I feel like as long as that patch of lighting is there, none of this setup matters at all.
The age old questions of “Max, what did you do for your master’s?”
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It gets really technical in parts so I’m gonna keep it super basic so that it’s a
little bit fun, a little bit interesting if you’re interested in it, um, and I can put
it in a nice condensed video, because if I talked about the whole thing, it would be
an hour long lecture and nobody’s here for that. Some of this might even sound a little
bit familiar to you in 2021, but I did finish this in 2018 and the current situation had
not arisen yet. The title of my master’s thesis was “The effect of inflammation on
toll-Like Receptor 8 expression in Human Immune Cells”. The thesis focused on asthma and
asthma being an allergic condition we wanted to see whether that inflammation affected
the expression of Toll-Like Receptor 8. Um, expression meaning how many of them are there.
36! Counted them myself. And we wanted to see whether that expression had an effect
on the asthma patient’s response to viral illness. So if you are an asthmatic person
or if you know somebody who is asthmatic, you might know that asthmatics generally suffer
with viral respiratory infections a little bit worse than non-asthmatics. So we wanted
to see if Toll-Like Receptor 8 could have played a role in making that worse. So what
are- what is Toll-Like Receptor 8? What are Toll-Like Receptors? Well. Toll-Like Receptors
are what we call Pattern Recognition Receptors or PRRs. They recognise these things called
Pathogen Associated Molecular Patterns. These Pathogen Associated Molecular Patterns, or
PAMPs for short, are basically just bits of RNA and DNA from invading bacteria or viruses.
This could be a whole hour on its own but Toll-Like Receptor 8, or TLR8, basically recognises
single stranded viral RNA, so yes, covid. And this single stranded RNA is very very
common in respiratory viruses. And these respiratory viruses wreak havoc on the asthmatic’s body.
They wreak havoc on everybody but asthmatics have it slightly worse, so this paper wanted
to find out whether the inflammation associated with asthma affected how many TLR8s were expressed
in our immune cells, and whether that was the reason why asthmatics experience respiratory
viruses worse. There are so many pathways in asthma, it’s not physically possible
in the space of a one year master’s course to assess them all, so what I did was to simulate
asthma conditions, I incubated cells in a selection of the “chemicals” that asthmatics
have more of in their body. So I chose Tumour Necrosis Factor Alpha, or TNFa, Interleukin
4 or IL-4, and Interleukin 5, or IL-5. IL-4 and IL-5 were chosen just to simulate a couple
of different branches of the asthmatic inflammation pathways. So interleukin 4 tends to play more
of a role in the mast cell side of things, and interleukin 5 generally plays a role in
the eosinophil side of things. TNFa is just sort of everywhere, it’s generally present
in all sorts of inflammation, and it just sort of acted as our positive control. We
chose THP-1 cells which are a monocyte like cell line derived from cancer cells. I chose
those because monocytes are heavily involved in the innate immune system and it seemed
the most appropriate white blood cell to pick, and also cheap, easy to get your hands on,
easy to cultivate, multiple reasons. How to slim this down. So I incubated the THP-1 cells
in TNFa, IL-4, and IL-5. I can’t remember how many days for, but then I tested them
using something called Quantiblue. And a Quantiblue assay works by measuring the product of TLR8
receptor activation. So instead of looking directly at how many receptors there are by
using something like a western blot (because our antibody failed multiple times and we
couldn’t figure out why), you measure the product of something. So we used something
called SEAP, which is Serum, something, Alkaline Phosphatase, but you can measure that using
a Quantiblue assay and it was quick, easy, ask me about it in the comments if you really
want to know the reason and how that works. Maybe I can make a video about it but this
is not that video. So we measure the amount of SEAP in our assay, and more SEAP meant
more TLR8 expression or activation. I’ve just had to quickly grab my poster which is
this bad boy. I’m not very good at posters but here it is. So by doing the assay we found
that the cells that we incubated in both the interleukins had less TLR8 activation. Which
indicated that there was likely less expression. How we differentiated lack of activation with
lack of expression, we treated with TLR8 agonists, which basically if you put one of those in
it will activate TLR8 and produce SEAP. But it didn’t. There was hardly any activation
