Alamar blue and Nerve Growth Factor were obtained from Invitrogen (Eugene, OR). Paclitaxel was obtained from Teva Pharmaceuticals USA (Sellersville, PA) as a 6 mg/ml solution containing 527 mg polyoxyl 35 castor oil, 2mg citric acid and 49.7% dehydrated alcohol /ml. CY-09 (SML2465) and cannabidiol were obtained from Millipore-Sigma.
The synthesis of KLS-13019 has been described previously in detail (Kinney et al. 2016). Verification of the structural identity for KLS-13019 was determined by 1H NMR, 13C NMR, HMBC, HSQC, COSY, NOESY, LC/UV, and LC/MS. The purity of KLS-13019 was 98.6% as determined by LC/MS.
Dissociated dorsal root ganglia (DRG) cultures derived from embryonic day 18 rats were employed as the primary assay system to explore anti-inflammatory mechanism of action for KLS-13019 in the context of reversing established inflammation associated with paclitaxel treatment. In brief, rat DRG were obtained commercially through Brain Bits (Springfield, IL) and cultures prepared according to methods described previously (Brenneman et al. 2019). Tissue was dissociated with a papain-based kit from Worthington Biochemical Corporation (Lakewood, NJ). The DRG cells were plated at low density (10,000 cells / well) in a 96-well format and maintained in serum-free medium consisting of Neurobasal Medium supplemented with B27, GlutaMAX (Gibco) and 25 ng/ml Nerve Growth Factor. Poly-D-lysine coated plates (BD Biosciences, Franklin Lakes, NJ) were employed for this culture system. Prior to the initiation of experiments between days 5 and 9 in vitro, a complete change of medium was performed in a working volume of 100 mL.
Anti-inflammatory actions of KLS-13019 were also studied in hippocampal cultures that were prepared by methods previously described (Brenneman et al. 2018). In brief, dissociated hippocampal cultures derived from embryonic day 18 rats were employed as a second test system to assess responses in a central nervous system preparation that exhibited the expression of GPR55, a drug-target candidate of KLS-13019. The primary purpose of using this culture system was to measure inflammatory responses produced by lysophosphatidylinositol arachidonate (LPIA), a recognized endogenous agonist of GPR55 (Gangadharan et al. 2013). Hippocampal tissue was obtained commercially through Brain Bits (Springfield, IL). Tissue was dissociated with a papain-based kit from Worthington Biochemical Corporation (Lakewood, NJ). The hippocampal neurons were platted at low density (10,000 cell/well) in a 96-well format and maintained in serum-free medium consisting of Neurobasal Medium supplemented with B27 and GlutaMAX (Gibco). Poly-L-lysine coated plates (BD Biosciences, Franklin Lakes, NJ) were used because of the preferential adherence and survival of neurons on this matrix support. Prior to the initiation of experiments between days 11 and 21 in vitro, a complete change of medium was performed in a working volume of 100 mL.
Culture treatments and inflammation
The primary purpose of these studies was to assess two relevant culture models for their GPR55-related responses to inflammation and then to test if our novel cannabinoid (KLS-13019) had anti-inflammatory actions on cultured neurons. Of clinical relevance to neuropathic pain, DRG cultures were treated with 3 mM paclitaxel, a chemotherapeutic agent with known inflammatory properties (Staff et al. 2020). The toxic level of paclitaxel (3 mM) used for all the present studies was based on both clinically relevant serum concentrations and a previously determined toxic concentration (3 mM) that also produced increased levels of reactive oxygen species as detected with 6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. KLS-13019 was dissolved in dimethyl sulfoxide (DMSO) to obtain a 1 mM stock solution and then serially diluted with sterile Dulbecco’s phosphate buffered saline (DPBS) from Gibco (Grand Island, NY). At the highest concentration tested (1 mM), the concentration of DMSO was < 0.1%. To study the potential effect of KLS-13019 in reversing paclitaxel-induced inflammation, DRG cultures were pretreated with either 3 mM paclitaxel or 1 nM LPIA to establish inflammatory responses with increases in IL-1b and NLRP3 immunoreactive (IR) spot area (inflammasome-3 marker) being demonstrated. After the establishment of an inflammatory response, KLS-13019 were added to the cultures for an additional 16 hours, with the paclitaxel remaining on the cultures. At the conclusion of the 16-hour treatment period, cultures were fixed and assays as described in the following immunofluorescent assay section. This sequence of 8-hour paclitaxel treatment followed by KLS-13019 treatment will be referred to as the “reversal” paradigm for DRG cultures throughout these studies.
