Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia, 2023, Sara Cavaria et al

[Mij]

Significance
We used a back-translational approach in mice to demonstrate the pronociceptive role of neutrophils in fibromyalgia. Adoptive transfer of neutrophils from mice with chronic widespread pain or from patients with fibromyalgia can confer mechanical pain to recipient naïve mice, sensitize evoked action potential firing of spinal cord neurons, and produce phenotypic changes in cell surface expression of neutrophil proteins that cause infiltration of neutrophils into dorsal root ganglia. These data provide the framework for an immunological basis of chronic widespread pain in fibromyalgia mediated by polymorphonuclear granulocytes.

Abstract
Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established.
Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.




https://www.pnas.org/doi/10.1073/pnas.2211631120

 

[Creekside]

A further question for them: do the neutrophils cause these changes themselves, or is it FM that changes the cells to react improperly to the neutrophils?

 

[rvallee]

Seems... significant. As they mentioned about the peripheral findings, research is aligning in some direction.

There was always a giant gap at the center of the central sensitization thing. Well, two if you count the central part, which was merely asserted. But, really: sensitized by what? How?

And of course the model never bothered, because it wasn't built coherently to account for something happening to cells. It was always the same old psychomagic.

 

[cassava7]

A side note that may or may not be relevant: I find it interesting that the recent results on autoantibodies and peripheral stimulation in fibromyalgia have involved British authors (from King’s and QMUL, respectively) but none of them have been published in high profile British journals such as the Lancet Rheumatology. They have only appeared in the Journal of Clinical Investigation and now PNAS, though both are well established journals to my knowledge.

 

[Hutan]

Some background information from the introduction that sets up their study - could be worth checking the references out:

Several studies point toward a peripheral drive fibromyalgia pain, e.g., peripheral administration of a localized lidocaine block of tender points reduces pain scores in patients with fibromyalgia (2). Moreover, abnormal activity-dependent slowing of unmyelinated nociceptors is observed in microneurography recordings (3), and pain-related evoked potentials reflect abnormalities in small fiber function (4).

Neutrophils are polymorphonuclear granulocytes that normally exist in circulation to function as primary mediators of rapid innate host defense, but are surprisingly found in higher number in the circulation of fibromyalgia patients, with a phenotype characterized by enhanced chemotactic and microbicidal properties (5–7).

Systemic expression of proinflammatory cytokines, such as IL-6, IL-8, and tumour necrosis factor alpha (TNFα), is increased in patients with fibromyalgia, and these same cytokines are otherwise released by neutrophils and are known mediators of nociceptor sensitization (8–10).

Polarization of resident macrophages in dorsal root ganglia and mitochondrial transfer from infiltrating macrophages to somata can confer sensitization of peripheral sensory neurons (11, 12).

(that's not about neutrophils - but might worth reading about. Have we heard about mitochondrial transfer?)

Neutrophils are not normally resident cells within intact nervous tissue, but infiltration into peripheral nerves has been reported in rodent models of nerve injury (16, 17), diabetic neuropathy (18), and degenerative disease (19).

(Neutrophils are a type of white blood cell, as are macrophages, monocytes and lymphocytes. They are part of the rapid response system to invading particles and microorganisms. When the body is under stress, reserves of neutrophils are released from the bone marrow.

Image from: Neutrophil: A Cell with Many Roles in Inflammation or Several Cell Types? 2018
That reference highlights the heterogeneity of neutrophils - clearly there's lots left to know about them.)

 

[Hutan]

Persistent Hypersensitivity in a Mouse Model of Chronic Widespread Pain
So, a model of chronic widespread pain?
What this seems to be is two injections of an acute inflammatory stimulus into muscle, 4 days apart - thus creating the 'Primed Mice'

The 'Control Mice' only had one injection. 7 mice of each.
Both sets of mice pretty much recovered after the first injection, in terms of sensitivity to pressure (50% mechanical withdrawal threshold), but the second injection caused the primed mice (in red in the chart below) to have much lower mechanical withdrawal threshold for the duration of the experiment, out to Day 28.



I'm not sure how relevant this is to fibromyalgia... but, onwards.

