Chronic fatigue syndrome or myalgic encephalomyelitis (CFS/EM) is currently a disease of unknown etiology, which appears suddenly in a previously active person and whose onset appears to be associated with an acute infection in most cases. Until now, CFS patients have been studied without being classified into pathogen subgroups. This article aims to show how from the viral cycle of Epstein Barr virus and its mechanism of immune evasion can be generated a CFS and what are the metabolic and physiological consequences that this entails, which could be responsible for the symptoms of chronic fatigue.
EBV expresses at least 44 miRNAs, most of them with unknown function, and two non-coding RNAs (EBERs). EBV-coded miRNAs control the expression of several cell genes with antiapoptotic functions, but also interfere with innate immune responses and inflammation. Some EBV miRNAs act by suppressing, in infected B cells, the release of pro-inflammatory cytokines such as IL-12, resulting in the suppression of differentiation from CD4 naive cells + to Th1 cells (important antiviral effects that activate macrophages and NK cells to eliminate intracellular pathogens).1
Several EBV miRNAs modulate the immune recognition of newly infected B cells (EBV target cells preferably). Viral miRNAs, in infected B cells, control gene expression of HLA class II and three lysosomal enzymes important for proteolysis and epitope presentation to CD4+ T cells. This allows them to interfere with the processing of peptides and on the class II HLA antigenic presentation. As a consequence of the decrease in the HLA II antigenic presentation, the activation of EBV-specific cytotoxic effector CD4+ T cells and the death of infected B cells is reduced.1
Also, to avoid detection of EBV-specific CD4 T cells, it was found that the latent membrane protein 2A (LMP2A) plays a critical role in negatively regulating the expression of class II MHC molecules in infected B cells. Functionally, LMP2A mimics constitutively activated BCR signaling; however, the LMP2A-activated PI3K pathway mediates the suppression of MHC class II and CD74 in EBV-infected B cells. Previous studies have revealed that CIITA is a major regulator of the expression of class II and CD74 MHC molecules. They demonstrated that LMP2A mediated the reduction of CIITA levels by decreasing the expression of PU.1 and E47.2
EBV-infected B lymphocytes generate a homologue of IL-10 (vIL-10), encoded by the BCRF1 gene of EBV during the prelatent phase and latent phase.3,4 vIL-10 can act on multiple cell types and inhibit the synthesis of cytokines in T cells (inhibits the production of IL-2 and IFN-g by Th1 cells) and NK.5 This cancels out the antiviral functions of effective CD4+ T cells and decreases NK cell-mediated death of infected B cells.6 In addition, it is a potent inhibitor of antigenic presentation, as it reduces the expression of MHC II and the accessory co-stimulation molecules CD80 and CD86 in dendritic cells.7
Other miRNAs interfere with the recognition and destruction of EBV-infected cells by CD8+ T cells. First, miRNAs go directly to TAP2, negatively regulate the entire TAP complex, and reduce class I HLA allotypes that present preferably TAP-dependent epitopes. Second, they repress EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8+ T cells. Thirdly, miRNAs decrease the release of IL-12 by infected B cells, since IL12B is directly suppressed by these miRNAs in infected cells. This repression of IL12B can not only reduce the differentiation of CD4+ T cells, it can also regulate the functions of effector T cells, decreasing the activity of CD8+ T cells specific to EBV.1,8
EBV can infect the CNS by infection with HBMEC. This leads to the rupture of adhesion molecules or narrow bonds of the EBV, achieving the passage of leukocytes (including EBV-infected B lymphocytes) through the capillaries into the surrounding tissue.9 B cells with latent EBV infection are able to release EBERs (two non-coding RNAs). Where the release of EBER1 induces the activation of TLR310 signaling resulting in an increase of pro-inflammatory cytokines (inflammation is generated in the tissue).
As in EBV infection of HBMEC, infection of epithelial cells of the intestinal mucosa by this virus leads to the rupture of the narrow junctions of the intestinal barrier, leading to the passage of bacteria and other substances.11 At the same time, EBV succeeds in infecting plasma cells of the mucosa. Therefore, B cells with latent EBV infection release EBERs. EBER1 activates the signaling of enterocyte TLR3, resulting in the induction of type I IFN and proinflammatory cytokines.10 This activation of TLR3 at the intestinal level reduces the activity of the serotonin transporter (SERT) in enterocytes, therefore, decreases the uptake of serotonin causing an increase of 5-HT extracellular in that tissue.11 This excess serotonin should be collected and transported by platelets, as platelets obtain 5-HT mainly from the intestine. However, platelets do not collect all this excess, as they also express TLR312,13 as enterocytes. As these receptors are activated by infection, serotonin reuptake from these cells decreases.14 This eventually allows serotonin to accumulate in the intestinal mucosa. Platelets activated via TLR3 also excrete the contents of their granules (dense granules contain serotonin).15 The serotonin released by platelets would increase vascular permeability and could favour inflammation in tissues16 where there are EBV-infected cells. All this generates a decrease in the levels of 5-HT in platelets.
