Macrophages and Evil Rubber Chickens

Hello everyone, welcome to the Solis Lab! This summer, we are working on understanding inflammation and potential factors affecting it. Inflammation is known to play a role in the onset of many diseases, such as Alzheimer’s disease, diabetes, and cancer. In order to understand the role of inflammation to a greater extent, it is essential to closely examine macrophages. Macrophages are differentiated cells that eat dead cells and bacteria, and are typically responsible for driving the inflammatory response.

We work with a mouse leukemia cell line called RAW264.7, and we have two ongoing projects this summer. Rose is working on understanding how macrophages respond to light and learning if/how it might play a role in the regulation of inflammation. Bryce is working on learning about the impact of heavy metals on inflammatory responses.

In addition to the work in lab, our workday typically includes a game of Wordle and Dr. Solis showing off his dabbing skills. Our work is strictly supervised by 6 miniature evil rubber chicks residing in an undisclosed location within the lab.

Meet The Team

Dr. Angel Solis

What do you find to be the most challenging part about research?

Most experiments will fail, either because you added something at the wrong time, or you misread an instruction, or most commonly, because it does not support the hypothesis you had. It can be demotivating to set up another experiment when your results are confusing or the hypothesis you were really excited about that explains all your data turns out to be incorrect. The only thing you can do is remind yourself that even when you disprove your hypothesis, you’re still learning something relevant, and hope your next experiment will be more clear!

What motivated you to become a scientist?

When I was in middle school, I was diagnosed with Crohn’s disease. That made me read as much as I could about what Crohn’s disease was, and why it was happening to me. Learning about all the different pathways that became activated in the cells, and how they can become dysregulated was so beautiful to me, and it made me want to learn as much as I can about cells and the molecules they had. During this time, I read a lot about the immune system as well. Reading about inflammation and immune cells was (and still can be) quite confusing, but still utterly mesmerizing.

What is your favorite amino acid?

(Incredulously) Lysine, obviously!

What type of bear is best?

That’s a ridiculous question.


Rose Ankeriasniemi

I am an international student from Finland, a member of class of 2024. My major is Biology and minor is Educational Studies. In my free time I like to do jigsaw puzzles, bike, play the violin and practice badminton with my friends. Right now I am working on the fifth puzzle of the summer; it’s a 1000-piece puzzle featuring a bookcase with cats playing on the shelves.

What motivated you to become a scientist?

I come from a country that has the highest rate of type 1 diabetes in the world, and a handful of my loved ones are affected by it. I myself have an autoimmune condition called localized scleroderma, which is caused by inflammation in certain parts of the skin. The exact causes of many autoimmune diseases, like T1D and scleroderma, are still unknown, and although we have a good idea of how the immune system works (or doesn’t), we often don’t know what has initiated such a response. I have always been invested in understanding the immune system; focusing on inflammation in particular is an excellent approach to learning more about the pathogenesis of certain conditions, because many conditions involve some level of inflammation. Working in the Solis lab has provided me with a way to discover my intellectual curiosity, and I look forward to continuing my work in the lab beyond this summer.

What do you wish was illegal?

Bullying or laughing at someone’s expense is never OK. Not at school, not at home, not at work. You never know what the other person is going through, and not everyone shares the same sense of humor. Please check in with your loved ones and make sure they know you care about them.

What do you value the most?

I am eternally grateful for the friendships I have developed over the years. I met some of the kindest people in high school when I first came to the USA. There were a few other new *international students at my school, whom I connected with immediately. It hits you differently when you are experiencing a culture shock, and you have someone to share those challenging feelings with.

*Feeney, if you are reading this, I love you! You’re famous now 😊

What are your two truths and a lie?

