Hypothesis: Increasing serum soluble CD40 ligand (sCD40L) may be a biomarker of ME/CFS and chronic Long COVID progression

by Vijay Iyer, Ph.D.[1,2]

1 Relevant affiliation: Independent Patient-Researcher
2 Other affiliation: Principal Neuroscience Specialist at MathWorks, Inc.

Cite as: Iyer, V. (2023). Hypothesis: Increasing serum soluble CD40 ligand (sCD40L) may be a biomarker of ME/CFS and chronic Long COVID progression. Patient-Generated Hypotheses Journal for Long COVID & Associated Conditions, Vol. 1, 44-48

Abstract

To date, no single blood lab test exists to diagnose or track ME/CFS or chronic Long COVID. Based on existing literature, this article brings together evidence that a molecule secreted by the immune system called sCD40L tends to become increasingly elevated in ME/CFS, Long COVID, and Multiple Sclerosis. These studies, along with what’s known about the role of sCD40L in health and other diseases, suggest sCD40L may be useful to track over time in ME/CFS and Long COVID patients.

Hypothesis

Many studies into myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID (LC) have sought to identify serum or plasma biomarkers. Most have focused on a single point in time, with a growing number identifying multi-marker “signatures” which can accurately classify patients versus healthy controls1-3. Some studies have investigated biomarkers at distinct time points with respect to condition, such as before and after exercise which is known to provoke ME symptoms4,5. Few if any LC and/or ME/CFS biomarker studies have been longitudinal, i.e., tracking the evolution of candidate biomarkers in individual patients over time.

One study from Hornig et al. (2015) explored candidate biomarker time-evolution at the group level, comparing plasma cytokine levels for ME/CFS patient cohorts that were early (< 3 years) and later (> 3 years) in their disease course6. They remarked on one cytokine in particular: soluble CD40 ligand (sCD40L) between the early and late stage ME/CFS patient cohorts, which showed a highly significant progression (p<0.00001 via null hypothesis testing).

ME/CFS is often a post-viral illness7, and recently further evidence has emerged for the progression of sCD40L levels in other post-viral illnesses:

  • Wu et al. (2021) reported on sCD40L as a marker of progression in multiple sclerosis (MS), with a clear increase between cohorts in the earlier (relapsing-remitting) and later (secondary progressive) forms of the disease8. Recent evidence indicates MS also has a viral trigger, specifically Epstein-Barr virus9
  • Patterson et al. studied sCD40L levels in chronic Long COVID cohorts (with predominantly ME/CFS-like symptoms), reporting significant but modest sCD40L elevation in an early study10 and a stronger significant correspondence of sCD40L levels to LC severity (across 4 of 5 symptom questionnaires) in a later study11.

These studies each point to increasing sCD40L correspondence with progression (time and/or symptom), giving rise to the hypothesis that sCD40L levels progressively increase on average during the course of chronic Long COVID and ME/CFS. Whereas Hornig et al. (2015) and Patterson et al. (2021) found opposing effects at an early timepoint for ME/CFS and LC, respectively, both showed evidence for a progressive increase in sCD40L levels. Such progression on average may give indications about the disease mechanism(s), while such progression individually may be an indicator of disease presence and/or severity.

Soluble CD40 ligand (sCD40L), alternatively known as CD154, is a mediator of CD40 receptor immune and inflammatory responses ubiquitous across immune cell types. The ligand form was originally found on the surface of activated T-cells12. But more recently it is recognized platelets are likely the largest source of soluble (circulating) CD40L; and that sCD40L may in turn be the most ubiquitous signaling molecule in the platelet repertoire13,14

The platelet origin of sCD40L may comport with multiple findings of abnormal platelet activation for LC and/or ME/CFS using various experimental methods:

  • Microscopic investigation of platelet-poor plasma from a cohort (n=80) of LC patients identified platelet hyperactivation as a candidate sign15, with similar findings in a report of a smaller cohort (n=25) of ME/CFS patients16.
  • Flow cytometry study of LC patients (n=24) with confirmed cardiopulmonary exercise test (CPET) findings found two markers of platelet activation—P-selectin and platelet-leukocyte aggregates (PLA)—persistently elevated at 6 months post-infection17.
  • RNAseq study of ME/CFS patients (n=30) found abnormally enriched gene sets for platelets (but not other immune cells) post-exercise18.

Beyond platelet activity dysfunction, several suspected pathological pathways for LC and ME/CFS have been linked to sCD40L signaling, including endothelial cell activation19, metabolism-associated cell danger signaling via monocytes20, pathogen-associated molecular pattern (PAMP) activation of B-cell adaptive immunity21, and neurocognitive impairment22.

Taken all together, sCD40L levels in LC and ME/CFS appear to be a readily achievable (via blood sample) and potentially fruitful longitudinal measurement, which may serve as a progressive biomarker and/or an indicator of underlying pathological mechanisms.

