2.1. Measurement of LPS-induced cytokine/chemokine levels in THP-1-derived macrophages
2.1.1. Human cell cultures
The THP-1 cell line was purchased from ATCC and maintained at 37°C under 5% CO2 in RPMI 1640 medium (Corning) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Genesee Scientific). THP-1 monocytes were differentiated into macrophages in the presence of 100 ng/mL phorbol-12-myristate 13-acetate (PMA; Sigma-Aldrich) for 48 h, followed by 24 hours of rest in PMA-free culture media prior to starting experiments. Only THP-1 cells cultured from passages 14 to 18 were used for experiments.
2.1.2. Preparation of compounds
Bacterial lipopolysaccharides (LPS) from Escherichia coli O111:B4 (Sigma-Aldrich) were dissolved in 1X phosphate-buffered saline (PBS; Cytiva) and subsequently diluted in culture medium. Omadacycline (Paratek Pharmaceuticals) and dexamethasone (MedChemExpress) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and diluted in culture medium to a final DMSO concentration of 0.1% (v/v). The glucocorticoid dexamethasone was included as a comparator because of its known anti-inflammatory properties [28]. For all in vitro assays, medium with 0.1% DMSO served as the vehicle control.
2.1.3. THP-1 cytotoxicity evaluation
THP-1 cells were seeded in triplicate at a density of 2 x 105 cells/well into 96-well culture plates (Genesee Scientific) in RPMI complete medium plus 10% FBS and differentiated as described above. After a 30-minute pre-incubation at 37°C with either the vehicle or omadacycline (20, 40, 60, 80, or 100 µg/mL), THP-1-derived macrophages were stimulated with LPS (100 ng/mL) for an additional 24 hours. Incubation with the positive control substance Triton X-100 (2%) was used as a positive control of cell death. The media was aspirated, and the samples were gently washed twice with PBS. A 10% (v/v) alamarBlue solution was prepared by diluting the alamarBlue reagent (ThermoFisher Scientific) in culture media, and 200 µL of the 10% alamarBlue solution was added to each well. Background wells without cells were also included for background subtraction. Plates were incubated for 2 hours (37ºC, 5% CO2), and their absorbance was measured at 570 nm, and 600 nm as a reference, using a Sunrise microplate absorbance reader (Tecan). Cell viability, expressed as a percentage of vehicle-treated control cells, was determined by calculating the percentage reduction of the alamarBlue reagent based on absorbance readings, following the manufacturer’s instructions.
2.1.4. Cytokine expression analysis
THP-1 cells were seeded in triplicate at a density of 2 x 106 cells/well into 6-well culture plates (Genesee Scientific) in RPMI complete medium plus 10% FBS and differentiated as described above. THP-1-derived macrophages were then preincubated at 37°C with vehicle, dexamethasone (1 µM), or omadacycline (20, 40, 60, 80, or 100 µg/mL) for 30 minutes and subsequently stimulated with LPS (100 ng/mL) for an additional 24 h. The cell supernatant was collected, aliquoted, and stored at -80ºC. The frozen supernatant was thawed and thoroughly mixed, and concentrations of tumor necrosis factor (TNF)-α, IL-1β, IL-6, chemokine (C-X-C motif) ligand (CXCL)-1, CXCL-2, and matrix metalloproteinase (MMP)-9 were analyzed by ELISA (ThermoFisher Scientific) per the manufacturer’s protocol and using a Sunrise microplate absorbance reader (Tecan). Concentrations in samples were determined by interpolation from standard curves.
2.2. Cytokine/chemokine mRNA expression levels in LPS-induced THP-1-derived macrophages
2.2.1. Cell processing and cDNA synthesis
To determine the effect of omadacycline on inflammatory gene expression, THP-1 cells were seeded, differentiated, treated with omadacycline, and stimulated with LPS as described above. After 4 hours, the cell supernatant was aspirated and total RNA was isolated using RNAzol RT (Sigma-Aldrich) according to the manufacturer’s protocol. Total RNA was treated with the Turbo DNA-free kit (Invitrogen) to remove genomic DNA contamination and purified by RNA Clean & Concentrator (Zymo Research). The first-strand cDNA was reverse transcribed using iScript Reverse Transcription Supermix (Bio-Rad). All mRNA processing steps employed RNase-free solutions and sterile, disposable labware, with surfaces routinely decontaminated using RNaseZap (Invitrogen).