The primary purpose of the hippocampal studies that investigated the responses produced by treatment with lysophosphatidylinositol arachidonate (LPIA), a recognized endogenous agonist of GPR55, was to elicit changes in inflammatory responses that were targets for reduction by KLS-13019. Based on preliminary experiments, it was determined that 1 nM LPIA produced the maximal increases in GPR55 after two hours of incubation. To assess hippocampal in a similar “reversal” paradigm for inflammation as that conducted for DRG, a similar but a shorter period of treatment (8-hour) was utilized. Hippocampal cultures were treated with LPIA (1nM) to establish inflammatory responses as measured with IL-1b and NLRP3 for four hours. After sister cultures were assay for viability with alamar blue and fixed, other cultures in the same experiment were treated with KLS-13019 to test for possible reversal of the inflammatory responses during an additional four-hour treatment. This later treatment with KLS-13019 was done without the removal of the LPIA. At the conclusion of this second 4-hr treatment period, the cultures were assayed for viability and then fixed for immunofluorescence assays of inflammation-related targets.
To assess the effects of various KLS-13019 treatments, immunofluorescent methods were used to measure neuronal responses in both DRG and hippocampal cultures. The goals for these assays included: 1) identification of neuronal structures with antibodies to type III beta tubulin; 2) to assess the immunoreactive spot area of selected molecular targets (IL-1b, GPR55 and NLRP3) with their respective primary antibodies and distinctively labeled secondary antibodies; and 3) to compare the relative responses of the molecular targets in both neuronal cell bodies and neurites. Prior to fixation, growth medium was removed and the wells were rinsed one time with 100 mL DPBS (37o C). This warm rinse is particularly important to maintain structural stability of neurites. After removal of the DPBS, cultures were fixed for 20 min at room temperature with 50 mL / well of 3.5% formaldehyde (Sigma-Aldrich: 252549) in warm (370C) DPBS that contained 5.5 mg/mL of Hoechst 33342 dye (Invitrogen: H3570) to label cell nuclei. After removal of the fixative, the cultures were rinsed twice with 100 mL of DPBS and then a permeabilization - blocking buffer containing 5% normal goat serum and 0.3% triton-X100 in DPBS was added to the cultures for 10 min. After removal of the blocking buffer, the cultures were rinsed twice with 100 mL of DPBS and then primary antibodies were added for one-hour incubation at room temperature. Neurons were identified with antiserum to type III beta tubulin (tuj 1) to measure changes in all neuronal structure parameters. The primary antiserum employed was a rabbit polyclonal obtained from Sigma –Aldrich (T2200) and used at 1:250 dilution. The secondary antibody was an Alexa Fluor 488-conjugated Fab fragment of goat anti-rabbit IgG obtained from Life Technologies (A11070) used at 1:600 for 30 minutes. After the secondary antibody treatment, cultures were rinsed 3 times with 100 mL of DPBS before performing high content fluorescent analysis. For storage, the wells were placed in 100 mL of sterile DPBS, with the plates wrapped in aluminum foil and maintained at 40 C. For the detection of inflammatory markers, the following primary antibodies were used: IL-1b (PA5-88078); NLRP3 (PA5-79740); and for GPR55 (ab203663). All primary antibodies for IL-1b and NLRP3 were obtained from Life Technologies. The GPR55 antibody was obtained from Abcam. All primary antibodies were diluted 1:250 and all secondary antibodies were used at 1:600. The secondary antibodies were obtained from Life Technologies. The following Alexa Fluor dyes labeled the secondary antibodies: Alexa Fluor 488 (A11070), 555 (A32732), 687(A32733) and 750 nm (A21039). By using secondary antibodies with differing dyes, the same culture wells were assayed for multiple molecular targets.