1. My dad has my name and birthday tattooed on his shoulder

2. I can solve the Rubik’s cube

3. I absolutely loathe seafood and cilantro

Good luck guessing 🙂


Bryce Weigartz

The Name is Bryce William Allen Weigartz and I’m from Gloucester Virginia, which is an hour south of Richmond. I am technically a rising senior who will be living in the depths of Apple with 5 other fellas come the fall/spring. As a guy with a major in Biology, and a minor in Chemistry, I guess you could say I’m a fan of learning. My other interests include the following: sleeping, showering, eating, gym, tennis, pottery, wood turning, reading obscure books on mechanics, bicycling (with the one wheeled variant), not cooking, tree climbing, piano, video games, and YouTube.

What has been the most injured you have ever been?

It’s a toss-up between having appendicitis in the third grade and being in a tree stand that fell and breaking my whole wrist/arm along with lacerating my spleen.

What has your diet consisted of during XSIG?

I keep it simple with plain chicken in a pan, maple oatmeal, chocolate bars, milk, water, mac n’ cheese, cheese crisps (a Weigartz classic) Pepsi, yogurt, and eggs. This is/has been my diet. I went home the other weekend and brought back actual food, a meatloaf and barbecue smoked pig butt, and it was stupendous.

What possession of yours is the most valuable?

Monetarily it is either my Stihl ms250 chainsaw with the bars and chains or it is my Hp omen laptop, in this market I don’t know which is worth more. To myself however, my most valuable object is my ”2-door hatchback 1996 Ford Escort Lx painted in the wonderful bright Calypso Metalic Green”. Her name is Hellen, after the Trojan’s, because this is a beauty to goto war over (look up what I put in quotations and, you’ll be forced to agree with me). I would say that this was my most valuable possession monetarily, but apparently the government estimates the value of this American Classic at a measly 500 pieces as per my property tax letter I got when I last visited home.

Glossary

  • Incubator: a closed cabinet-like space for cells to grow in where the temperature and CO2 are controlled for optimized cell growth
  • Cell culture media: cell food (contains glucose, glutamine, calf serum and pen strep)
  • Macrophage: a differentiated cell that eats dead cells, bacteria, and mediates parts of the immune response
  • ELISA: a technique to measure the amount of proteins or other substances
  • Cell plate & wells: a flat plastic piece that has indents on it where the cells and cell culture media are placed in for cells to grow
  • LPS & Poly I:C: substances that initiate an immunological response. We use these substances to activate our cells, which results in a production of cytokines
  • Cytokines: cytokines are produced by macrophages to act as signals for the rest of the immune system to recruit more immune cells when an invading pathogen is detected
  • Pathogen: a virus, bacteria or other organism that is harmful to the body and can cause disease
  • Antibody: a protein in blood produced in response to foreign substances like a pathogen

Techniques In the Lab

ELISA (Enzyme-linked immunoassay)

Cytokines are indicators of inflammation, and to quantify the cells’ immune response, cytokines can be analyzed. They are produced by the cells (macrophages) and their presence can be tested for with ELISA from the culture media in which the cells are growing. ELISA is a multi-step process that uses antibodies and other molecules to bind to the cytokines, eventually indicating the amount of cytokines present through the strength of a colored reaction product.

We use 96-well plates for ELISA (see below what it looks like—it’s a lot of tiny wells!!), where one well is dedicated for one sample. Below on the left is an illustration on how the cytokine is detected in each well. The more cytokines there are in the sample, the more of these structures are present, which leads to a greater production of the colored product in that well. The absorbance of each well is then read on a spectrophotometer. The data is compared to standard proteins, which we always include on the plate. The process from harvesting cells to collecting the samples is described in detail in Rose’s light project section.

Isolation of RNA

One might isolate RNA from cells to learn how well the cell performs its job. Rose has been isolating RNA from her cells that have been growing in the dark and the light environments. Specifically to compare the two environments, her cells are divided into small wells on plates after which those plates are placed in an incubator overnight for cells to acclimate. The next day, the cells are treated with immunological response initiators (LPS and Poly I:C). The plates are then placed in the corresponding light treatment in incubators. We specifically used an LED light taped in the incubator. After six hours, the plates are taken out of the incubators and the cell culture media is discarded with a pipette. The cells are lysed with TRIzol reagent, after which the liquid from the wells is pipetted into tubes. In short, the mixture is separated by polarity using chloroform, the RNA precipitate is formed by presence of isopropanol, and the formed RNA pellet is resuspended in water and then heated up on a heat block.