 

[Hutan]

In vivo electrophysiology

Electrophysiological recordings were performed by an experimenter blind to experimental groups. Briefly, mice were anesthetized with isofluorane (4%; 0.5L/min N2O and 1.5 L/min O2) and secured in a stereotaxic frame. Anesthesia was reduced and maintained at 1.5% isoflurane for the remaining duration of the experiment. A laminectomy was performed to expose L3–L5 segments of the spinal cord, and extracellular recordings were made from wide dynamic range neurons in the deep dorsal horn (lamina III–V, 200 to 600 μm) using parylene-coated tungsten electrodes (A-M Systems, USA). Mechanical and thermal stimuli were applied to the peripheral receptive field of spinal neurons on the hindpaw glabrous skin and the evoked activity of neurons was visualized on an oscilloscope and discriminated on a spike amplitude and waveform basis using a CED 1401 interface coupled to Spike2 software (Cambridge Electronic Design, UK). Natural stimuli (dynamic brush, vF hairs 0.16 g to 60 g, noxious prod 100 g cm−2mechanical stimulation; thermal water jet 35 to 50 °C) were applied in ascending order of intensity to receptive fields for 10 s and the total number of evoked spikes was recorded.

The mice were anaesthetised,and the spinal cord exposed. Then the researchers applied stimuli (pressure and heat/cold) to the hind paw and measured the evoked activity of neurons. These measurements were done by an experimenter blind to which sort of mouse they were working on. I'm not sure when these measurements were done.

The researchers found that the neurons of the primed mice fired more than the neurons of the control mice when stimulated mechanically (prodded), but not when stimulated thermally. That does seem to be true, particularly for the measurements done with von Frey hair stimulus - which seems to be basically thin plastic rods of various stiffness which are poked at the mouse.

So, I think so far this only proves that inflamed tissue is more tender, which equates to the neurons firing at lower pressure thresholds.

 

[Hutan]

Circulating Neutrophils Are Essential for the Development of Hypersensitivity in a Mouse Model of Chronic Widespread Pain
So, now the researchers wanted to find out what is causing this sensitivity of the nerves. This works was done in 4 control and 4 primed mice.

We first transferred isolated blood cells and serum from primed and control mice to recipient naïve mice. Transfer of blood cells, but not serum, from primed mice induced transient mechanical hypersensitivity lasting up to 2 h

They transferred blood cells only and serum only from the control and primed mice to new mice. The top chart is for the blood cells, the bottom chart is for the serum - again it's the sensitivity to pressure analysis (50% mechanical withdrawal threshold). The p values are for the area under the curve for the full time course - only the blood cells from the primed mice caused a reduction in pressure thresholds in the new mice. There does seem to be good separation in the pressure thresholds for up to one day after injection into the new mice.

These observations suggest that circulating cells constitute the algogenic etiology of chronic widespread pain in this murine model.

That's a new word for me - "algogenic" = "inducing pain".

At this point, the researchers don't know which blood cells are causing the pressure sensitivity. They sorted the cells into T cells, B cells, neutrophils and monocytes for both the primed and control mice, and repeated the experiment of transferring them into the new mice.

Mice that were administered neutrophils derived from primed mice displayed robust, transient mechanical hypersensitivity in the von Frey assay for up to 1 d compared to mice that were administered neutrophils derived from control mice (Fig. 2 D, iii).

That looks to be true, (although the T cells from the primed mice also look to be having an effect when transferred, although it seems that that difference wasn't statistically significant).

Then the researchers removed the mice's neutrophils (using an antineutrophil antibody) prior to the day 4 second carrageen injection that created the primed mice. The neutrophil depletion lasted at least 24 hours; the researchers say that the application of the antibodies didn't affect pain behaviour. The mice treated in this way didn't subsequently show a lowered pressure threshold. The researchers also found that administering the antineutrophil antibody to the primed mice at day 28 reduced the hypersensitivity to the pressure for at least a day. Figure 3B - it looks pretty convincing.

Our observations suggest that neutrophil depletion can delay or attenuate the development of persistent and widespread pain, without affecting gross measures of inflammation and its resolution

 

[Jonathan Edwards]

This is all a nonsense. If you create an animal 'model' that seems to produce the right symptoms by some arbitrary experimental intervention you will find that it is due to that arbitrary intervention. It tells us nothing whatever about human disease.

high profile British journals such as the Lancet Rheumatology.