2.2.2. PCR primers
Primer sequences for human tumor necrosis factor (TNF)-α, interleukins (IL)-1β, IL-6, chemokine ligand (CXCL)-1, CXCL-2, matrix metalloproteinase (MMP)-9, and ubiquitin C (UBC) were designed using GeneBank database sequences from the National Center for Biotechnology Information (Supplementary Table A1). To further avoid the contamination of genomic DNA, primers were designed to cross exon-exon junctions. Primers were synthesized by Integrated DNA Technologies.
2.2.3. Real-time qPCR
Real-time quantitative polymerase chain reaction (qPCR) was performed using the QuantStudio 12K Flex Real-Time PCR System (ThermoFisher Scientific) using 384-well plates with 10 µL of total reaction volume. The SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) was used as the qPCR master mix. The thermal cycle profile was as follows: initial denaturation at 95°C for 2 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing and elongation at 60º for 1 minute. Each reaction was run in technical triplicates. The delta-delta Ct method was employed to quantify gene expression [29]. Relative expression levels of all genes were calculated relative to the housekeeping gene, UBC [30]. Gene expression was scaled relative to the control, where untreated control samples were set at a value of 1. Statistics were performed on log2-transformed data.
2.3. Measurement of IL-8–induced chemotaxis of primary human neutrophils
2.3.1. Neutrophil isolation and culture
Human blood collection was approved by the University of Southern California’s Institutional Review Board (HS-14-00294). All methods were carried out in accordance with relevant guidelines and regulations. Whole blood was collected from a single healthy volunteer by venipuncture into Vacutainer collection tubes containing the anticoagulant K2EDTA (BD Biosciences). The isolation of peripheral blood neutrophils was performed using the EasySep™ Direct Human Neutrophil Isolation Kit (STEMCELL Technologies). Following isolation, neutrophils were centrifuged (350xg, 5 minutes), resuspended in phenol red-free RPMI 1640 (Gibco) supplemented with 1% (w/v) bovine serum albumin (Sigma-Aldrich), counted using a hemocytometer, and adjusted to a density of 1.25 x 107 cells/mL. Neutrophil purity and viability were > 95%, as assessed by differential counts and trypan blue dye exclusion, respectively. Neutrophils were then seeded in triplicate at a density of 1 x 106 cells/well into a 96-well culture plate (Genesee Scientific) and pre-incubated (37ºC, 5% CO2) with either the vehicle or omadacycline (100, 250, 500, 750, or 1000 µg/mL). After 1.5 h, these neutrophils were either used for cytotoxicity evaluation or introduced into the chemotaxis assay.
2.3.2. Neutrophil cytotoxicity evaluation
Following pretreatment with omadacycline, 50 µL of neutrophil cell suspension (5 x 105 cells) were supplemented with RPMI 1640 (1% BSA) and alamarBlue reagent (ThermoFisher Scientific) to achieve a 10% (v/v) concentration of the alamarBlue solution. Incubation with Triton X-100 (2%) was used as a positive control of cell death. Background wells lacking cells were included for background subtraction. Plates were incubated for 4 hours (37ºC, 5% CO2), and their absorbance was measured at 570 nm, and 600 nm as a reference, using a Sunrise microplate absorbance reader (Tecan). Cell viability, expressed as a percentage of vehicle-treated control cells, was determined by calculating the percentage reduction of the alamarBlue reagent based on absorbance readings, following the manufacturer’s instructions.
2.3.3. Chemotaxis assay
Chemotaxis experiments were performed in 96-well HTS Transwell plates with 3 µm pores (Corning). The chemotactic factor Interleukin 8 (IL-8) was used as the chemoattractant. The bottom well of the chemotaxis chamber was filled with 50 µL of IL-8 (20 ng/mL; PeproTech) or medium alone (negative control). The upper filter was then carefully placed on top of the plate and visually inspected to confirm the membrane's contact with the fluid in each well's bottom compartment and ensure no air bubbles were present between the filter and the media in the lower wells. Then, 25 µL of the omadacycline pre-treated neutrophils (2.5 x 105 cells) was carefully pipetted directly onto the membrane and the lid was put on the plate. The neutrophils were seeded in technical duplicates and allowed to migrate for 1.5 hours (37ºC, 5% CO2).
