Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 1
Microglial Cells
Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 Microglial
Cells
Brianna Cyr
Department of Psychology
Honors Thesis
April 2019
2 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells
Abstract
Microglial cells are responsible for the immune response in the brain. When they come in contact with an entity that requires such a response, the microglia become activated and can phagocytose it. This activated state can potentially lead to neuroinflammation, if sustained for long enough. Neuroinflammation is seen in neurodegenerative diseases such as Parkinson’s Disease and Alzheimer’s Disease. Antioxidants may be able to decrease this inflammation, and an antioxidant of interest is L-carnitine.
SIM-A9 microglial cells were activated with LPS and administered L-carnitine.
We hypothesize that activated cells rely more on glycolysis than oxidative phosphorylation, ramified cells rely more on oxidative phosphorylation than glycolysis, and the cells that were activated and treated with L-carnitine rely more on oxidative phosphorylation than glycolysis. This study was not able to get usable data in order to conclude if L-carnitine is a potential treatment for neuroinflammation. Further studies need to be conducted to assess the metabolic rates of LPS and L-carnitine treated cells to determine if L-carnitine can be used as a treatment for neuroinflammation.
Introduction
Microglial cells are one of many types of cells found in the brain that have a
number of functions including immune response, phagocytosis, matrix remodeling,
and lipid transport. The function most commonly associated with microglia is immune
response in the central nervous system. The cells constantly monitor their surrounding
environment and can change their morphology depending on the stimuli they Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 3
Microglial Cells
encounter (Boche et al., 2013). The two morphologies of interest are the ramified form
and the amoeboid form. The ramified form is the typical form microglial cells are found
in. The ramified microglia survey the environment with small projections they send out.
The amoeboid form is an activated state, these cells lack processes and become
phagocytotic. It is thought that when the ramified microglia come across anything that
would constitute an immune response, it transforms into the activated amoeboid form
seen in Figure 1 (Boche et al., 2013).
Figure 1: From Gill et al., 2018. The cycle of ramified microglia transforming into its activated state and back to ramified.
It has previously been reported that there are many forms of activated microglia,
but the two forms of interest are the M1 and M2 states (Boche et al., 2013 & Orihuela
et al., 2015). Generally, M1 is thought of as a pro-inflammatory state whereas M2 is
associated with neuroprotection. When a microglial cell is activated to the M1 state, it
triggers the activation of mitogen-activated protein kinase (MAPK), causes the 4 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells
upregulation of inducible nitric oxide synthase, and the secretion of pro-inflammatory
cytokines and reactive oxygen species (ROS) (Bhat et al., 1998). The pro-
inflammatory cytokines are released by activated microglia, the cytokines can also
cause microglia to become activated therefore perpetuating the activation (Orihuela et
al., 2015). In contrast, the M2 state is characterized by the expression of heparin-
binding lectin, cysteine-rich FIZZ-1, and arginase 1 (Freilich et al., 2013). The M2
phenotype is also thought to help bring the M1 phenotype back to a ramified state
(Orihuela et al., 2015). It can release anti-inflammatory factors to resolve inflammation
and re-establish homeostasis.
In many neurodegenerative diseases an increase in neuroinflammation is
observed. This neuroinflammation can be caused by an abundance of activated
microglia. The neurodegenerative disease of interest for this study is Parkinson’s
Disease (PD). Symptoms of PD include tremors, dystonia, ataxia, and cognitive
impairment. In PD, there is chronic inflammation and the release of pro-inflammatory
cytokines which causes death of neurons in the substantia nigra pars compacta
(Long-Smith et al., 2009). There is ultimately a loss of both acetylcholine and
dopamine, both of which help to inhibit tumor necrosis factor alpha and the
phosphorylation of MAPKs (Shytle et al., 2004). As mentioned previously, MAPK is
activated when microglial cells are in the M1 activated state. Microglia can be
activated by administration of many different agents such as complement 3,
fractalkine, and phosphatidylserine but lipopolysaccharide (LPS) which is a
neurotoxin, is commonly used for this purpose (Nagamoto-Combs et al., 2014, Peña-
Ortega, 2017, Orihuela et al., 2015; Gill et al., 2018, Wang et al., 2019). LPS binds to Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 5
Microglial Cells
4 receptors: CD14, scavenger receptor A, Toll-like receptor 4, and complement receptor 3 (Peña-Ortega, 2017). LPS mainly acts on the Toll-like receptor 4 and there is evidence that this receptor is only on microglia or due to the presence of microglia
(Peña-Ortega, 2017). This means that the confidence that the activation of microglia is higher than if another agent was used and why LPS was chosen over other agents.