Lancet Rheumatology is not a high profile journal - it is a joke pseudo posh journal, like all the 'Nature' mini journals. Nobody would publish anything decent in it. A rheumatologist would publish in Arthritis and Rheumatism or Annals of Rheumatic Disease - or - if they thought they had something scientifically exciting Journal of Clinical Investigation or PNAS!! JCI has always been way more prestigious than any clinical journal. Trouble is that they take garbage too now.

 

[Jonathan Edwards]

Some background information from the introduction that sets up their study - could be worth checking the references out:

I checked out the references. They are mostly a handful of insignificant studies in poor quality journals that don't even say what the authors say they say.

The introduction reads like a naive first year PhD student writing a conference abstract - just a jumble of stuff gleaned from here, there and everywhere.

 

[Jonathan Edwards]

And I do not understand how it can be ethical to subject mice to things like this when it tells us nothing.

 

[Hutan]

I checked out the references. They are mostly a handful of insignificant studies in poor quality journals that don't even say what the authors say they say.

I'm not surprised. I can't recall any reliable reports of elevated cytokines in ME/CFS or fibromyalgia, although perhaps there are some.

I'm not sure, has it been known before that neutrophils are associated with peripheral pain? And that removing them seems to prevent the pain? That bit seemed interesting and as convincing as samples of 8 mice in total could be.

 

[Hutan]

Neutrophils Derived from Mice with Chronic Widespread Pain Infiltrate Sensory Ganglia

To test the hypothesis that circulating neutrophils in primed mice have the capacity to sensitize peripheral sensory neurons, we measured whether neutrophils were directly interacting with somata within dorsal root ganglia. We observed significant de novo infiltration neutrophils into L4 DRG of primed mice compared to control and naïve animals (Fig. 4A).

Somata? Plural of soma - the body of the neuron (and a few other cell types).


So, they found neutrophils had infiltrated the somata of the dorsal root ganglion in the mice that had had two injections of the inflammatory substance, whereas they had not in the controls that had only had one injection. The somata are a relatively long way away from the site of the induced inflammation.


Figure 4 ai.
It looks as though some neutrophils were in the dorsal root ganglion somata of the control mice (one injection) and the new mice (no injections), but there were a lot more in the primed mice (two injections). They say they also used FACS (fluorescence activated cell sorting) to confirm that.

They looked at cell surface markers - they found the neutrophils in the blood and in the neurons looked different in the primed versus those in the control animals. That's not surprising as the primed animals presumably have inflammation and the control animals don't. They also found that the neutrophils in the primed animals looked different in the blood compared to those in the neurons - that perhaps means something.

So, yeah, still waiting to see how this might be relevant to fibromyalgia.

 

[Jonathan Edwards]

Messing about with a mouse's neutrophils in this sort of way is probably a bit like giving them a transfusion reaction or a quick dose of septic shock. If you are then put into a torture chamber and prodded with a stick I suspect it is not very surprising that your responses might change.

People who can have so little insight into the material they summarise in an introduction are likely to have equally little insight into the biases entering their experiments or the complete meaninglessness of any of it in relation to people whose pain is not yet explained.

 

[Hutan]

Neutrophils Derived from Patients with Fibromyalgia Syndrome Induce Mechanical Hypersensitivity in Mice

The researchers then took neutrophils from people diagnosed with fibromyalgia (9) and healthy controls (5), and injected them into new healthy mice (4 mice for each person).

So, this is the key chart, I think, Fig 5c. The measure again is the mechanical withdrawal threshold of the injected mice when prodded with the rods of varying stiffness.



After an hour, the thresholds in the healthy mice given the fibromyalgia neutrophils were a lot lower than in the healthy mice given the neutrophils from healthy people. A small number of samples, sure, but that looks interesting, doesn't it?

Neutrophils derived from patients with widespread pain conferred robust mechanical hypersensitivity in recipient naïve mice compared to neutrophils from healthy control subjects (Fig. 5C)

The neutrophils infiltrating sensory ganglia were both endogenous mouse neutrophils and exogenous human neutrophils, suggesting tissue-restricted innate mechanisms for the recruitment of endogenous and exogenous neutrophils into nervous tissue in chronic pain states

 

[Jonathan Edwards]

The somata are a relatively long way away from the site of the induced inflammation.

I have no idea whether there is any real biology here but injections of carrageenan produce pretty dreadful local inflammation often with cell death. If the stuff is toxic enough to kill nerve endings in muscle then there may well be death of cell bodies in dorsal root ganglia. So you would get neutrophils. But people with fibromyalgia do not have a kilogram equivalent of seaweed extract injected into their muscles.