CRISPR

The CRISPR method is used to delete a specific gene from the cell line DNA, and a knockout (KO) cell line is created by targeting the macrophages with plasmids. An inflammatory response is induced with the KO cell line, and the aspects of the response are measured by qPCR and ELISA. For instance, if a KO cell line that is missing a gene called RABGGTB presents a different inflammatory response from cells that have the gene, it can be suggested that the gene RABGGTB plays a role in inflammation. In that case, we would want to learn about the function of the gene in order to understand its exact role in inflammation.

Western Blot

A western blot is very similar to the aforementioned ELISA, but where an ELISA detects proteins outside of the cell, a western blot detects proteins WITHIN the cell. The process in our lab is a 3 day one. First the media the cells are grown in is discarded, leaving the cells stuck to the bottom of the plate. Then the cell membrane gets popped open by a solution called RIPA. Once this occurs, the cells are centrifuged down to get rid of any cellular debris, leaving, among other things, proteins that can then be targeted. the samples are loaded into a gel which then has a current applied to it that draws the proteins down through the gel. All of the proteins end up separating out by size within this gel. From here the proteins are transferred over to an easier to handle membrane, where they are then stained for whatever protein you are looking for. This staining is done through the use of protein specific antibodies, which bind to the protein. The result of this process is a membrane that, hopefully, has a few marks where you expect to have seen the protein you stained for.

Rose’s Light Project

The role of light in inflammation can be interpreted as a natural stimulant; when one gets a wound, light has easier way to access the contents under the skin when compared to the gut, for instance. In my project, I am specifically interested in learning the response macrophages produce in response to light. Light therapy is commonly used to treat conditions involving inflammation, such as rheumatoid arthritis and depression; light has been shown to reduce the amount of proinflammatory cytokines, reducing inflammation and furthermore the symptoms associated with these conditions. The goal of this project is to eventually identify new genes that are responsible for mediating macrophages’ response to light.

What are macrophages?

In short, macrophages are immune cells that get activated to kill pathogens. Some of the common ways for macrophages to get activated are ‘communication’ with a T-cell and the presence of cytokines. Once activated, macrophages are able to recognize the pathogen that needs to be destroyed. Macrophages also produce more cytokines to mediate the inflammatory response. Cytokines act as signals for the rest of the immune system to recruit more immune cells when an invading pathogen is detected. When macrophages are activated by exposing them to light (and LPS & Poly I:C), we would expect them to release more anti-inflammatory cytokines, or less proinflammatory cytokines when compared to the dark environment, our control. This hypothesis is based on the use of light therapy to reduce the production of proinflammatory cytokines during low-grade inflammation.


Now I explain to you what the process of detecting cytokines produced by the macrophages actually looks like. This experiment specifically involved a drug called vardenafil, which I will talk more about in another section. The macrophage cells are grown on petri dishes with cell culture media (cell food). After a few days of letting the cells sit in the petri dish in an incubator (a warm place for cells to grown in), a small portion of the cell population on the petri dish is transported onto two new dishes for more space to grow. The leftover cells are placed onto 12-well plates: each well has 0.5 million cells and 1 mL of red cell culture media:

The two new petri dishes were placed in an incubator overnight to give the cells some time to acclimate to their environment. The next morning, two of the plates were treated with vardenafil (the drug I was testing for) and the plates then sat in the incubator for 30 minutes to again acclimate for the presence of the drug.

I took those two plates out, in addition to two other plates I had prepared the previous day and treated one row of wells in each plate with LPS and one row with Poly I:C (remember, these substances activate the immunological response in the cells, which causes a production of cytokines). 2 plates, vardenafil-treated and non-treated, were placed in the same incubator the cells spent the previous night, and the other 2 plates were placed in another incubator that had a LED light taped above the plates as seen in the image below.

