Our body evolved an immune system to fight infections. Our bodies also evolved the feeling of pain to avoid injury and make us aware of the site of infection. But is it possible that the nerves which sense pain, also prepare distant sites in the body to prepare for impeding infections and injuries? Let's find out in today's journal club.

Illustration showing personification of a pain-sensing afferent sensory neuron, which has encountered an enemy (a pathogen) and is waring others by announcing it on loudspeaker
Image adapted from max pixel | CC0

The paper

The paper I am going to put up for discussion this week is titled - Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity. It was published this month in Cell by Cohen et al., from Daniel Kaplan's lab.

What is so interesting about it? What is the take-home message?

Well, before this paper we had some idea that the nerves in the skin, that play a role in pain sensation, also play a role in initiating an immune response to some pathogens. However, what was not known was if these nerves are sufficient in isolation to any other cell type to initiate the immune response. The authors say they are sufficient for kick-starting type 17 (aka type 3) immunity. Moreover, the paper also discovers a mechanism for anticipatory immunity. That means if you get an infection at one site in the skin the nervous system is capable of initiating an immune response at distant sites as well as if it is preparing the body for upcoming danger. Plus, they use a pretty cool technique to do so, called optogenetics, which I will talk about soon.

A tat bit of background

TRPV1+ afferent neurons

TRPV1+ afferent neurons (transmit information from periphery to spinal cord) are a subset of nerve fibres that innervate the skin. Their major job profile over there is of the nociceptor, which are the neurons which sense pain. They were initially discovered as neurons which respond to capsaicin, the compound in chilli peppers such as capsicum. However, with time they have also been shown to convey the feeling of pain associated with heat, acidic pH, cellular damage related molecules and even pathogen-related molecules (Szallasi et al., 2007). They are also the neurons which makes you itch when your body senses an irritant (Kittaka et al., 2017). Moreover, these neurons when activated also secrete neuropeptides such as substance P and CGRP, which can cause inflammation (Roosterman et al., 2006). It has also been shown that chemically induced dermatitis (the itchy inflammation of skin), kind of a model for psoriasis, requires TRPV1+ neurons (Riol-Blanco et al., 2014).

Even though it has been shown that molecules from pathogens like - staphylococcus aureus, Candida and Streptococcus pyogenes, can activate TRPV1+ neurons and skin and induce type 17 immune response; it is not clear if these neurons are sufficient to explain the immune response or if they are just part of stuff that happens (Cohen et al., 2019).

A brief intro to types of immune responses

The immune response of our body can be put in 3 broad categories type 1, type 2 and type  3 (aka type 17). They are classified based on subtypes of T cells and cytokines that are recruited in mediating the immune response. For instance, type 1 response is categorised by activation of T helper 1 (Th1), and T cytotoxic 1 cells, and natural killer cells which produce interferon-γ, TNF and LTa. These cytokines also activate phagocytic cells and cause B cells to produce  IgG, IgM and IgA antibodies. Type 1 immunity hence helps the body to fight against intracellular pathogens. On the other hand, the type-2 immune response is devoted to fighting against worms, venom and other allergens.  Type 2 immunity is characterised by the presence of Th2 and Tc2 cells, which produce IL4, IL5 and Il13. These cytokines cause B cells to produce IgE and recruit mast cells, eosinophils and basophils (Anunziato et al., 2015).

But, the one important to us for today is the third type of immune response aka type 17, is activated by extracellular by bacteria and fungi, such as those that live on the skin. It is activated by cytokines such as IL-23, TNFa, IL-6, IL1B. It is characterised by Th17, Tc17 and γδT-cells which produce 1L17 and IL22 as signature cytokines of type 3 immunity. These cytokines also target the epithelial the cells to produce anti-microbial peptides which target the bacteria on the skin. This is the kind of immune response that is linked to TRPV1+ cell activation. However, are these sensory neurons enough to kick start type 17 response to skin pathogens, is the question of the day.