At the end of the experimental period, the Transwell plate and membrane were centrifuged (350xg, 1 minute) to dislodge any migrated cells adherent to the underside of the filter membrane. The filter membrane was then removed and the number of migrated neutrophils was quantified with the CellTiter-Glo 2.0 assay (Promega) according to the manufacturer’s instructions. In brief, an equal volume (125 µl) of the CellTiter-Glo 2.0 Reagent was added to each well. The plate was shaken for 2 minutes to induce cell lysis and subsequently incubated at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was measured using the BioTek Synergy HTX plate reader (Agilent) equipped with the BioTek Gen5 Software and an integration time of 1 second. The percentage of maximal chemotaxis was calculated by establishing the migrated cell count in the presence of chemoattractant alone as 100% after subtraction for the mean number of cells undergoing random migration as previously described [31]. The response of omadacycline-treated cells under chemoattractant was normalized to the untreated control.
2.4. Comprehensive assessment of pulmonary inflammation, edema, and acute lung injury in murine lungs
2.4.1. Animals and husbandry
All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Southern California (Protocols 21249). Male, 6–8 week old BALB/c mice (Charles River Laboratories), were housed under standard temperature (20–24°C) and humidity (60–65%) laboratory conditions, with a 12:12-hour light-dark cycle. The animals were provided standard laboratory chow and water ad libitum.
2.4.2. Reagents
LPS from E. coli O111:B4 (Sigma-Aldrich) was dissolved in sterile 1X PBS (Cytiva). Omadacycline (Paratek Pharmaceuticals) was dissolved in DMSO (Sigma-Aldrich) and subsequently diluted in sterile PBS to final concentrations of 0.25, 0.75, 1.5, and 3 mg/mL (0.3% v/v DMSO). Dexamethasone (MedChemExpress) was dissolved in DMSO and subsequently diluted in sterile PBS to a final concentration of 0.1 mg/mL (0.1% v/v DMSO). Azithromycin (Selleck Chemicals) was dissolved in DMSO and diluted in sterile PBS to a final concentration of 6 mg/mL (50% v/v DMSO). This formulation was essential due to azithromycin's high insolubility in water alone. It should be noted that DMSO may have potentially contributed synergistically to azithromycin's effects, as DMSO has recently been explored for potential immunomodulatory properties itself [32]. For all in vivo assays, PBS with 0.1% DMSO served as the vehicle control.
2.4.3. Experimental procedures
Several experiments were performed in groups of mice to assess the pharmacokinetics, efficacy, and safety of omadacycline in a murine model of LPS-induced ALI. Acute neutrophilic airway inflammation and lung injury were induced in mice by intranasal instillation of bacterial lipopolysaccharide as previously described [25–27]. Mice were sedated with isoflurane (VetOne) delivered via a vaporization and anesthesia device. Acute lung inflammation was induced by intranasal administration of E. coli O111:B4 LPS. The solution of LPS was prepared at 3 µg per µL of saline and 1 µl per gram of body weight was introduced (e.g. 25 µl for a mouse of 25 g) with a siliconized pipette tip during the inspiration. Efficacy was then determined in two dose-ranging studies for the prevention and treatment of ALI. No mortality was observed in the disease model.
In the dose-ranging prevention study, mice were randomly assigned saline (SC), dexamethasone (1 mg/kg; IP), azithromycin (30 mg/kg; SC), or omadacycline (2.5, 7.5, 15, or 30 mg/kg; SC) one hour prior to LPS challenge (n = 6/group). The omadacycline doses and administration route were selected based on prior published data [33]. The antibiotic azithromycin and the glucocorticoid dexamethasone were chosen as comparators due to their established anti-inflammatory properties. Their doses and administration routes were based on a prior published study and produced reproducible effects in the LPS-induced neutrophilia model [34]. Twenty-four hours after LPS administration, mice were euthanized by intraperitoneal injection of a lethal overdose of Euthasol (Virbac).