After 6 hours of sitting in the incubators, the cell culture media (the red stuff, cell food) is carefully taken out of the wells into microcentrifuge tubes by pipetting. At this point, the live cells are stuck on the bottom of the wells, but the cytokines they produce are in the cell culture media. The tubes are centrifuged to push all the extra stuff we don’t need anymore, like dead cells, to the bottom of the tube forming a separate ‘pellet’. The liquid is then taken out of the tubes by pipetting and placed in -20 degrees celsius. ELISA is performed on the samples to determine the cytokine concentration in each sample, and the ELISA results are analyzed on Excel. The absorbance of each sample is compared to the absorbance of the standard proteins. Because we know the concentration of cytokines in the standard proteins, we can extrapolate the concentration of cytokines in each sample.

Rose’s ELISA results for illustrative purposes

TNF and IL-6 are both proinflammatory cytokines. The hypothesis of these light experiments was that with exposure to light, macrophages produce less proinflammatory cytokines. Below are selected results from this experiment. It is evident that proinflammatory cytokine production was not decreased with exposure to light, which does not support our hypothesis. Vardenafil treatment seemed to reduce the production of TNF, but it had the opposite effect on IL-6. LPS and Poly I:C both activate the immunological response; we juts wanted to see which one might be more efficient to work with in these experiments.

Vardenafil is a drug that inhibits a cascade that uses light to moderate the transmembrane Na+ channels. I experimented with this drug to learn if the phototransduction cascade of the cell can be disrupted and what that would look like in presence and absence of light. Below is an illustrative diagram of what that cascade looks like; In short, light stimulation causes PDE to break down cyclic GMP (cGMP) into GMP. Reduced concentrations of GMP cause the sodium channels to close. Vardenafil inhibits PDE, therefore inhibiting the closure of the Na+ channels.

(Neuroscience, Purves et al., 2001)

Why is this research important?

Some genes are known to show a greater prevalence in inflammatory macrophages, although their function is still unknown; knowing the specific function of these genes will help us accept the role and significance of inflammation on human health to a greater extent. I will keep working on this project during the academic year, and ultimately I want to learn how light affects macrophage gene expression that results in an inflammatory response. I have identified genes of interest from literature as well as from a large data set based on their prevalence in neutrophils and macrophages. Primers for those specific genes are waiting in the freezer to be used in our experiments.

The CRISPR method will be used to delete a specific gene from the cell line DNA, and a knockout (KO) cell line is created by targeting the macrophages with plasmids. Right now we are optimizing volumes of puromycin we can use to initiate pyroptosis, cell death, on the cells that were not transfected with the KO DNA; we only want to have cells that have the KO DNA. An inflammatory response is induced with the KO cell line, and the aspects of the response are measured by qPCR and ELISA. For instance, if a KO cell line that is missing a gene called RABGGTB presents a different inflammatory response from cells that have the gene, it can be suggested that the gene RABGGTB plays a role in inflammation. In that case, we would want to learn about the function of that gene in order to understand its exact role in inflammation. This part of the research will be conducted during the academic year.

Bryce’s Heavy Metal Project

Light is cool and all, but whats even cooler is HEAVY METAL. Well, that’s a bit of a click bait to get you interested, in reality I’m only looking at two regular old metals, nickel and aluminum. Similar to how Rose’s main experiment is seeing if light affects the macrophage immune response, my experiment is seeing if nickel or aluminum affect macrophages immune response. I think that this path of research is important to study because with there are many possible routes in which nickel and aluminum can make it into the body. Just to name a few: cooking with aluminum foil, medical equipment/implants with nickel infused in as an anti-corrosive, or mining releasing high levels of both compounds into the air and water. Because these metals are getting into the human body through the aforementioned ways, I think it is both interesting and important to see how they may affect our immune cells.

