So the thing is the current Covid19 crisis has kept some of us really on our toes at work. Also, since Sars-CoV-2 is all we are allowed to work on for now, that is all I have been reading. So I thought why not share some of the papers that have been running through my head. So here is the 1st journal club on this topic.
The story of mammals that fly and
why they don't fall sick!
Source | pixabay
Given, the current Coronavirus crisis, we must have all heard a lot about bats as of now. One of the things that you must have heard that Bats are reservoir to many zoonotic viruses. Even the current virus that has caused the crisis SARS-CoV-2 shows 96% similarity to a bat coronavirus RaTG13 (Zohu et al., 2020). It is true that bats do harbour a lot more zoonotic viruses, than any other known mammal (Olival et al., 2017). But, how is it that bats don't keep getting sick and falling dead on the ground?
Well, this is exactly what this paper published by Ahn et al., 2019 talks about. How bats carry this virus and still don't fall sick? Can lessons from bats help in reducing Covid19 related deaths, or for that matter other viral related deaths in humans?
The idea of the paper revolves around a specific kind of immune response that is heightened in humans but dampened in bats. And, since the excessive immune response is what causes many of the symptoms and even mortality in many infectious diseases; it might be possible to carry the virus safely by dampening some of the more violent immune responses.
The NLRP3 Inflammasome
To be more specific, the NLRP3 Inflammasome response is what we are talking about. NLRP3 is a protein in cells, which gets expressed when another protein NfKb is activated due to a stress signal - when molecules from damaged cells or external pathogens bind to toll-like receptor (TLR) on certain immune cells. Once there is enough NLRP3 in the cells, another stress signal opens up potassium ion channels which in turn causes a change in confirmation of NLRP3 protein. This activated form of NLRP3 then forms megastructures by aggregating together. Another protein called ASC binds to this mega structure created by NLRP3 aggregation and forms ASC filaments. These aggregated filamentous structures can be seen as specs inside the cells. The ASC filaments then recruit a protein called Caspase1 via its card domain. A lot of caspase1 molecules in close proximity cleave each other exposing their catalytic domain.
This active caspase1 is now ready to cleave other proteins - it cleaves pro-Il1b and pro-Il18 to yield Il1b and Il18 inflammatory cytokines. Caspase1 also cleaves pro-Gasdermin-D. The cleavage of Gasdermin-D releases its N-Terminal region, which then aggregates in cell membrane creating pores that eventually kills the cells. The cell leaks out inflammatory cytokines Il1b and Il18 into the surrounding tissue along with other cellular contents. While cellular contents may act as a signal for another nearby cell to induce inflammation, the inflammatory cytokines recruit immune cells at the site.
Here is a link to a nice animation of NLRP3 inflammasome
The story
Ahn et al. took peripheral blood mononuclear cells (PMBCs) - lymphocytes (T-cells and B-cells) and monocytes (precursors to macrophages) from human bat blood. As a first signal, they primed the cells with lipopolysaccharide, a component of the bacterial membrane. Then as a second signal, either via ATP (extracellular ATP signals cellular damage) or via Nigericin a toxin and an antibiotic derived from Streptomyces hygroscopicus - they activated the NLRP3. So ATP acts as if it is a signal from the body, while nigercin act as a signal from the pathogen. Nevertheless, in both cases, bat PMBCs showed a lower amount of ASC specs and reduced IL1b secretion as compared to human PMBCs. The authors also compared the response of macrophages and dendritic cells derived from the bone marrow of either bats or mice. And yet again looks like mice cells had a stronger inflammasome response as compared to bat cells.
So what the hell is going on here? Why is it that bat cells don't show such high inflammasome activity? Is there a problem with NFkB activation to begin with? Turns out, other genes such as Il6 and TNFa under NFkB regulation just do fine. Nevertheless, expression of NLRP3 was lower in bat cells as compared to human or mouse cells. They tried multiple signals known to activate NFkB pathway - bacterial, cellular damage and even analogue of viral DNA. But nada, NLRP3 expression was weak in all cases.
On top of that they found that when it comes to binding to ASC protein, the human and mouse NLRP3 was much more potent than bat NLRP3. Moreover, in bats, NLRP3 comes in 4 flavours due to alternative splicing - a process by which cell create multiple flavours of proteins from the same transcript of gene - by removing different portions from the gene transcript for a different flavour. Of these 2 flavours lacked a sequence of a gene - exon 7 - and was even worse than the other 2 flavours when it came to binding to ASC.
**A model of dampened inflammasome response in bat cells. ** In bat cells there is lower expression of NLRP3 in response to NFkB activation. Moreover, even the expressed NLRP3 is much less potent in binding to ASC than human NLRP3. This, in turn, would hinder with Caspase1 clustering and activation. Hence, everything downstream from mature inflammatory cytokine production, to cell death via membrane pore formation would be affected.
Illustrated by
They finally tested how different viruses - Influenza A, PRV3M , and MERS coronavirus affect bat cells compared to humans or mouse. It turns out that these viruses don't activate NLRP3 inflammasome in bat cells.
Well, at least from these in vitro cell culture studies it looks as if bats cells are not oversensitive to the virus, at least when it comes to NLRP3 inflammasome mediated inflammation. It is likely that because of dampened NLRP3 inflammasome response bats don't become as sick from these viruses as other mammals do. I mean even Ebola don't fucking make them sick nor does the other coronaviruses. And it's not as if they eliminate the virus very fast, they carry it around in high titer without falling sick (Swanepoel et al., 1996, Munster et al., 2016). The question, however, is why did bats evolve such an ability? Like did they really evolve to be viral reservoirs? Or being a viral reservoir is a byproduct of something else?
You must have noted in our discussion that the NLRP3-Inflammasome response is dampened in bats not only to pathogenic signals but also to cellular damage signals. Now bats are the only flying mammals. Which means their metabolic rate is pretty high. And, a high metabolic rate implies a lot of cellular wear and tear because of reactive oxygen species generated during cellular respiration, for example. If the immune cells were oversensitive to every cellular damage then they would have wreaked the havoc inside the bats. So its likely bats evolved NLRP3 pathway in a way that its immune cells don't overreact to metabolic damage. And, becoming a virus reservoir was just a side effect.
Also, if NLRP3-inflammasome is what is causing the symptoms and damage in humans when it comes to human viral diseases, esp the current crisis of Covid19, will dampen this help? I mean it's worth a shot to try it on animal models at least for now.
Nevertheless, there are a couple of things I think authors could have done more is - one, they should have tested their hypothesis in-vivo. It would be ideal to make a transgenic bat with human NLRP3 gene and test this hypothesis, but they could have tried it in mouse model at least. What if we make a transgenic mouse with bat NLRP3? Would it be able to tolerate more viral load without getting sick? Will NLRP3 pathway inhibitors reduce severe symptomatic viral infections in mice and to what extent?
The second thing I wish authors did was to find why NFkB doesn't cause high expression of NLRP3 in bat cells? Is the sequence of NLRP3 promoter different or is there a mutation in bat NfKb?
Well that's it for today.
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References
Zhou et al., 2020. A pneumonia outbreak associated with a new coronavirus of probable bat origin
Olival et al., 2017. Host and viral traits predict zoonotic spillover from mammals