in all of the samples, we extrapolated that that means there’s less TLR8 here because
if there was TLR8, it would’ve been activated by the agonist. There are so many tangents
that we came across in this thesis, there’s layers and layers and layers. It’s 13,000
words just FYI. To sum up, we found the inflammation that is present with asthma reduces the amount
of TLR8 expression because it wasn’t there to be agonist, it wasn’t there to be turned
on and make the SEAP. And there was something like a P value of less than 0.01. So what
does that mean for asthma? Why does that explain why asthmatics suffer from viral illnesses
worse. Well one of the arguments that I presented in my thesis was if you have less TLR8, you
have less opportunity to recognise a viral infection. So it’s repeate- so it’s replicating
itself so you’re having a bigger viral load until maybe it hits a TLR8 receptor and it’s
like oh hey no, actually, we have an infection, bad. You’ve already got a lot of virus in
there, compared to someone without asthma who has more of the TLR8 being expressed,
and more opportunity to be like oh hey there’s a virus here and then you’ve only got like
3 or 4 more particles, and you have more of a chance to mount your immune defence. I could
talk about that immune defence but I’m looking at my viewfinder and we’re already at 16
minutes and I don’t want this to be an hour. So what does this mean for future work? Well
it hinted at lots of things. It hinted at opportunity for further study towards a preventative
drug, um so for example, maybe an interleukin 4 blocker or modulator because we have to
remember that these interleukins exist for a reason, and by blocking them outright you
might be supressing the immune system which is like the opposite of what you want to do.
So an interleukin 5 blocker wouldn’t really be helpful because you kind of need that so
you can have eosinophils migrate to the area to deal with the infection. They do that for
a reason. But interleukin 4 might be a safer thing to modulate or block slightly because
its role is sort of duplicated by other chemicals, and might not have so much of an effect on
TLR8 expression. It also hinted at things like mRNA vaccines, or single stranded RNA
vaccines, um, similar to the covid one. Ugh, the way that works is much more complicated
to explain in this video, it’d be longer than this whole video so far. But one of the
TLR8 agonists was also used as a vaccine adjuvant which can help to boost the effect of a vaccine,
so it hinted at a role of TLR8 in RNA vaccines which I think is pretty cool. Especially in
the current situation. I’d love to make a video about TLR8 signalling but I really
don’t know if anybody’s interested in that. I think I will make one for next month
because that will help explain this video better. But here is my posted, you can’t
really see it, it’s big, bigger than me. That poster does quite a good job of summing
it up. I’m just gonna read my conclusions and future work to you because I think it
really sums up what my masters thesis actually found out. Conclusion and future work. Interleukin
4 causes a decrease in TLR 8 mediated NF-kB activation due to its activation of STAT6
which out competes NF-kB binding to DNA. This decrease in TLR8 efficacy increases the likelihood
of virus induced disease exacerbation in asthmatic patients, as viruses are able to progress
past TLR mediated defences. Future work seeks to investigate whether IL-4 antagonism would
be a viable treatment for asthmatic patients to reduce disease exacerbation. Furthermore
IL-5 also causes a decrease in TLR8 mediated NF-kB activation though the precise mechanisms
aren’t known. However similarly to IL-4, IL-5 would increase virus induced disease
exacerbation in the same way. IL-5 antagonism however may not be suitable as treatment due
to its vital role in eosinophil recruitment. So the end product of TLR8 activation is um,
it removes the inhibition of NF-kB and NF-kB basically makes other stuff, not- I don’t
want to go into it too deep because it’s just so much. So much. Um, but NF-kB is basically
what allows SEAP to be produced but also a whole bunch of other things including cytokines
and things that kick your immune system into gear to fight the virus. NF-kB being the end
product of TLR8 signalling um, you don’t want anything to really interfere with that,
uh but because it does because there’s no TLR8 there’s less NF-kB, there’s less
viral defences, basically. I hope I’ve managed to edit this into some sort of usable video
because I worked really hard on this for a year and got my master’s with commendation
for it yay, uh so don’t forget to like this video, drop a comment down below if you have
any questions, if you want me to make a video explaining stuff, I definitely will because
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