So what's the story

The blues after adding channelrhodopsin to TRPV1+ neurons

Illustration showing how the researchers made the mice expressing channel-rhodopsin (which can be activated via blue light) in TRPV1+ neurons. They took mice which had a Channelrhodopsin gene in Rosa26 locus. Which means that it has a stop codon inserted in the middle of the gene. Hence, even though this mouse carries the As it won't make the protein for it. However, the stop codon is flanked by loxP sites. An enzyme called Cre recombinase can cut the DNA at these sites. They then crossed their mice with another which carried Cre recombinase gene driven by TRPV1 promoter. As a result, any offspring from the cross would cause the TRPV1 expressing cells to remove the stop codon in the channelrhodopsin gene, only in TRPV1+ cells.
Illustrated by @scienceblocks using the following images -
Mouse cartoon 1 by da5id1 | public domain
Mouse cartoon 2 | Pixabay

In order to test whether TRPV1+ neurons are sufficient to activate type 17 immunity, the authors used an optogenetic approach. Which means that they expressed this protein called channelrhodopsin specifically in TRPV1+ neurons of the mice. So now, these neurons can be activated just by shining blue light on them. Sadly, the first thing that happens when you shine blue light on one of the ears of these mice is that it feels pain. Also, when they did this for 30 mins per day for 5 days, the entire ear became thick, inflamed and displayed psoriasis-like features, indicative of type 17 inflammation.

Schematic of the experiment and findings. Illuminating ear of the mouse having ChR2 in TRPV1+ neurons causes inflammation as compared with a mouse with no ChR2. Inhibiting TRPV1+ neurons with RTX blocks this response.
Illustrated by @scienceblocks
Mouse cartoon 2 | Pixabay
Syringe by pitr | public domain

When they dissociated the ear and passed the cells through a flow cytometer, they could detect the presence of γδT-cells, T helper cells and neutrophils - characteristics Type 17 response. Moreover, the authors also observed that 6 hours after they said -"let there be light", there is also an increase in expression of inflammatory cytokines - IL-23, IL-6, TNFa - the activators of type 17 immunity. And then 48 hours later IL-17 and IL-22, the signature cytokines of type 17 immune response went up. However, none of this was observed in the ear not exposed to blue light or in control animals with channelrhodopsin exposed to light. Moreover, inhibiting TRPV1+ neurons with a chemical called RTX, also inhibited the inflammation of the ear and recruitment of immune cells.

Neuropeptide CGRP mediates the effect of TRPV1+ neurons.

The activation of channelrhodopsin in neurons causes them to secrete neuropeptide CGRP. Moreover, blocking vesicle fusion and hence secretion of CGRP using Botulinum neurotoxin A (BoNTA), also blocked the immune response.
Doodled by @scienceblocks

Since, CGRP, the neuropeptide secreted by these neurons, also went up when the neurons saw the blue light, the authors wanted to know if it is required for mediating the immune response. To test this they injected botulinum neurotoxin A into the dermis of mice. This neurotoxin blocks the vesicle fusion in neurons which is required for neuropeptide secretion. In the mice injected with this neurotoxin and photostimulated with blue light, the expression of cytokines did not go up.

But why neurons?

Now given that TRPV1+ neurons can be directly activated by molecules of the pathogens, and the experiments above showing that they are sufficient to activate the immune response - it means that these neurons are part of the first line of defence against extracellular microbes (of course after the skin barrier). But why did neurons need to evolve something that could have been taken care of by other resident immune cells, or for that matter even by some cells that form the skin? Why in the freaking world neurons need to work extra hours for this?

If you think about it neurons can do something other cells don't. They can transmit the signal in a matter of milliseconds using electric impulses. They can even relay the information to the brain. In a way, they can warn the body about the infection (which they do by transmitting pain) much faster, and it is possible that they can even start an immune response at other places before the infection reaches there. So like when infection happens at one place, and then start spreading to a nearby place, the immune cells there would be like - "haha, we were expecting you". It would be an ambush. So does it happen?

Because they spread the message

One thing with the neurogenic inflammatory response is that it also increases the blood flow from nearby vessels. So to test if activation of these neurons also activated nerve fibres in nearby non stimulated regions, the researchers used 2D doppler ultrasound to measure the blood flow in different regions. They would shine blue light at one region in back skin and measure the blood flow in that area as well as nearby area. And, voila! The blood flow increased not only in the stimulated area but also in the adjacent non-stimulated area. They also found CGRP being released both at the site of stimulation and in nearby areas. The same help true for expression of inflammatory cytokines - IL-23, IL-6 and TNFa. Moreover, when they photostimulated the skin before infecting it with Candida, the Candida infection found it hard to spread to adjacent regions, in mice expressing channelrhodopsin vs control mice.