I test the cells by starting out a 12 well plate with newly added cells, and giving them a few hours to acclimate to the new plate, around 6. Then, I bask the cells in a small amount of metal, around 10uM. The cells then get over night to acclimate again, this time to the metal environment, where then the next morning they activated by either LPS, Poly I:C or both. 6 hours after that, the plates are ready to be processed. Depending on the proteins that I want to look for, I will either collect the supernatent to form an ELISA, as seen in our glorious descriptions above, or I will lyse the cells for a Western blot, again way up there, you’ll see it I can wait whilst you re-educate if you need to.

Awesome, so for the ELISA I am looking at Il-6, TNF, Il-10, and Il-1b. Unfortunately, the Il-1b ELISA test hasn’t worked all semester, and the Il-10 doesn’t differ in treated vs non treated groups. This leaves me with either Il-6 and TNF which do interesting things. In the beginning we saw super low levels of Il-6 in nickel exposed cells, with non-significance in TNF. That was dope. We figured that since TNF was the same with the non-treated cells as the metal cells, that the cells were not dying. Boy were we wrong. We started going through this rabbit hole of experiments, specifically western blots, trying to figure out how Il-6 would go down, while TNF stays the same. We found that Nickel affected a protein called IkBz, and were ecstatic. Then I had to start thinking about the poster, and in preparation for it, me and Dr. Solis were like ” I guesssss we can do a test to make sure the cells aren’t dying or whatever”. And uh yeah, turns out the cells are totally dying. Its okay though don’t get too worried, not all is lost. I didn’t waste the whole summer. Think about it, if before we thought that Il-6 was going way down and TNF was the same, then now what we can see is that Il-6 may not be going down, but something is causing the TNF to spike up, making it appear at the same levels as nice and healthy cells, even though the amount of cells are less with nickel, because they are dead, and much more stressed out, because they are dying. The revelation happened at the end of week 6 beginning of week 7, and we are still trying to come to terms.

We still have pieces to the Nickel puzzle, it is just that before we thought we were making a nice picture of a cat during a family picnic. Whereas now, we have pieces of a cat, a basket, some foods, a family, trees, and the words “What a lovely Picnic”, but we know that it is NOT a picnic. Yeah I know sucks right, but as Dr. Solis likes to say, that’s science for ya. You think you got a nice picnic going so you start doing westerns for a pathway that would potentially reduce il-6 and not affect TNF, through a protein called IkBz, then wham, even after you find out that pathway is true and nickel does indeed influence it, you also find out the cells are dying and you are left with a bunch of broken puzzle pieces that look like they should be a picnic but you know cant be. Sad I know, but that’s Science.

Anyways, enough of that sad talk, how is lab usually? Well great question, the first 6 weeks of experimenting were all ELISAs, which was a LOT of pipetting. I am not saying I like doing westerns more, but there is only like 107 instances of pipetting, versus the ELISA at like 2400 pipettes if I do a whole plate. Unfortunately I have nothing too bad to say about the western that the ELISA doesn’t also have. Proteins, check. Lots of waiting? Check. Cold room over night? Check. Nerve wracking feeling of wondering if you messed up a 3 day protocol because you cant tell if you did the assay correct until the last 5 minutes of the entire test? Check. As you can see, they are pretty much the same, though I guess the western does have gel. One thing I can say however is at the end the ELISA gives a nice quantitative answer. See this graph of il-6 for nickel induced cells, great awesome.

 

Look how pretty that is, now compare it to this western I have looking at IkBz, a protein that is one of the few ways to decrease IL-6 while keeping TNF the same.

Just two small lil pencil lines. That’s all a Western blot gives you. I mean it was great for our hypothesize that nickel decreased the amount of IkBz, but I am now on my second attempt at replication, so here is to hoping.
To to end off this blog, I want to make a list of all the things I do when I’m NOT actively Sciencing:

  • Autoclaving
  • Talking to the Craig lab
  • And the Powell Lab
  • Plus the Meiss Lab
  • And you know, Justin
  • And my Lab mate Rose
  • Also who could forget my men tor Dr. Solis

now that I typed it out, I guess I do a lot of talking to peoples…… Oh well that’s Science, thanks for stopping by

Selected pictures from the summer

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