Literature has shown that microglial cells that have become activated modify their primary course of producing energy (Wang et al., 2019). Ramified microglia predominantly use oxidative phosphorylation for energy production and activated microglia predominantly use glycolysis. Specifically, there is an increase in the upregulation of the glucose transporter 1 and the glycolytic capacity when microglial cells become activated (Wang et al., 2019). Since neuroinflammation is a factor in the death of neurons, it may be possible to treat this issue by helping the microglia revert back to their ramified state via the use of antioxidants.
Antioxidants have been used to decrease neuroinflammation for a variety of diseases (Verlaet et al., 2018; Langley et al., 2017; Yang et al., 2016). Antioxidants scavenge ROS and free radicals. A particular antioxidant of interest is L-carnitine. L- carnitine is a substance that is biosynthesized but also supplemented dietarily (Gulcin,
2005). It plays a role in carrying long chain fatty acids across the mitochondrial membrane for beta-oxidation and then carrying products (such as acetyl-CoA) from beta-oxidation to enter the citric acid cycle. L-carnitine also has a number of antioxidant effects including reducing power, superoxide radical scavenging, hydrogen peroxide scavenging, and metal chelating activities (Gulcin, 2005). In addition, it can prevent free radical formation through inhibition of certain enzymes (Gulcin, 2005). L- 6 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells
carnitine also has other forms, one of which is acetyl-L-carnitine. This form of carnitine
is found primarily in the brain and helps the transfer of acetyl groups for acetylcholine
synthesis (Ribas et al., 2013). L-carnitine can convert to this form in the brain through
a reversible acetylation reaction. A previous study has shown the effects of direct
administration of acetyl-L-carnitine into rats who have been administered LPS to have
a positive effect on reducing the number of activated microglia (Kazak & Yarim, 2017).
SIM-A9 microglial cells are an immortalized cell line that spontaneously
developed from a primary murine culture and behave the same as primary cultured
microglia (Nagamoto-Combs et al., 2014). These cells were found to express
microglia-specific proteins and variations in morphology, produce cytokines and nitric
oxide when stimulated with either LPS or tumor necrosis factor alpha, display
phagocytotic activity, express either M1 or M2 phenotypes, and generally retain the
microglial characteristics after many passages (Nagamoto-Combs et al., 2014). These
characteristics allow us to activate the cells to the M1 state with our chosen agent of
LPS, produce its normal factors when in the activated state, and are able to change
morphology back to ramified or to the M2 state. These cells are effective in testing our
hypothesis and allow us to have a better level of translation as opposed to virally
transformed microglia.
Our lab previously published a study investigating the effects of LPS and L-
carnitine on SIM-A9 cells (Gill et al., 2018). In this past study, SIM-A9 cells were
effectively activated (Figure 2) with 2.5 g/mL of LPS and concentrations of 10 and 15
mM of L-carnitine had a significant effect on the cells (Figure 3). Activation was
recorded based on nitric oxide production measured by a Greiss analysis. There was Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 7
Microglial Cells
a significant difference in nitric oxide production from cells activated with LPS and the
cells treated with 10 and 15 mM of L-carnitine. We can infer that L-carnitine played a
role in potentially reverting the activated cells back to its ramified form or that it
prevents the activation in the first place. The present study aims to take it a step
further and try to understand the mechanisms of how the LPS and L-carnitine are
affecting the SIM-A9 cells.