The message is related via electrical impulses in the nerve fibre. The activation of a neuron at one site causes a release of CGRP at other sites too

To test if it is indeed the neurons that are spreading the message via electrical impulses, they blocked the impulses using a drug called bupivacaine. Bupivacaine is a voltage-gated sodium channel blocker and is hence used to stop neuronal communication. When they injected bupivacaine in dermis before shining the blue light, the CGRP and inflammatory cytokines were released at the site of stimulation but not in nearby areas. Moreover, when they injected the botulinum neurotoxin A (to block CGRP neuropeptide release), they found that the inflammatory cytokine expression was only blocked at site of injection and stimulation but not in nearby areas. Confirming that the signal to activate the immune response in adjacent areas travel via electrical impulses.

Finally, to test if the neurons can also respond the same way to Candida, they infected one of skin site with heat-killed fungus (so that the fungus can't spread on its own but its molecules can activate the neurons). The heat-killed fungus kick-started the type 17 immune response both at the site of infection and in nearby areas. The immune response in nearby areas even this case could be blocked by bupivacaine. Moreover, if the mice pretreated with heat-killed fungus were now infected with live fungus, they live fungus found it hard to spread to adjacent areas. Well, except with these mice were also treated with bupivacaine.

These experiments make a strong case not only about TRPV1+ neurons being sufficient for kick-starting the immune response to pathogens but also for the warning the nearby areas to prepare for war. Of course like any other papers, there are things that one need to be careful about - for instance TRPV1+ expression is not only limited to neurons, it can also be expressed by other immune cells and epithelial cells. However, authors do address this and show that on their model the expression of channelrhodopsin was limited to neurons. However, why that might be the case in their model is not very clear. Then, in their model, the channelrhodopsin got expressed in both peptidergic and non-peptidergic neurons TRPV1+ neurons. Though they talk about this, it might need further investigation to confirm their results in a more specific model.

Nevertheless, their results are interesting not only in terms of anticipatory immunity to pathogens; but also makes you wonder about other scenarios such as wound healing. Like, can getting wounded at one site prepare you to respond faster to would at the adjacent site? How far and how fast can this immune response prepare the body for incoming pathogens and injuries? What if this system is activated without any reason at one site - say via tumour, can this also make inflammatory environment-friendly or detrimental to tumour metastasizing to another site. Can this system also explain the psoriasis flare-up by psychological stress (well because type 17 immunity is implicated in the pathology of psoriasis)?

A bit about journal club series

This is a new series I am starting. In this series rather than discussing an entire field say about diabetes, CRISPR or ageing, I will summarize that paper I liked a lot, and we can have a discussion on it. We often do this in the lab, so I thought why not on steemstem? In comments do let me know if you liked the idea and approach, and if you think I should add or change something. It would be nice of you to put up questions on the paper and fuel discussion. Also, if you like it, I would encourage you to write about your favourite papers.

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Cohen et al., 2019. Cutaneous TRPV1+ Neurons Trigger Protective Innate Type 17 Anticipatory Immunity

Nociceptor

Szallasi A, Cortright DN, Blum CA, Eid SR. The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov. 2007 May;6(5):357-72. Review. Erratum in: Nat Rev Drug Discov. 2007 Jun;6(6):442.PubMed PMID: 17464295.

Kittaka et al., 2016. The molecular and cellular mechanisms of itch and the involvement of TRP channels in the peripheral sensory nervous system and skin

Roosterman et al., 2006. Neuronal Control of Skin Function: The Skin as a Neuroimmunoendocrine Organ

1: Riol-Blanco L, Ordovas-Montanes J, Perro M, Naval E, Thiriot A, Alvarez D, Paust S, Wood JN, von Andrian UH. Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Nature. 2014 Jun 5;510(7503):157-61. doi: 10.1038/nature13199. Epub 2014 Apr 23. PubMed PMID: 24759321; PubMed Central PMCID: PMC4127885.

Annunziato F, Romagnani C, Romagnani S. The 3 major types of innate and adaptive cell-mediated effector immunity. J Allergy Clin Immunol. 2015 Mar;135(3):626-35. doi: 10.1016/j.jaci.2014.11.001. Epub 2014 Dec 18. Review. PubMed PMID: 25528359.