4R-Cembranoid Treatment Alters Gene Expression of RAW264.7 Macrophages in Basal and Inflammatory Conditions

Inflammation is considered an important target for stroke therapy because it induces secondary brain damage after the initial ischemic insult. Peripheral monocytes migrate to the brain parenchyma after a central insult. They then differentiate to macrophages in a positive feedback fashion contributing to damage instead of ischemic resolution and inflammation control. A cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R), decreases neurodegeneration after ischemia with central anti-inflammatory activity. This study aims to determine whether the central anti-inflammatory effect of 4R is effective against peripheral inflammation triggered by brain ischemia. To investigate the anti-inflammatory effect of 4R, we treated macrophages with lipopolysaccharide (LPS) as an inflammatory model, followed by treatment with 4R. Microarray transcriptome analysis of over 30,000 genes identified the differential expression of 393 genes. Genes related to inflammation, cell adhesion, and Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 16 April 2020 doi:10.20944/preprints202004.0278.v1 © 2020 by the author(s). Distributed under a Creative Commons CC BY license.


Introduction
Stroke is medically defined as a disease that affects arteries conducting to or in the brain, affecting the proper supply of blood. It is the second leading cause of death and adult disability worldwide [1]. Neuronal death initiates glutamate excitotoxicity that causes a toxic effect on the surrounding tissue, propagating an inflammatory signaling cascade responsible for increasing the ischemic damage caused by stroke [2][3][4][5]. A few days after the initiation of ischemia, brain inflammation causes the recruitment of peripheral monocytes, which are responsible for regulating the transition from acute inflammation to chronic inflammation [6]. The inflammatory factors produced by macrophages influence the activation, migration, and effector function of partner inflammatory cells by changing the inflammatory response, either in the resolution of inflammation or the propagation of tissue damage [7].
Macrophages are essential producers of cytokines, chemokines, and adhesion molecules, as well as other inflammatory factors that are critical signaling mechanisms for host defense from pathogenic infection to traumatic injuries [8].
Unfortunately, effective drugs that target chronic inflammation in the central nervous system (CNS) have not yet been discovered. Therefore, there is an urgent need to identify new safe molecules that can modulate central inflammation after an ischemic insult. Peripheral immunity depends on a set of signaling and adhesion 3 of 28 molecules such as integrins and cell adhesion molecules (CAMs) that permit the infiltration of macrophages into the affected tissue.
A cyclic diterpenoid, (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol, termed 4R, has been found to provide CNS protection in neurodegenerative diseases [9,10] and exposure to an analog of sarin, the neurotoxic gas [11]. Moreover, 4R has been shown to decrease expression of intercellular adhesion molecule 1 (iCAM-1) and vascular cell adhesion molecule 1 (vCAM-1) in a murine brain-derived endothelial cell line, bEND5, that serves as a blood-brain barrier model under normal and pathologic conditions such as stroke, Parkinson's disease, and inflammation [9,10]. The direct central neuroprotection and decreased reactivity of astrocytes, which can be considered a decrease of inflammation [12], provided by 4R is triggered by processes that involve the Akt pathway [11]; however, little is known about the modulatory mechanisms of peripheral inflammation. Olsson (1993) reported that 4R inhibits prostaglandin synthesis in seminal bovine microbiomes with an IC50 of 390μM [13], supporting the notion that 4R possesses the capacity of modulating inflammation. These changes in the inflammatory microenvironment will influence the activation of astrocytes, as well as the recruitment of the peripheral immune system. In this study, we examine the peripheral inflammatory modulatory capacity of 4R and its role in the adhesion of macrophages to human brain microvascular cells, a model of the blood-brain barrier, thus expanding and reproducing its effect in bEND5 endothelial cells [9]. non-essential amino acids (100x, Thermo Fisher formulation) and 2mM L-glutamine] and maintained at 37°C with 5% CO2. The medium was changed every 2-3 days, and cells were passed using a cell scraper before they reached 95% confluence. For experiments, 2x10 6 RAW264.7 cells were plated in 60 mm tissue culture-treated dishes and left overnight to recover. Macrophages were treated with 5μM of 4R (stock 20mM), 0.025% polyethylene glycol/ ethanol at a ratio of 90%/10% v/v, respectively (PEG/Eth ), and/or 100ng/mL LPS (stock 0.1mg/mL).
Then the cells were washed with cDMEM twice and treated with 5μM 4R or vehicle (0.025% PEG/Eth) for 1hr. The treatment was followed by two washes with cDMEM, and cells were left to rest for 2hrs in fresh cDMEM, allowing time to respond to the given stimuli. After treatment, cells were collected using the cell-scrapper and pelleted by centrifugation at 500 x g for 10minutes. The cell pellet was stored at -80°C. Samples were processed for comprehensive transcriptome analysis at the University of Puerto Rico Medical Sciences Campus (San Juan, PR, Core Facility) using the GeneChip WT Pico Kit (902622; Affymetrix) and the GeneChip Mouse Transcriptome Pico Assay 1.0 (902663; Affymetrix).