In the dose-ranging treatment study, mice were randomly assigned saline (SC), dexamethasone (1 mg/kg; IP), azithromycin (30 mg/kg; SC), or omadacycline (15 or 30 mg/kg; SC) six hours after LPS challenge (n = 6/group). Forty-eight hours after LPS administration, mice were euthanized by intraperitoneal injection of an overdose of Euthasol (Virbac).
2.4.4. Terminal blood collection
Immediately following euthanasia, blood was collected via intracardiac puncture in EDTA-coated tubes. Plasma was promptly obtained through consecutive centrifugation steps at 1200g, 4ºC for 10 minutes, followed by a second centrifugation at 2000 rpm, 4ºC for another 10 minutes, and subsequently stored at -80ºC until analysis.
2.4.5. Bronchoalveolar lavage fluid collection and cytological analysis
Bronchoalveolar lavage was collected by flushing the lungs 3 times with 1 mL of cold PBS (Cytiva) without Ca2+/Mg2+. Collected BALF samples were immediately placed on ice, followed by clarification through centrifugation (400xg, 10 minutes, 4ºC). BALF supernatant was aliquoted and stored at -80ºC. The resulting cell pellets were then resuspended in 250 µL PBS. Airway total white blood cell (WBC) counts were determined for each BALF sample using Turk blood diluting fluid (Ricca Chemical) and a hemocytometer. Approximately 100 µL of cell suspension was added to a slide chamber, spun at 700 rpm for 10 minutes in a Shandon Cytospin 3 cytocentrifuge (ThermoFisher Scientific), and stained using the Differential Quik III Stain Kit (Polysciences) according to the manufacturer’s instructions. Differential cell counts were performed with a light microscope and 40X objective lens for polymorphonuclear neutrophils (PMNs) and macrophages. Neutrophil-to-macrophage ratios were calculated using a deep learning AI (Biodock.AI) and averaged from five random fields per sample.
2.4.6. Lung tissue sampling
After bronchoalveolar lavage, whole mouse lungs were removed, separated into left and right lobes, snap-frozen in liquid nitrogen, and stored at − 80°C until further analysis. Frozen lungs were thawed and homogenized in T-PER Tissue Protein Extraction Reagent (ThermoFisher Scientific) supplemented with Complete Mini protease inhibitor tablets (Roche). Samples were then mechanically homogenized using a Tissue-Tearor (Polytron). Blades of the homogenizer were sterilized with 10% bleach and subsequently rinsed with ethanol and PBS before beginning and after processing each sample.
2.4.7. LC-MS/MS analysis of omadacycline
Omadacycline concentrations in mouse plasma and BALF were determined using LC-MS/MS (Institute of Clinical Pharmacodynamics, Schenectady, NY). For plasma, the LC-MS/MS method utilized was linear (1/x2) over the concentration range 0.020–5.000 mg/L and the correlation coefficient (r2) values ranged from 0.9995–0.9996. For the method quality control plasma samples, the inter-assay coefficient of variance (%CV) ranged from 2.48% − 4.64%, and the inter-assay percent recovery ranged from 99.6% − 110.6%. For BALF, the LC-MS/MS method utilized was linear (1/x2) over the concentration range 0.001–0.250 mg/L and the correlation coefficient (r2) values ranged from 0.9993–0.9997. For the method quality control plasma samples, the inter-assay coefficient of variance (%CV) ranged from 0.97% − 9.23%, and the inter-assay percent recovery ranged from 99.0% − 103.7%.
2.4.8. Pharmacokinetic studies in mice
Pharmacokinetics was performed in healthy mice receiving a single dose of 2.5, 7.5, 15, or 30 mg/kg omadacycline subcutaneously. Plasma and BALF samples were collected at the terminal time points of 0.25, 0.5, 1, 2, 6, 12, and 24 hours (n = 3/group). Plasma and BALF time-concentration profiles were analyzed by noncompartmental pharmacokinetic analysis using a sparse sampling and Bailer-Satterthwaite approach in WinNonlin Phoenix version 8.4 (Certara Inc.). Linear regression analysis was conducted using the maximum plasma and BALF concentrations (Cmax) and total drug exposure (AUC0 − 24) to assess dose linearity and proportionality.