Figure 2: From Gill et al., 2018. SIM-A9 cells that (A) have been activated with LPS and (B) have not been activated with LPS. 8 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells
Figure 3: From Gill et al., 2018. SIM-A9 cells that have been pretreated with (A) 1 mM, (B) 5 mM, (C) 10 mM, and (D) 15 mM L- carnitine for 24 hours before LPS administration. Photos taken 24 hours after LPS administration.
In the current study, we aim to investigate the rate of oxygen consumption of
ramified microglial cells, activated microglial cells via LPS stimulation, and activated
microglial cells via LPS stimulation and L-carnitine treatment. To record this measure,
a Seahorse XF96 Extracellular Flux Analyzer will be used. The Seahorse analyzer has
been used in numerous studies to measure cellular respiration in a variety of cells
(Orihuela et al., 2015; Rubio-Araiz et al., 2018; Voloboueva et al., 2013; Wang et al.,
2019). The Seahorse analyzer measures the Oxygen Consumption Rate (OCR) and
the Extracellular Acidification Rate (ECAR) of the cells. OCR is an indicator of Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 9
Microglial Cells
oxidative phosphorylation since oxygen is required for this process whereas ECAR is
an indicator of glycolysis which emits protons during the process.
The assay of interest to use with the Seahorse analyzer is the Cell Mito Stress
Test. The Cell Mito Stress Test includes oligomycin, trifluoromethoxy carbonylcyanide
phenylhydrazone (FCCP), and rotenone/antimycin A in the kit. Each of these reagents
test the cells’ ability to perform oxidative phosphorylation (Figure 4).
Figure 4: From Agilent Technologies website. Diagram of what measures are obtained via OCR from the Cell Mito Stress Test.
Oligomycin inhibits ATP synthase which causes a decrease in the flow of
electrons in the electron transport chain (ETC). This causes a decrease in the oxygen
consumption which is linked to cellular ATP production. FCCP disrupts the proton
gradient and mitochondrial membrane potential causing unhindered flow of electrons
through the ETC. Oxygen consumption reaches its maximum and allows the spare
capacity to be calculated. The spare capacity is the difference in the maximum OCR 10 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells
and the basal OCR. It is a measure of the cells’ ability to respond to an increase in
energy demand or stressful conditions. Antimycin A inhibits complex 3 of the ETC and
rotenone inhibits complex 1. The combination of these two will completely shut down
the ETC and enable the calculation of nonmitochondrial respiration. The reagents will
be administered in the order listed at different time points during the assay.
Methods
Cell culture
SIM-A9 microglial cells were purchased from ATCC. The cells are passaged
every 2-3 days until they reach about 80% confluence in a T25-flask with complete
media (50/50 Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F-12
supplemented with 10% fetal bovine serum, and 5% horse serum). Cells and media
are extracted from the flask and are put in a 15 mL centrifuge tube. The flask is rinsed
with phosphate buffered saline (PBS) and cells are lightly scraped from the bottom of
the flask. The cell suspension is centrifuged for 5 minutes at 110 x g at 25C. Once
the centrifuge is completed, the supernatant is removed leaving only the pellet of cells.
The pellet is resuspended in 1 mL of the complete media. In a new flask, 250 L of the
cell suspension and 4.75 mL of complete media are added. The cells are incubated at
37C at 5% CO2 and 95% air environment.
Cellular respiration experiments
To investigate the energetic phenotype of SIM-A9 microglia, a Seahorse XF96
Extracellular Flux Analyzer will be used. The Seahorse has several assays that can be
run, each measuring some metabolic process of the cell. The assay used here is the Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 11
Microglial Cells
Cell Mito Stress Test. This assay measures OCR and ECAR which are indicative of
oxidative phosphorylation and glycolysis respectively.