Ingenuity Pathway Analysis (IPA)
For the analytical section, data processing and differential expression analysis were performed using the Bioconductor software packages oligo and limma. The microarray raw intensity values were processed in the Affymetrix CEL file format using the Robust Multi-array Average (RMA) algorithm from the oligo package.

Real-time quantitative PCR
Total RNA was isolated from samples treated according to the LPS ± 4R microarray timeline ( Fig. 1)    Quantitative PCR was performed in duplicates in a total volume of 20μL containing 100ng of RNA reverse-transcribed to cDNA, 10μL of SYBR Green PCR Master Mix (Applied Biosystems), and 300nM primer concentration. Cycling conditions for the quantitative RT-PCRs were as follows: 95°C for 10 min, followed by 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds. Analysis of the melting curves demonstrated that each pair of primers amplified a single product. Relative changes in mRNA expression for the genes were assessed by the 2 −ΔΔCt method using the corresponding mRNA expression for vehicle-treated samples (PEG/Eth, and LPS+PEG/Eth) as a calibrator (set to a value of 1). Samples were classified into two groups of basal and LPS stimulation, and then an unpaired t-test was performed for the samples for each gene and group using Prism7. Table 1. Primers used for qRT-PCR analysis. according to the manufacturer's protocol. ImageJ software was used to detect the intensity value reading for each sample within the sample blot. Using Prism7 samples were classified and divided into groups of basal drug treatment and LPSstimulated macrophages, then an unpaired t-test performed detected any changes in protein intensity.

Adhesion Assay
The hCMECD3 human cerebral microvascular endothelial cells were incubated for 24hrs with 100ng/mL LPS incubation. After 24hrs, 4R (5μM) or PEG/Eth (0.025%) was added for the remaining 24hrs period. The 4R-treated cells were stained with

4R treatment alters gene expression in murine macrophages responsible for canonical pathways involved in inflammation and cell survival.
Treatment with 4R after inflammatory treatment with LPS regulates an array of genes involved in mediating select cell responses, including inflammation, viral protection, and proliferation when compared to the vehicle groups, PEG/Eth, or LPS + PEG/Eth. A total of 393 genes (302 up-regulated and 91 down-regulated) were identified and color-coded in a volcano plot (Fig. 3A-B). Genes that were not regulated by 4R treatment are shown in black, and these are considered natural variation within cell samples and not due to the treatments.
Of the 302 genes that were upregulated, 33% are associated with inflammation, implying activation of the pathways involved. Of the 91 down-regulated genes, 86% and cell migration (CXCR4 and S1PR1, respectively). Specific fold changes for selected genes are shown in Table 2. Notably, post-inflammatory 4R-treatment upregulated genes involved in inflammatory responses in peripheral macrophages after LPS. However, this did not translate into the propagation of cell damage in previously published in vivo experiments [9][10][11]14,15]. The post-inflammatory response to 4R could mean that 4R modulates inflammation in a manner that provides additional immediate support, but not prolonged enough to produce damage. Also, it strongly suggests that the inflammatory mechanism of action is through a signaling pathway that includes NF-kB. Under basal conditions (Fig. 1) (Figs. 3A and 4A). Only iNOS expression was up-regulated 2-fold by 4R treatment for 1hr (Fig. 5A). Induction of mRNA for all genes was evident after LPS stimulation for 2hrs before drug treatment when compared to basal conditions (Table S1-

B.
In contrast, 6hrs stimulation of macrophages with 4R alone decreased mRNA of IL-1Beta (FC= 0.74±0.073, mean±SEM) when compared to the PEG/Eth group; the remaining genes showed no differences in mRNA expression (Fig. 5A).

