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EBV/HHV-6A dUTPases contribute to ME/CFS pathophysiology by enhancing TFH cell differentiation and extrafollicular activities, 2022, Cox et al

Discussion in 'ME/CFS research' started by Andy, Apr 29, 2022.

  1. Andy

    Andy Committee Member

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    Full title: EBV/HHV-6A dUTPases contribute to Myalgic Encephalomyelitis/Chronic-Fatigue-Syndrome pathophysiology by enhancing TFH cell differentiation and extrafollicular activities

    Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic, debilitating multisystem illness of unknown etiology for which there is no cure and no diagnostic tests available. Despite increasing evidence implicating EBV and human herpesvirus-6A (HHV-6A) as potential causative infectious agents in a subset of ME/CFS patients, there are few mechanistic studies to address a causal relationship.

    In this study we examined a large ME/CFS cohort (n=351) and 77 controls and demonstrate a significant increase in activin A and IL-21serum levels, which correlated with seropositivity for antibodies to the EBV and HHV-6 protein deoxyuridine-triphosphate nucleotidohydrolase (dUTPase), but not CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation, generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH-function in ME/CFS. In vitro studies revealed that virus-dUTPases strongly induced activin A secretion while in vivo, EBV-dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells.

    Altogether, our data indicate abnormal germinal center (GC) activity in ME/CFS subjects and highlight a mechanism by which EBV and HHV6-dUTPases may alter GC and extrafollicular Ab responses.

    Open access, https://insight.jci.org/articles/view/158193
     
  2. Hutan

    Hutan Moderator Staff Member

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    This looks interesting, looks like quite a few of us missed it. Brandon Cox is presenting at the 2022 IACFS/ME conference.
     
  3. Hutan

    Hutan Moderator Staff Member

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    Figure 1 - measures of activin A, IL-21 and CXCL13 in the sera of ME/CFS, GWI and healthy controls

    Screen Shot 2022-07-26 at 6.59.05 pm.png

    Those results look very interesting. Clearly, the increases in Activin A and IL-21 aren't happening in every person diagnosed with ME/CFS and GWI, but there does seem to be a significant subset. Note the y axis scales - these levels are very definitely different.

    And the sample size is very good. So, is the result something that can be explained by different lifestyles or is it a real clue? And would some of the other ME/CFS and GWI people show similar increases if the blood sample was taken on a bad day?
     
  4. Hutan

    Hutan Moderator Staff Member

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    So, who has high activin A levels?
    Serum activin A and B levels predict outcome in patients with acute respiratory failure: a prospective cohort study 2013
    These authors thought high activin A levels were predictive of bad health outcomes and suggested they indicate inflammatory challenges:
    Serum immunoreactive activin A levels in normal subjects and patients with various diseases 1996
    This 1996 paper suggests normal levels of activin A are marginally higher in men than in women.
    But, there were a lot more women in the ME/CFS (83%) and the GWI (93%) cohorts than in the healthy control cohort (51%) in the 2022 study, so gender would suggest that the disease samples should have had lower, not higher levels.

    The 1996 paper also suggests that activin A levels increase with age. But, the average ages of the disease cohorts (47 and 44 years) were a lot lower than the average of the healthy control cohort (67 years). So age would suggest the disease cohorts should have had lower levels of activin A.

    The 1996 paper reports increases in pregnancy and in some diagnosable health conditions, but these seem unlikely to account for differences in the 2022 paper findings.
    The units in the 2022 paper are pg/mL (picograms per millilitre), so I think we need to divide by 1000 to get to micrograms per litre. The mean for the controls in the 2022 paper was around 500, so 0.5 micrograms/L whereas the means for ME/CFS and GWI were around 10,000, so 10 micrograms/L. I don't know if the methods are comparable, but even with different methods, differences between healthy and unhealthy levels should be comparable. The 1996 paper was suggesting that unhealthy levels are about 2 to 3 times the healthy levels. But here, we have a lot of people in the disease cohorts having activin A levels that are 20 or more times higher than those of the healthy controls.

    Given people with chronic renal failure and advanced solid cancer are probably not very active, that would seem to rule out the possibility that a sedentary lifestyle is somehow causing the higher activin levels.

    So, I think the idea of higher activin A levels is interesting.
     
    Last edited: Jul 26, 2022
  5. Hutan

    Hutan Moderator Staff Member

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    One problem is that another study of activin A found normal levels in ME/CFS cases:

    We discussed that Lidbury paper here: Activin B is a novel biomarker for chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) diagnosis, 2017, Lidbury et al
    and here:Rethinking ME/CFS Diagnostic Reference Intervals via Machine Learning & Utility of Activin B for Defining Symptom Severity (2019) Lidbury et al.
    and here: Potential of Activin B as a Clinical Biomarker in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), 2021, Gravelsina et al

    Maybe the Lidbury group had technical issues? (Or maybe this group did...)
     
    Last edited: Jul 26, 2022
  6. Hutan

    Hutan Moderator Staff Member

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    So, what about IL-21, and the subset this 2022 study found with the stunningly high values? (around 100 pg/mL for the healthy cohort; with the disease subsets with around 20,000 pg/mL).