Given its progression appears in MS, one caveat is in order: sCD40L is not apt to be a specific marker for LC and/or ME/CFS. It has also been implicated as a marker of cardiovascular disease including stroke23,24 and for several other neurological disorders including Alzheimer’s disease25,26. Any studies of this marker for LC and/or ME/CFS should be designed and interpreted accordingly.


References

1 Klein, J. B., Wood, J. R., Jaycox, J. R., Lu, P., Dhodapkar, R. M., Gehlhausen, J. R., Tabachnikova, A., Tabacof, L., Malik, A. A., Kamath, K., Greene, K., Monteiro, V. V. S., Peña-Hernández, M. A., Mao, T., Bhattacharjee, B., Takahashi, T., Lucas, C., Silva, J. G., McCarthy, D., . . . Iwasaki, A. (2022). Distinguishing features of Long COVID identified through immune profiling. MedRxiv. https://doi.org/10.1101/2022.08.09.22278592

2 Sato, W., Ono, H., Matsutani, T., Nakamura, M., Shin, I., Amano, K., Suzuki, R., & Yamamura, T. (2021). Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome. Brain Behavior and Immunity, 95, 245–255. https://doi.org/10.1016/j.bbi.2021.03.023

3 Schultheiß, C., Willscher, E., Paschold, L., Gottschick, C., Klee, B., Henkes, S., Bosurgi, L., Dutzmann, J., Sedding, D., Frese, T., Girndt, M., Höll, J. I., Gekle, M., Mikolajczyk, R. T., & Binder, M. (2022). The IL-1β, IL-6, and TNF cytokine triad is associated with post-acute sequelae of COVID-19. Cell Reports Medicine, 3(6), 100663. https://doi.org/10.1016/j.xcrm.2022.100663

4 Tsilioni, I., Natelson, B., & Theoharides, T. C. (2022). Exosome‐associated mitochondrial DNA from patients with myalgic encephalomyelitis/chronic fatigue syndrome stimulates human microglia to release IL‐1β. European Journal of Neuroscience, 56(10), 5784–5794. https://doi.org/10.1111/ejn.15828

5 Germain, A., Giloteaux, L., Moore, G. H., Levine, S. C., Chia, J., Keller, B. A., Stevens, J., Franconi, C. J., Mao, X., Shungu, D. C., Grimson, A., & Hanson, M. R. (2022). Plasma metabolomics reveals disrupted response and recovery following maximal exercise in myalgic encephalomyelitis/chronic fatigue syndrome. JCI Insight, 7(9). https://doi.org/10.1172/jci.insight.157621

6 Hornig, M., Montoya, J. G., Klimas, N. G., Levine, S. C., Felsenstein, D., Bateman, L., Peterson, D. A., Gottschalk, C. W., Schultz, A. J., Che, X., Eddy, M. L., Komaroff, A. L., & Lipkin, W. I. (2015). Distinct plasma immune signatures in ME/CFS are present early in the course of illness. Science Advances, 1(1). https://doi.org/10.1126/sciadv.1400121

7 Choutka, J., Jansari, V., Hornig, M., & Iwasaki, A. (2022). Unexplained post-acute infection syndromes. Nature Medicine, 28(5), 911–923. https://doi.org/10.1038/s41591-022-01810-6

8 Wu, Q., Wang, A., Yang, J. H., Martens, J. W., Mills, E., Saad, A., Chilukuri, P., Dowling, C. A., & Mao-Draayer, Y. (2021). Elevated sCD40L in secondary progressive multiple sclerosis in comparison to non-progressive benign and relapsing remitting multiple sclerosis. Journal of Central Nervous System Disease, 13. https://doi.org/10.1177/11795735211050712

9 Bjornevik, K., Cortese, M., Healy, B. C., Kuhle, J., Mina, M. J., Leng, Y., Elledge, S. J., Niebuhr, D. W., Scher, A. I., Munger, K. L., & Ascherio, A. (2022). Longitudinal analysis reveals high prevalence of Epstein-Barr virus associated with multiple sclerosis. Science, 375(6578), 296–301. https://doi.org/10.1126/science.abj8222

10 Patterson, B. W., Guevara-Coto, J., Yogendra, R., Francisco, E. B., Long, E., Pise, A., Rodrigues, H., Parikh, P., Mora, J., & Mora, R. (2021). Immune-based prediction of COVID-19 severity and chronicity decoded using machine learning. Frontiers in Immunology, 12, 700782. https://doi.org/10.3389/fimmu.2021.700782

11 Patterson, B. W., Yogendra, R., Jose, N., Rodrigo, N., Osgood, E., Bream, J. B., Parikh, P., Kreimer, M., Kaplan, G. B., & Zgoda, M. (2022). Targeting the monocytic-endothelial-platelet axis with maraviroc and pravastatin as a therapeutic option to treat Long COVID/post-acute sequelae of COVID (PASC). Research Square. https://doi.org/10.21203/rs.3.rs-1344323/v1

12 Lederman, S., Yellin, M., Krichevsky, A., Belko, J. S., Lee, J. A., & Chess, L. (1992). Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help). Journal of Experimental Medicine, 175(4), 1091–1101. https://doi.org/10.1084/jem.175.4.1091