Simultaneous treatment with 4R and LPS promotes an early increase in the activation of NF-kB through the Ser536 phosphorylation of p65
Detecting phosphorylation of p65 at the TAD site indicates that p65 has moved to the nucleus and has begun gene transcription. The expression of phosphorylated Ser536 p65 was not significantly different from vehicle PEG/Eth under basal conditions (p65 Ser536 116.1±15.82%, compared to the mean value for PEG/Eth normalized to 100). Stimulation with LPS for 1hr increased overall protein expression, and co-incubation with 4R enhanced the increase (120.7±7.77%, mean±SEM vs. control) (Fig. 6A). Total p65 protein was significantly increased under basal conditions with 4R treatment for 6hrs (p65 total 4R mean±SEM, 113.9±5.003%) when compared to the vehicle-treated group. In contrast, total p65 was unchanged when incubated with LPS ± 4R or vehicle for 6hrs (total p65 =106.3±7.911% vs. normalized control, mean±SEM) (Fig. 6B) for the p65 Ser536 and p65 total for each treatment were divided to determine the ratio of activation for each treatment. Under basal conditions, 4R treatment did not modulate the ratio of p65 phosphorylation (p65 ser536/total p65 after 4R, 104.7±20.72%, mean±SEM), but the ratio of phosphorylated ser536 to p65 did increase following LPS stimulation with 4R treatment for 6hrs (mean±SEM of ratios of normalized values, 126.9±11.07%) (Fig. 6C).
The 4R compound has been shown to modulate α4β2 and α7 nicotinic receptors activity [16]; these receptors are known to have a downstream activation effect on p50/p65 activation. Additionally, LPS activates toll-like receptor 4, ultimately activating the NF-kB pathway. Therefore, the simultaneous activation of these pathways may explain the increased expression of the p65 Ser536 phosphorylation in macrophages co-treated with 4R and LPS (Fig. 6D). Activation of this pathway is known to lead to an inflammatory response of macrophages.

4R decreases the adhesion of peripheral murine macrophages to the hCMEC/D3 cells, a model of the blood-brain barrier (BBB) after 48hrs of LPS stimulation.
Macrophage cells are key modulators of inflammation in the host. However, when it comes to CNS inflammation, they have to adhere, and then infiltrate the BBB to mediate any injury. Excess peripheral infiltration of macrophages to the CNS will prolong and disseminate damage in the brain. In the presence of an insult, the BBB increases in adherent proteins that act as anchors for peripheral immune cells, and this phenomenon also occurs in macrophages via integrins and cell adhesion molecules [17].
Validated 4R promoted downregulation of ITGB5 for microarray samples, indicates a modulation in macrophage tissue invasion at a genetic phase. To test the functional effect of 4R on macrophage adhesion, DiI + stained macrophages were exposed to hCMEC/D3 cells treated for 48hrs with LPS or left in basal conditions. After the first 24hrs of inflammation insult, 4R or PEG/Eth was added for the remaining 24hrs (Fig.   7A). Macrophages that remained adhered to hCMEC/D3 after intensive washes were counted, and the overall ratio was determined as adhesion. Basal conditions showed no change in adhesion of DiI + stained macrophages when comparing 4R and PEG/Eth groups (macrophages per DAPI, 0.12±0.015, and 0.17±0.02, respectively, mean±SEM) (Fig. 7B). As expected, incubation with LPS and PEG/Eth increased the adhesion of DiI + macrophages to hCMEC/D3 cells (0.36±0.04 macrophages/DAPI mean±SEM). The co-incubation of 4R with LPS significantly decreased the adhesion of macrophages compared with incubation of LPS with PEG/Eth (adhesion/DAPI, 0.23±0.03, mean±SEM) (Fig. 7C), to a level similar to control conditions (control adhesion, 0.21±0.02, mean±SEM).