High content image analysis
The immunofluorescent assays were conducted on the Cell Insight CX5 high content imaging system (Thermo Fisher Scientific). The system is based on an inverted microscope that automatically focuses and scans fields of individual culture wells using a motorized stage at predetermined field locations. Fluorescent images from individual fields (895mm x 895mm) were obtained with a 10 x (0.30NA) Olympus objective and Photometrics X1 CCD camera, with analysis by HCS Studio 2.0 Software. The light source was LED with solid state five-color light engine used with filter sets that had the following excitation/emission: 386/440, 485/521, 560/607, 650/694 and 740/809). With this capability, multiple fluorescent assays in a single well were conducted. Images were acquired in a low-resolution mode (4 x 4 binning). Image analyses for neuronal cell bodies and neurites were performed with the Cellomics Neuronal Profiling BioApplication. For analysis of neurons, objects were identified as cells if they had valid nuclei and cell body measures based on size, shape and average intensity. Acceptable ranges were determined in preliminary studies to ensure that aggregated cells and non-cellular objects were excluded from the analysis.
For both the DRG and hippocampal cultures, the goals were to examine the immunoreactive spot areas for all the analytes of neurons only. Because the neuronal morphology was different between the two cultures, unique size and shape parameters for cell bodies and neuritic arbors were empirically determined for each culture type in preliminary studies. Once these parameters were determined, the analyses for each culture type were used throughout their respective experiments. Important to these analyses, an essential goal was to compare the immunoreactive spot areas for all analytes in both cell bodies and neurites. Type III beta tubulin immunoreactivity was used to identify the neurons for each culture type (Brenneman et al. 2019). For DRG cultures, twenty predetermined fields of view were sampled in each of six replicate wells per plate, with two replicate plates from different cellular preparations used for each assay. This extensive sampling of the low-density cultures was conducted as some fields contained 1-3 neurons while other fields contained complex networks of 10 or more neurons. The age of the DRG cultures at the time of analysis was 8-10 days after plating. Because primary cultures exhibit a variety of neuronal phenotypes and a range of morphological complexities, extensive sampling was employed to obtain an average neuronal response with the inflammatory markers. For measuring parameters of type III beta tubulin immunoreactivity and spot analysis for the inflammatory markers, the Cellomics Neuronal Profiling Bioapplication was used that combined spot analysis on neurons that resided within this bioapplication. For analysis of spot immunoreactivity with this bioapplication, a key parameter was the empirical establishment of fluorescent thresholding that permitted the use of a dynamic range that optimized the measurement of fluorescent differences among the treatment groups as well as distinguishing the fluorescent signal from background. This thresholding level was set based on our previous experience with antibody-based assays and the smallest distinguishable size of immunoreactive spots (radius:1.5 m) under our imaging conditions. With this algorithm, the immunoreactive area was a relative measure that was characterized by an effective computerized spot analysis in a rapid screening mode. Importantly, the same imaging parameters for neurons from all treatment groups were employed for the DRG studies. The key comparisons in these studies were aimed at measuring the changes in immunoreactive area that was associated with KLS-13019 treatment. Due to the observed differences in the cellular distribution among the analytes, the cellular locations of all inflammatory markers in DRG were determined, thus distinguishing the relative changes between cell bodies and neurites. This capability and experimental focus were obligatory aspects of measuring the inflammatory markers by image analysis. Because dissociated hippocampal cultures exhibited more abundant neurons and had more extensive neuritic arbor than the DRG cultures, ten predetermined fields of view were sampled in each of six to eight replicate wells per plate, with two replicate plates from different cellular preparations used for each assay. The fluorescent thresholding parameters for the immunoreactive spot analysis for hippocampal cultures were similar to that employed for the DRG cultures. Again, the goal of the studies was to obtain measures of the relative changes in immunoreactive area for each of the analytes after KLS-13019 treatment. In contrast to the DRG cultures, the experiment with hippocampal cultures were conducted 14-20 days after plating. As in the case of DRG cultures, field sampling was extensive in order to obtain an assessment of the average neuronal response to the pharmacological treatments. For each experiment, the values are the mean from 6 wells with 10 fields per well being analyzed. An estimate of 400-600 neurons were assessed for each treatment. In all cases, the results were expressed as immunoreactive area of each of the analytes per neuron.
Since CBD had played a prominent role in the discovery and mechanistic history of KLS-13019, preliminary studies also were conducted in hippocampal cultures to investigate the possible activity of this cannabinoid on GPR55 immunoreactive area after treatment with 1nM LPIA. Treatment with 10 mM CBD for 6 hours has no detectable effect on LIPA-induced increases in GPR55 IR area in either neurites or cell bodies of hippocampal neurons (data not shown). Furthermore, CBD treatment produced only low efficacy protection (40% of maximum) from LPIA-mediated decreases in cellular viability as assessed by the alamar blue assay. Since these screening studies with CBD and GPR55 were ineffective, further studies in DRG cultures were not performed.