    We have looked at this paper with a POTS cohort:
    Inflammatory Biomarkers in [POTS] with Elevated G-Protein-Coupled Receptor Autoantibodies, 2021, Gunning, Grubb et al
    Screen Shot 2022-07-26 at 8.21.34 pm.png
    Those values reported for the POTS study are also in pg/mL and seem to fit. Less than 200 pg/mL is reported as normal, and in this case, levels in the POTS cases were around 10 times higher at around 2000 pg/ml.



    Serum IL-21 levels are elevated in atopic dermatitis patients with acute skin lesions 2016
    This 2016 study found as the title suggests for this allergic skin disease, with higher IL-21 levels being associated with higher severity of acute skin lesions.
    So those levels also seem to fit with a normal value of less than 200 pg/mL (although I start to wonder if some of the levels in the 2022 ME/CFS +GWI study are possibly unbelievably high).
    The atopic dermatitis study authors say:
    . They also say that IL-21 levels are raised with mechanical injuries to the skin. They note that serum levels of IL-21 were found to be lower in children with atopic dermatitis, unlike the adults in the 2016 study. So, I don't know. Maybe measuring these substances accurately is difficult? Maybe the levels of them change with the time of day or with storage?

    Plasma interleukin-21 levels and genetic variants are associated with susceptibility to rheumatoid arthritis 2021
    There seems to be a substantial body of findings linking IL-21 to RA. The unit here is nanograms/mL - to convert that to the picograms/mL used in the 2022 ME/CFS+GWI study, we multiply by 1000. So, they are suggesting the healthy controls had 2000 pg/mL and the RA patients had around 20,000 pg/mL. That healthy control level in the RA study is higher than what the 2022 study found, but it's still in the ball park, as the subset of the disease cohorts had IL-21 levels of around 20,000 pg/mL. The RA study found an exceptionally good relationship between RA severity (DAS 28 score) and IL-21 levels.

    The authors mention a number of studies in different countries that have found similar results in RA. But they also note some studies that didn't find the relationship.

    So, it looks to me as though the finding of increased IL-21 could tell us something about ME/CFS and GWI. But there is variability in studies of IL-21 in other diseases, which makes me wonder if there are technical difficulties. A study of Kawasaki disease noted
    Therefore, it is encouraging to note that this 2022 study used an ELISA method:
     
  7. Hutan

    Hutan Moderator Staff Member

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    To me, it looks as though this team have some results in the Activin A and IL-21 levels well worth investigating further. But I'm not convinced by their suggestion that seropositivity of cases to antibodies against herpes virus dUTPases is related to action A and IL-21 levels:

    Screen Shot 2022-07-26 at 10.19.13 pm.png
     
  8. Hutan

    Hutan Moderator Staff Member

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    The next bit of the study - testing to see what the ME/CFS sera can do!
    It took me a while to work this out. I didn't find the text very clear, but the Figure 2 caption helps a lot.

    So they took human naive CD4+ T cells and stimulated them with anti-CD3/CD28–coated beads
    1. with nothing else [negative control]
    2. + control sera
    3. + ME/CFS sera
    4. + control sera +IL-12
    5. +ME/CFS sera + IL-12
    6. + Activin A
    7. + IL-12
    8. + Activin A +IL-12 [positive control]


    Screen Shot 2022-07-26 at 10.36.10 pm.png

    The chart shows the percentage of PD-1+CXCR5+ cells after 3 days. (Yes, that's PD-1: Programmed cell death protein, recently discussed elsewhere on the forum.)

    That's a pretty remarkable result in Figure 2b. ME/CFS sera, and especially ME/CFS sera helped along with IL-12, produces the same sort of percentages of T cells expressing PD-1 and CXCR5 as a combination of Activin and IL-12 do.

    What does this mean; is it important? I don't know yet, I'll have to read on. But one thing it tells us is, if this study is right, the ME/CFS sera is not like the control sera under these particular conditions. Could this finding be wrong? They have done quite a lot of replications (as evidenced by the dots).
     
  9. Hutan

    Hutan Moderator Staff Member

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    And the ME/CFS serum isn't just good at making PD-1 + CXCR5+ cells. The authors found that it was considerably better at making T cells expressing CD4+BCL6+ and BCL6+CXCR5+ than the control serum. The results are very nicely clustered, and there's a decent number of replicates.

    Screen Shot 2022-07-26 at 11.04.20 pm.png
     
  10. Hutan

    Hutan Moderator Staff Member

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    The next part of the study was to begin to find out 'what's in the ME/CFS serum that makes it different'? They looked at whether activin A was part of the story by adding follistatin-315 (FST), an activin inhibitor. Figure 3 B shows that the addition of FST did reduce the percentage of those T cells expressing PD-1 and CXCR5 produced by the ME/CFS serum by a lot, as it did for the positive control (Activin A + IL-12).

    Screen Shot 2022-07-26 at 11.16.39 pm.png

    Figure 3c looks at how much IL-21 the cells incubated for 3 days were secreting.