Before beginning with the experiments, cell density and FCCP concentration must
be optimized. In the 96 well plate, there are four blanks: one in each corner. The cell
density was varied by row while the FCCP concentration was varied by column. This
allowed for replicates for each concentration of FCCP crossed with each density of
cells. The optimization assays included only ramified microglia. The LPS concentration
was also optimized to see which concentration was the most effective for activating
the cells without killing them entirely. The last assay includes L-carnitine and LPS.
There will be a column of LPS only treated cells followed by the same LPS
concentration and the addition of L-carnitine, which will be administered at a
concentration of 10 mM at the same time as the LPS: 22 hours before the Seahorse
assay. This concentration of L-carnitine was chosen based on the results from Gill et
al.
Cell imaging
Cells will be imaged under a Lecia DM IL microscope. The software used to capture the images on the computer is Motic Images Plus 2.0. The cell images were captured soon after the assay was completed.
Data analysis
The data we collect will be analyzed by using the Wave software from Agilent
Technologies.
12 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
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Results
Cell and FCCP optimization
In order to effectively use the Seahorse machine, cell density and FCCP concentration needed to be optimized. In the first assay, cell densities of 5,000, 10,000,
15,000, 20,000, 30,000, and 40,000 and FCCP concentrations of 0.25 M, 0.5 M, 1.0
M, and 2.0 M were tested. Both of these ranges were recommended by Agilent. From this, we looked at the graphs made from the Wave software and saw the best results from 10,000 and 15,000 cells and FCCP concentrations of 1.0 M and 2.0 M highlighted in Graph 1. These were chosen because they all fall within the suggested basal OCR of 20-160 pmol/min from Agilent for cell seeding density.
Graph 1: Each of the groups bolded fall within Agilent's suggested OCR range of 20-160 pmol/min for optimal cell seeding density. Those above the range have too many cells and those below the range have too little cells for the Seahorse to accurately measure the OCR and ECAR.
To determine which group is best since they all fall within the suggested range,
Agilent suggests looking at the groups’ basal ECAR which should be between 10-90 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 13
Microglial Cells mpH/min. As seen in Graph 2, none of the groups fall within this suggested range for the first data point so a second round of optimization was needed.
Graph 2: The first data point for basal ECAR for all of the groups is below 0. The suggested basal rate for optimal cell seeding density is 10-90 mpH/min, which none of the groups here meet with the first data point but do for the other basal points.
The second optimization focused in on 10,000 and 15,000 cells as well as FCCP concentrations of 1.0 M and 1.5 M. 1.5 M was chosen instead of 2.0 M because it was thought that the 1.5 M would be a more effective concentration than 2.0 M.
Again, all the groups are within the recommended basal OCR range (Graph 3) and this time, all the groups were within range of the recommended basal ECAR (Graph 4). 14 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells
Graph 3: All groups are within the recommended basal OCR like the previous optimization.
Graph 4: All the groups are within the recommended ECAR range.
Since these were the only two factors given by Agilent to find the best cell seeding density, the actual visualization of the cells was taken into account. The wells that had 15,000 cells looked to have the most uniformity in cell adhesion and the least clumps of cells (Figure 5). The OCR and ECAR trends for both of the 15,000 cell groups are also more similar between each other than the 10,000 cell groups.
Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 15
Microglial Cells
A. 15,000 cells, 1.5 FCCP B. 15,000 cells, 1.0 FCCP
C. 10,000 cells, 1.0 FCCP D. 10,000 cells, 1.5 FCCP Figure 5: 15,000 cells (A and B) versus 10,000 cells (C and D). There is more clumping in the 10,000 cell groups and less uniformity across the well.