Mitochondrial GPR55 localization
Exploratory studies were conducted on day 21 hippocampal cultures to assess the possible localization of GPR55 within some mitochondria. These studies were undertaken because of the central importance of GPR55 in the inflammatory responses reported in the present study as well as our findings that both DRG and hippocampal cultures respond to increases produced by the GPR55 agonist: LPIA. The primary antibody used as a marker for mitochondria was GT6310 for cytochrome c oxidase 4 [COX4] (Life Technologies). GT6310, used at 1:300, is a mouse monoclonal antibody that was made to a recombinant fragment between amino acid 1-169 in COX4. The secondary antibody (A-21037) for the COX4 assay was goat anti-mouse labeled with Alexa Fluor 750 (Life Technologies) that was used at 1:1000. For measuring parameters of type III beta tubulin immunoreactivity and spot analysis for COX4, the Cellomics Neuronal Profiling Bioapplication was used that combined spot analysis on neurons that resided within this bioapplication. Conditions for detecting neurons and GPR55 were identical to that described previously. For analysis of spot immunoreactivity with this bioapplication, a key parameter was the empirical establishment of fluorescent thresholding which was based on our previous experience with antibody-based assays and the size of the spots which were set at the lowest limit for spot radius (1.5 m). Under these imaging parameters, the spot overlap function was set a 100% in order to detect spot overlap with the highest stringency. The goal of these studies was to use the highest exclusionary conditions to estimate if such colocalizations may exist within in the limits of detection for this system. With the use of pseudo color imaging, 100 % overlapped spots were reported with a blue color, while COX4-positive spots alone were reported as yellow and GPR55-positive spots alone were reported red.
At the conclusion of some experiments, sister cultures to those assessed by image analysis were evaluated first with the viability dye alamar blue. Because of our interest in mitochondria, we noted with interest the suggestion (Iuchi et al. 2019) that the alamar blue dye “mainly” consisted of an assessment of succinate dehydrogenase reductive activity, an enzyme that participates in both the electron transport chain and the citric acid cycle. The preferred dye was alamar blue as this assay was conducted first and then the dye washed off the cultures for subsequent fixation and follow-up immunocytochemical assays. On every plate, wells without cells were used to provide a blank reading that was used to subtract background fluorescence. For the alamar blue viability assay, 10 ul of the dye was added directly to the culture well that contained 100 ul of nutrient medium. Incubation times with the dye ranged from 2-4 hours. Fluorescence was measured at an excitation of 530nm and an emission of 590 on a Cytofluor plate reader.
A commercial assay for b-arrestin was obtained from Eurofins that provided a means of testing human GPR55 in a cell line (93-024C2) that had a background of CHO-K1. In this assay, agonist-induced activation of GPR55 stimulated binding of b-arrestin to the Pro-Link-tagged GPCR and forces complementation of two enzyme fragments that resulted in the formation of an active b-galactosidase enzyme. The Pro-Link fragment of b-gal was a low affinity enzyme donor that was stably expressed with b-Arrestin tagged with an enzyme acceptor. This interaction leads to an increase in enzyme activity that was measured using chemiluminescent PathHunter detection reagents. For our application, the GPR55 agonist lysophosphatidylinositol (LPI) was tested from 0.1 nM to 30 mM to increase the relative luminescent signal relative to that of control. Incubations were conducted for 90 min at 37 degrees C in 5% CO2. Lysophosphatidylinositol (LPI) produced a robust and reproducible signal at 10 mM in this transfected system. For the GPR55 antagonism assay, KLS-13019 concentrations ranging from 0.1 nM to 30 mM were pre-incubated with the cells for 10 min prior to the addition of 16 mM LPI. Concentrations of KLS-13019 ranging from 0.1 nM to 30 mM were tested alone for possible effects of agonistic activity on b-arrestin. The data indicated that KLS-13019 had no detectible agonist activity in the b-arrestin assay.
All statistical comparisons were made by ANOVA, with normality of values tested by the Shapiro-Wilk test followed by a multiple comparison of means test with the Holm-Sidak method as performed through Sigma plot 14. All EC50 and IC50 values were generated by the curve-fitting procedure provided by the 4-parameter logistic analysis.