Discussion
These studies demonstrate that 4R, a neuroprotective compound that easily crosses the BBB and accumulates in the brain, can be used as a lead compound for future anti-inflammatory therapy after an ischemic stroke. We have shown that 4R decreases macrophage adhesion to a cell model of the blood-brain barrier following inflammatory stimulation. 4R also downregulates NF-kB. Currently, there are no therapeutic measures that target the post-ischemic inflammatory response.
However, several agents are under clinical studies, and some already licensed to treat other inflammatory diseases are being evaluated for their capability to reduce inflammation after brain ischemia.
After brain ischemia, the live neurons in the penumbra tissue suffer from deleterious signals, deteriorate, and die. The damaged or dead neurons release a series of inflammatory and death factors such as caspases and molecules, which change the microenvironment to initiate a local immune response from microglia and astrocytic cells. Local immunity will target the inflammatory insult via the production of more signaling molecules, phagocytosis, and the activation/recruitment of peripheral immunity [18][19][20][21]. Local inflammation induces a breakdown in the blood-brain barrier to permit peripheral leukocytes to migrate into the brain parenchyma. Primary macrophage and neutrophil infiltration commence a series of responses that contribute to a preliminary protective effect in the brain. However, studies have shown that prolonged activation of macrophages proves to be detrimental to the brain by the engulfment of healthy neurons [22,23].
4R is a natural cembranoid derived from the tobacco leaves and flower. It does not have a noxious effect when used in rats up to 98mg/kg [16]. Olsson and colleagues (1993) tested the anti-inflammatory effect of 4R on the activity of cyclooxygenase type two (COX2) in seminal bovine vesicle microsomes [13]. A decrease in COX2 activity was produced by 4R at a concentration of 397μM, considerably higher than the EC50 at 0.8μM at which 4R induces neuroprotection in ex vivo hippocampal slices [24,25]. In the present work, treatment with LPS and 4R at 1.53μg/mL (5μM) differentially regulated close to 400 genes related to canonical pathways from the cell cycle to inflammation. Microarray analysis determined that among the inflammatory canonical pathways, genes associated with the NF-kB signaling pathway were positively modulated by 4R treatment.
The NF-kB family is comprised of 5 subunits (RelA/p65, cRel, RelB, p50, p52), forming dimers that are generally inactive before post-translational modifications (PTM). The PTMs lead to conformational changes and detachment of inhibitor of kappa B proteins (I Bα, IkBß, IkBε, Bcl3, and IkBζ), permitting nuclear translocation and transcriptional activity [26]. The NF-kB cascade transcribes genes for diverse cell functions ranging from apoptosis, proliferation, survival, adhesion, invasion, and inflammatory responses [27]. Among the dimers formed, the most abundant and directly related to inflammation is the p65/p50 subunit found in different cell types and hosts [26,28]. In this study, 4R treatment showed gene modulation that correlates with those transcribed by the p65/p50 dimer, the predominant dimer that mediates inflammation [29], and genes directly involved in the NF-kB pathway, such as the upregulation of the NFKB1 gene.    Post-inflammatory 4R treatment upregulates genes involved in inflammatory responses of macrophages. However, this did not translate in damage propagation when using in vivo experiments previously published [9], suggesting that 4R modulates inflammation in a manner that provides additional immediate support, but not prolonged enough, validated by the adhesion of macrophages to the BBB.
Inhibition of the NF-kB signaling cascade, inhibits the adhesion and chemoattraction of monocytes/macrophages [12], determining that any modulation of the NF-kB pathway will affect the infiltration of peripheral macrophages. However, the confirmed 4R treatment increase of p65 activated form, translates to a reduced adhesive capacity of macrophages to the BBB after an inflammatory insult, suggesting that 4R minimizes the invasion of macrophages to the CNS. Reducing the immune cells readily activated and providing a wave of inflammatory molecules could help the CNS restore homeostasis by not prolonging the inflammatory response in ischemic stroke. Direct neuroprotection by 4R in stroke provides an immediate benefit for the host [9], and now there is evidence that suggests that this protection can also include a decrease of inflammation.