13 Dewitte, A., Tanga, A., Villeneuve, J., Lepreux, S., Ouattara, A., Desmoulière, A., Combe, C., & Ripoche, J. (2015). New frontiers for platelet CD154. Experimental Hematology & Oncology, 4(1). https://doi.org/10.1186/s40164-015-0001-6

14 Cognasse, F., Duchez, A. C., Audoux, E., Ebermeyer, T., Arthaud, C., Prier, A., Eyraud, M., Mismetti, P., Garraud, O., Bertoletti, L., & Hamzeh-Cognasse, H. (2022). Platelets as key factors in inflammation: Focus on CD40L/CD40. Frontiers in Immunology, 13, 825892. https://doi.org/10.3389/fimmu.2022.825892

15 Pretorius E., Venter, C., Laubscher, G. J., Kotze, M. J., Oladejo, S., Watson, L. R., Rajaratnam, K., Watson, B. W., & Kell, D. B. (2022). Prevalence of symptoms, comorbidities, fibrin amyloid microclots and platelet pathology in individuals with Long COVID/Post-acute sequelae of COVID-19 (PASC). Cardiovascular Diabetology, 21(1), 148. https://doi.org/10.1186/s12933-022-01579-5

16  Nunes, J. M., Kruger, A., Proal, A., Kell, D. B., & Pretorius E. (2022). The occurrence of hyperactivated platelets and fibrinaloid microclots in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Pharmaceuticals, 15(8), 931. https://doi.org/10.3390/ph15080931

17 Camera, M., Brambilla, M., Canzano, P., Becchetti, A., Conti, M., Agostoni, P. G., Pengo, M., Tortorici, E., Mancini, M. E., Andreini, D., Bonomi, A., & Parati, G. F. (2022). Long COVID-19 syndrome: Association of cardiopulmonary impairment with a persistent platelet activation. European Heart Journal, 43(Supplement_2). https://doi.org/10.1093/eurheartj/ehac544.3038

18 Ahmed, F., Vu, L. T., Zhu, H., Iu, D. S. H., Fogarty, E. A., Kwak, Y., Chen, W., Franconi, C. J., Munn, P. R., Levine, S. C., Stevens, J., Mao, X., Shungu, D. C., Moore, G. E., Keller, B. A., Hanson, M. R., Grenier, J. K., & Grimson, A. (2022). Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation. BioRxiv. https://doi.org/10.1101/2022.10.13.512091

19 Henn, V., Slupsky, J. R., Gräfe, M., Anagnostopoulos, I., Förster, R., Müller-Berghaus, G., & Kroczek, R. A. (1998). CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature, 391(6667), 591–594. https://doi.org/10.1038/35393

20 Dai, J., Fang, P., Saredy, J., Xi, H., Ramon, C., Yang, W. H., Choi, E. T., Ji, Y. G., Mao, W., Yang, X., & Wang, H. (2017). Metabolism-associated danger signal-induced immune response and reverse immune checkpoint-activated CD40+ monocyte differentiation. Journal of Hematology & Oncology, 10(1), 141. https://doi.org/10.1186/s13045-017-0504-1

21 Boeglin, E., Smulski, C. R., Brun, S., Milosevic, S., Schneider, P., & Fournel, S. (2011). Toll-like receptor agonists synergize with CD40L to induce either proliferation or plasma cell differentiation of mouse B cells. PLOS ONE, 6(10), e25542. https://doi.org/10.1371/journal.pone.0025542

22 Davidson, D., Hirschman, M. P., Sun, A., Singh, M. V., Kasischke, K. A., & Maggirwar, S. B. (2012). Excess soluble CD40L contributes to blood brain barrier permeability in vivo: Implications for HIV-associated neurocognitive disorders. PLOS ONE, 7(12), e51793. https://doi.org/10.1371/journal.pone.0051793

23 Shami, A., Edsfeldt, A., Bengtsson, E., Nilsson, J., Shore, A. C., Natali, A., Khan, F., Lutgens, E., & Gonçalves, I. (2021). Soluble CD40 levels in plasma are associated with cardiovascular disease and in carotid plaques with a vulnerable phenotype. Journal of Stroke, 23(3), 367–376. https://doi.org/10.5853/jos.2021.00178

24 Li, M., Wang, H., & Gao, Y. (2021). Association of MMP-9, ADMA, and sCD40L with ischemic stroke and correlations with stroke severity: A case-control study. Research Square. https://doi.org/10.21203/rs.3.rs-821626/v1

25 Buchhave, P., Janciauskiene, S., Zetterberg, H., Blennow, K., Minthon, L., & Hansson, O. (2009). Elevated plasma levels of soluble CD40 in incipient Alzheimer’s disease. Neuroscience Letters, 450(1), 56–59. https://doi.org/10.1016/j.neulet.2008.10.091

26 Ots, H. D., Tracz, J. A., Vinokuroff, K., & Musto, A. E. (2022). CD40–CD40L in neurological disease. International Journal of Molecular Sciences, 23(8), 4115. https://doi.org/10.3390/ijms23084115