 

[Hutan]

Messing about with a mouse's neutrophils in this sort of way is probably a bit like giving them a transfusion reaction or a quick dose of septic shock. If you are then put into a torture chamber and prodded with a stick I suspect it is not very surprising that your responses might change.

People who can have so little insight into the material they summarise in an introduction are likely to have equally little insight into the biases entering their experiments or the complete meaninglessness of any of it in relation to people whose pain is not yet explained.

For sure, it's a bad sign when the introduction makes claims that aren't supported by the references given for them. But, I think I said just the other day, it seems to happen in most papers.

It's the last section that I find most interesting. The healthy mice were treated identically, except that the human neutrophils were either from people with fibromyalgia or people without fibromyalgia. The pain response didn't change from baseline in the mice with neutrophils from healthy people, but it did change in the mice with neutrophils from people diagnosed with fibromyalgia.

I haven't had time to think about what might be wrong with this, other than bias in the researchers who were prodding the mice.

 

[Jonathan Edwards]

The researchers then took neutrophils from people diagnosed with fibromyalgia (9) and healthy controls (5), and injected them into new healthy mice (4 mice for each person).

Human neutrophils are incompatible with mouse endothelium because basic lectin adhesion systems use different molecules. Human neutrophils should also immediately be coated with mouse antibodies directed (relatively non-specifically) against non-self material - especially IgM. Most of the neutrophils will embolise at first pass in the lung microvasculature. Few if any are likely to migrate into sites of inflammation. I have never heard of anyone trying to interpret xenogeneic neutrophil infusion, although there may well be a literature somewhere.

I did my doctorate in the department that became there William Harvey Institute, where these people work. I am sorry to say that I would not have great confidence in any of the experimental work.

Confirming neutrophil infiltration with FACS rather than histology seems to me barmy.

Most people doing doctorates in medical science labs discover that their project involved some fatal technical mistake that their supervisor was blissfully unaware of. The secret is to learn from that. Sadly, a high proportion of career scientists never do. Like Fiona Watt not learning that studies need to be blinded if the data are subjective. Things were always bad but a few people learnt to do things better. I worry that nobody does now. Most recent progress in medical research has been in terms of DNA - where the machines do not make many mistakes.

 

[Jonathan Edwards]

But, I think I said just the other day, it seems to happen in most papers.

Yes, most papers now are garbage. This is the problem. The proportion of papers worth citing twenty years later has gone down from about 30% to about 0.3%. It is pretty horrific.

 

[Hutan]

Human neutrophils are incompatible with mouse endothelium because basic lectin adhesion systems use different molecules. Human neutrophils should also immediately be coated with mouse antibodies directed (relatively non-specifically) against non-self material - especially IgM. Most of the neutrophils will embolise at first pass in the lung microvasculature. Few if any are likely to migrate into sites of inflammation. I have never heard of anyone trying to interpret xenogeneic neutrophil infusion, although there may well be a literature somewhere.

Ha. I wondered whether human neutrophils would work in mice, it seemed a bit amazing. I haven't read the discussion of this paper yet (or much of the Method - which is at the end of the paper. Surely, they will address that issue, it's pretty fundamental.

I have no idea whether there is any real biology here but injections of carrageenan produce pretty dreadful local inflammation often with cell death. If the stuff is toxic enough to kill nerve endings in muscle then there may well be death of cell bodies in dorsal root ganglia. So you would get neutrophils. But people with fibromyalgia do not have a kilogram equivalent of seaweed extract injected into their muscles.

Not to minimise the impact of these studies on the poor mice, but this set of results seems to suggest more nuanced impacts than just across the board dreadful inflammation.

For example Figure 3: Baseline pain threshold is about 1 to 1.2. There might be some habituation over time, as by day 28 the thresholds were typically a bit higher.

The controls in Fig 3A just had the one injection of carageen, and seemed to bounce back from it ok, with their pain thresholds at least as high as at baseline, whether or not they had their neutrophils depleted at day 3 (clone versus PBS saline)
The primed animals in Fig 3B had two injections of caragreen (black arrows). The pain threshold of the ones that had their neutrophils depleted at day 3 bounced back up. But the animals that didn't have their neutrophils depleted clearly were very tender throughout.

(The Ipsi and Contra are just different locations where the pressure was applied.)