat:1863005) to make the reaction droplets. The emulsified samples were carefully transferred to PCR plates (Bio-Rad, Cat. 12001925) and amplified using the cycling: 95 °C for 10 minutes, 40 cycles of a two-step cycling protocol (94 °C for 30 seconds and 60 °C for 1 minute), and 98 °C for 10 minutes. Next, the post-cycling plate was placed into the QX200 Droplet Reader with the FAM/HEX setting. The absolute quantity of DNA per sample (copies/µL) was processed using QuantaSoft Analysis Pro Software. For the data analysis, the ratio of quantified Ace2 to Pgk1 was used.
Capillary-based Western Blot
The capillary-based western assay was performed using a Jess Protein Simple system (San Jose, CA, USA) as manufacture suggested protocol. Briefly, samples were lysed with RIPA buffer containing a protease inhibitor cocktail (Sigma, Cat. P8340). Cell debris was removed after 10 min centrifugation at 16,000 x g. Protein concentration was determined by BCA assay (Thermo Fisher Scientific, Cat. 23225) and 1 µg used/ reaction. Fluorescent Master Mix was mixed with 40 mM DTT, and the mixture was added to each sample to provide a denaturing and reducing environment. Samples were heated at 95 °C/5 min, and 3 µL of each sample were loaded. The 12–230 kDa cartridge (Protein Simple – #SM-W004) was used. Primary antibodies were diluted in antibody diluent 2 buffer (Protein Simple, #042-203) while the working solution of secondary antibodies was provided by the company (Protein Simple, #042-206). Then, the filled plate was spin-down for 10 min at 1,000 x g to remove bubbles and plate, and capillaries were loaded into the Jess machine. For data analysis, the area of spectra that matched the molecular weight of the target protein was used. To reduce the coefficient variant, we analyzed the GAPDH for each capillary. The ratio of the targeted protein to GAPDH was used for statistical comparisons. For visualization, the artificial lanes generated from spectra volume was used.
High-throughput qPCR using Biomark™ HD
Quantitative PCR was performed with the Biomark HD system (Fluidigm, San Francisco, CA, USA). Briefly, 200 ng of RNA was reverse-transcribed using iScript Reverse Transcription Supermix (Bio-Rad), and the cDNA was pre-amplified using the PreAmp Master Mix (PN 100-5580; Fluidigm). The cDNA was then subjected to Exonuclease I treatment and diluted 5 times in TE Buffer. The qPCR reaction mixture and primer reaction mixture were made and loaded into the Biomark 96.96 IFC™ (Integrated Fluidic Circuit). The enzyme reaction was mixed using Juno™ Controller (Fluidigm) and run using the cycling program of (i) 70 °C for 40 minutes followed by 60 °C for 30 seconds, (ii) hot start for 1 minute at 95 °C, (iii) 30 cycles of denaturation at 96 °C for 5 seconds, and annealing at 60 °C for 20 seconds, and (iv) melting curves between 60 °C and 95 °C with 1 °C increments/3 seconds. The Ct value of target genes was normalized to the house-keeping genes Gapdh, Hprt1, and Tfrc before normalized to the vehicle group. Relative fold changes from the ΔΔCT calculation was used to make the graph. Primersequences are provided in Table 2.
Data are expressed as mean ± SD. The data were analyzed by 1-way ANOVA followed by Dunnett's multiple comparisons post hoc test at 95% confidence level with the vehicle as reference. All graphs were made using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA) with the mean ± SD while all statistical analyses were done using built-in function of Prism 7.