LPS optimization
With LPS, there is a point in which the concentration is high enough to fully kill the cells instead of putting them in an activated state. To avoid killing the cells, the LPS concentration was optimized as well. We chose concentrations of 1.0 M, 1.5 M, 2.0
M. and 2.5 M. It was expected to see the OCR be diminished, but that was not the result of the assay. All of the groups showed normal OCR and most had higher OCR’s than the control cells (Graph 5). 16 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
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Graph 5: All of the LPS treated cells had higher basal OCR than the control cells and show normal OCR trends. This indicates that the cells in the LPS treated wells did not get affected by the LPS. According to previous literature, LPS is an effective agent to activate microglial cells and so it is thought that these results are abnormal. The unexpected results required that this experiment be repeated.
L-carnitine and LPS optimization
Since the outcome from the LPS optimization was not expected, it needed to be done again. To better utilize the plate space, we included L-carnitine in the same plate as the LPS. However, results from this run show the basal OCR is low for all groups, below the recommended basal OCR for cell density optimization (Graph 6). The groups that included L-carnitine did not follow the general trend of the OCR graph. Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 17
Microglial Cells
Graph 6: The OCR of all of the groups is abnormally low, the maximum point being below 40 pmol/min and some OCR points being in the negative range. The previous assays had maximum points of 300 and upwards. Upon further examination it was observed that there was a discrepancy of cells and a lack of sufficient cells. There was a disparity of cells in almost all of the wells and almost no cells at all in the groups that contained L-carnitine (Figure 6).
18 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
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A. 1.0 LPS only B. L-carnitine only
C. Control cells D. 1.5 LPS + L-carnitine Figure 6: Cells in all of the groups did not adhere well to the well plate.
Discussion
The ultimate aim of this study was to investigate the possible anti- neuroinflammatory mechanisms of L-carnitine on SIM-A9 cells. Microglia are the resident immune cells of the central nervous system and so constitute the first line of defense from pathogens via inflammatory signaling pathways. The inflammatory response includes the release of pro-inflammatory signals and factors such as nitric oxide, cytokines, and ROS. Microglia also serve in neuroprotective functions to regulate inflammatory conditions via promotion of anti-inflammatory factors and cell repair, Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 19
Microglial Cells thereby playing a role in destruction and safeguarding of cells. Thus, homeostasis is maintained via a feedback loop and an imbalance results in several neuroinflammatory diseases.
It has been reported by our lab and other groups (Kazak & Yarim, 2017, Ribas et al., 2013, Rubio-Araiz et al., 2018, & Wang et al., 2019) that treatment with LPS induces a proinflammatory response with the release of cytokines in microglial cells and that a subsequent suppression of the proinflammatory profile with antioxidants and small molecules can have neuroprotective effects. In a previous study, we showed that the inflammatory response of SIM-A9 cells was induced via administration of LPS and the quenching of this response from treatment with L-carnitine. In this study, we aimed to demonstrate a difference in the metabolic response of SIM-A9 in its different states in an attempt to pin down the mechanisms behind this process.
We were able to optimize the cell density and FCCP concentration for use with the Seahorse which was used to measure the OCR of the cells. Cells were plated at densities of 5,000, 10,000, 15,000, 20,000, 30,000 and 40,000 cells along with FCCP concentrations of 0.25 M, 0.5 M, 1.0 M, 1.5 M, and 2.0M. Cell density optimization was found to be 15,000 cells per well and FCCP concentration optimization was found to be 1.5 M. SIM-A9 cells were induced with LPS concentrations of 1.0, 1.5,
2.0, and 2.5 M but the treatment failed to activate the cells.
Future studies will need to be performed to ensure that LPS mediated SIM-A9 activation can be achieved reproducibly in vitro and to be able to record its metabolism.
Potential future studies could look into using different antioxidants, since they can all have different functions. There is also potential to expand this investigation by looking at 20 Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9
Microglial Cells other methods of metabolism, such as fatty acid oxidation, and see if these are affected as well. To move closer towards translatable results, animal models could be used. This model is closer to modeling what is actually happening in human brains which increases the potential for L-carnitine to be a treatment for neuroinflammation.
Understanding the Metabolic Profile of Lipopolysaccharide Activated SIM-A9 21
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