    Screen Shot 2022-07-26 at 11.23.42 pm.png
    The cells exposed to 3 days of control sera weren't secreting much IL-21. Relatively speaking, the cells exposed to 3 days of ME/CFS sera were secreting a lot more IL-21. Adding the FST (the activin blocker) to the ME/CFS sera reduced IL-21 secretion down to nearly control sera levels.

    Look at the relatively huge amount of IL-21 secreted by the T cells after three days of ME/CFS sera and IL-12. Again, the activin blocker knocks IL-21 levels down.
     
  11. Hutan

    Hutan Moderator Staff Member

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    Gosh, this paper has a lot in it.

    Next up - can EBV and HHV-6A dUTPase proteins induce the secretion of regulators of the T cell differentiation?
    Well, yes, it seems they can.

    At this point, I think I need to know more about viral dUTPase. Brandon Cox and some of the other co-authors of this paper have co-authored
    Viral dUTPases: Modulators of Innate Immunity 2022 Ariza at al
    It's mostly a survey of the various sorts of viral dUTPases - I think this is the key part for our current purposes:

    The idea of viral dUTPases in ME/CFS is covered in a recent paper discussed on the forum:
    Article: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: The Human Herpesviruses Are Back! 2021, Ariza

    I'm not sure that things are so 'demonstrated fairly conclusively' as this team suggests, but their work has been funded by the NIH for some time.

    I'll call it a night for now, but I hope I or someone else can sift through the rest of the paper at some point.
     
    Last edited: Jul 26, 2022
  12. NelliePledge

    NelliePledge Moderator Staff Member

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    I looked up IL 21 on Wikipedia. As a non scientist what jumped out to me was the use in immunotherapy for cancer and flu like symptoms being a side effect https://en.m.wikipedia.org/wiki/Interleukin_21

    in all the time skimming through trying to get the gist at my low level of understanding over the last few years the results plots rarely if ever? have differentiated between controls and PWME with clear blue water
     
  13. Hutan

    Hutan Moderator Staff Member

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    Carrying on with this - Figure 4

    Screen Shot 2022-07-27 at 12.37.58 pm.png

    Monocyte-derived human dendritic cells were stimulated with three different dUTPases for a day:
    1. human dUTPase
    2. HHV6-dUTPase
    3. EBV-dUTPase
    and then the culture supernatant was assessed for activin A.
    The viral dUTPase clearly made the cells produce activin A in a way that human dUTPase did not.

    They note that the herpesviruses’ dUTPase proteins also substantially increased the expression of B cell–activating factor (BAFF) and pentraxin-3 - figures are given to support the claim. They say
    It looks convincing.
     
  14. Hutan

    Hutan Moderator Staff Member

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    Figure 4B
    Screen Shot 2022-07-27 at 12.57.56 pm.png

    This team have been busy.

    So this experiment was back to the naive CD4+ T cells, cultured with anti-CD3/anti-CD28-coated beads (why those beads? I'm not sure at the moment).

    Then various things were added:
    1. nothing - control
    2. media that has had dendritic cells incubating in it (DCM) with nothing (plus or minus IL-12)
    3. DCM +HHV-6A dUTPase (plus or minus IL-12)
    4. DCM+ EBV dUTPase (plus or minus IL-12)

    After 3 days, the T cells were examined for T cell follicular helper markers - PD-1 +ICOS+ CXCR5+
    The viral dUTPases clearly increases the conversion of naive T cells to TFH cells, with the addition of IL-12 increasing the response further.
     
  15. Hutan

    Hutan Moderator Staff Member

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    So, the media that has had human dendritic cells incubating in it (DCM) with viral dUTPase can make T cells differentiate to TFH cells. But do these TFH cells do anything much?
    Figure 5

    Screen Shot 2022-07-27 at 1.19.28 pm.png

    Yes, they do. They produce a lot more IL-21.

    If they are re-stimulated with phorbol 12-myristate 13-acetate plus ionomycin (PMA/I), then they produce even more.

    (What's PMA/I? It's not explained in the results section of this paper.
    So, I'll just accept it as an inducer of cells to produce cytokines for now.)

    These cells also produced a lot more IFN-y (interferon gamma), CXCL13 and IL-10.
     
    Last edited: Jul 30, 2022
  16. Snow Leopard

    Snow Leopard Senior Member (Voting Rights)

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    Activin A secretion is associated with other infections as well, it is not specific to EBV.

    Warning speculation about hypothetical pathophysological role of Activin:

    Activin A has been known to sensitise afferent nerves (in conjunction with nerve growth factor) and is also involved in neuroregeneration.

    https://www.sciencedirect.com/science/article/pii/S0012160604002465
    https://www.sciencedirect.com/science/article/abs/pii/S0306452207011827
    https://www.sciencedirect.com/science/article/pii/S0012160606010943

    https://academic.oup.com/stmcls/article/27/6/1330/6402444
    https://www.sciencedirect.com/science/article/abs/pii/S0065242306430043
     
  17. Hutan

    Hutan Moderator Staff Member

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    Abstract of that last paper that @Snow Leopard linked above.
     

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