2.4.9. Measurement of total protein and cytokines levels in BALF
The frozen BALF supernatant was thawed and thoroughly mixed, and the total protein concentration for each sample was determined using a BCA protein assay (Pierce, ThermoFisher Scientific). Concentrations of TNF-α, IL-1β, IL-6, CXCL-1, CXCL-2, and MMP-9 were analyzed by ELISA (ThermoFisher Scientific) per the manufacturer’s protocol and using a Sunrise microplate absorbance reader (Tecan). Concentrations in samples were determined by interpolation from standard curves.
2.4.10. Urea correction
Epithelial lining fluid (ELF) concentrations (CELF) were determined by utilizing the urea correction methodology according to the following formula: CELF = CBAL × ([urea]plasma/[urea]BAL). In this equation, CBAL is the concentration of protein measured in the BALF, [urea]plasma is the concentration of urea in plasma, and [urea]BAL is the concentration of urea in the BALF [35]. The urea concentrations in plasma and BALF samples collected simultaneously at the time of bronchoscopy were analyzed by QuantiChrom Urea Assay Kit (BioAssay Systems) according to the manufacturer's instructions and using a Sunrise microplate absorbance reader (Tecan). Concentrations in samples were determined by interpolation from standard curves.
2.4.11. Measurement of total protein and cytokines levels in lung tissue
After homogenization, concentrations of TNF-α, IL-1β, IL-6, CXCL-1, CXCL-2, and MMP-9 from lung homogenate supernatant were analyzed by ELISA (ThermoFisher Scientific) per the manufacturer’s protocol and using a Sunrise microplate absorbance reader (Tecan). Concentrations in samples were determined by interpolation from standard curves. The measured concentrations were normalized to total protein content, as determined by BCA protein assay (Pierce, ThermoFisher Scientific).
2.4.12. Measurement of lung wet/dry ratio
In separate studies, the severity of pulmonary edema was assessed by the wet-to-dry ratio (W/D). For the prevention and treatment studies, mice were treated as previously described (n = 3/group). Immediately after euthanasia, the lung wet weight for each mouse was recorded. The lungs were then incubated in an oven (80°C for 24 hours) to obtain a dry weight. The W/D ratio was calculated using the wet and dry weights.
2.4.13. Lung histopathology
In separate studies, the effects of omadacycline on the severity of LPS-induced acute lung injury were investigated. Mice were treated as previously described for both the prevention and treatment studies (n = 3/group). Immediately after euthanasia, the lungs were transcardially perfused with ice-cold PBS containing 10 U/mL of heparin to remove blood. They were then infused with 10% formalin buffered solution (Sigma-Aldrich) until fully inflated [36]. Subsequently, the lungs were resected, placed into cassettes, fixed in 10% formalin buffered solution for 24–48 hours, then dehydrated by gradient ethanol, embedded in paraffin, and sliced into 3 um sections. Tissue processing, embedding, and sectioning were conducted by the USC Mann Histology Laboratory. The slides were stained with hematoxylin and eosin by the USC Norris Comprehensive Cancer Center Translational Pathology Core and scanned at 40x magnification with a Hamamatsu NanoZoomer S60 digital whole slide scanner. Analysis software NDP.view2 was used for image processing (Hamamatsu Photonics). Lung injury scores were evaluated by a pathologist who was blinded to the identity of the slides following a previously described lung injury scoring system [37].
2.4.14. Data and statistical analysis
Statistical and graphical analyses were performed using GraphPad Prism version 10.1 (GraphPad Software, Inc.). Data normality was evaluated through visual inspection, Q-Q plots, histograms, and by measuring skewness and kurtosis. Non-normal data was then log-transformed and re-evaluated using the same methods. Statistical differences between treatment groups were determined by one-way ANOVA with Tukey's multiple comparison post-hoc tests. The strength and direction of associations were determined using Pearson's correlation coefficient. A significance level of P less than 0.05 was determined a priori.