Add that under fasting conditions the plasma 5-HT levels of these patients are the same as those of healthy patients. This occurs thanks to alternative transports that eliminate free serotonin from the portal blood17 (prevents excess 5-HT at the intestinal level from reaching the systemic circulation). But in post-prandial conditions, the further stimulation of serotonin release (especially with carbohydrate consumption) saturates transport systems, thus increasing plasma levels of free 5-HT, as has been seen in patients with IBS-D, along with an increase in plasma levels of their 5-HIAA metabolite compared with healthy subjects.18
In addition, the rupture of the narrow junctions of the intestinal barrier allows bacteria and other harmful substances to pass from the lumen into the bloodstream by activating TLR4, which also decreases SERT activity.11Activation of the various serotonin receptors would lead to increased intestinal motility11 , malabsorption problems along with vitamin deficiencies (vitamin A, E, D, K and B12)19 , diarrhea11 , dysautonomia due to communication of the vagus nerve between the enteric and cardiovascular systems20 , significant increase in wakefulness and a reduction in slow-wave sleep21 along with cognitive problems.22 In addition to problems in temperature regulation23 and hormone secretion24.
It should be noted that the environmental factor (EBV infection) not only influences the occurrence of CFS, but also the age at which the first infection occurs and the genetic susceptibility to this infection. That is to say, those patients with genes of MHC class I and II molecules susceptible to develop diseases associated with EBV will have difficulties in fighting infection with this virus. As most of these diseases have numerous polymorphisms of these susceptibility genes, there is a great genetic heterogeneity among patients who develop one of these diseases, which manifests itself as a great phenotypic variability among the different patients who suffer from the same disease. All this is due to the fact that in all vertebrate species the MHC molecules present a high polymorphism. This polymorphism reflects an immune system strategy to prevent the evasion of pathogens from the immune system. By possessing different MHC molecules, individuals are confronted with microbes in a different way, with individuals in a given population being more susceptible and more resistant to a given disease.25
With a view to future research, it would be interesting to begin to classify patients into subgroups according to the possible pathogens involved on the basis of understanding and focusing a possible treatment. Rituximab is currently being studied for this disease and the explanation of why it works in some patients and not in others may be due to the type of pathogen involved. On the other hand, it is necessary to look for common markers based on being able to diagnose the disease. At the metabolic level it behaves in a way similar to a cancer, all the antioxidants (vitamin C, Q10, E…) are reduced to compensate for the high oxidative stress and the appearance of cachexia and constitutional syndrome is common due to the high energy expenditure that the Warburg effect entails. In the most advanced disease there may be a decrease in levels of glutamine, cysteine together with high production of urea and glutamate, this could be a marker of severity and a key point to consider on the basis of supplementing them before implementing any therapy. It should be borne in mind that the role of NK is key in viral infection and, therefore, key in CFS. Above all it is essential to evaluate which pathogen is involved in the clinical picture, how the immune system eludes and therapeutic strategies to reverse the process and return the system to its initial state, for example in this case infection by Epstein Barr the rituximab could be crucial, as the virus acts generating latency mainly in B lymphocytes. Therefore, if infected B lymphocytes were lysed we would reduce the Warburg effect and consequently chronic fatigue, as well as immunological problems (there would no longer be a deficit in the expression of MHC class II molecules). But perhaps rituximab treatment should be given together with antivirals, since the immunosuppressing the patient the EBV could be reactivated and continue to infect. Currently there is a more promising treatment without significant adverse effects, adoptive immunotherapy specific to the Epstein-Barr virus. Where death of EBV-infected B cells is achieved by CD8+ T cells transferred adoptively. This treatment has had promising preliminary results in progressive multiple sclerosis by EBV, with improvements in the patient’s symptoms and signs.
Therefore, several markers are proposed to be performed in patients with post-infection EBV CFS:
- Activated T lymphocytes (CD3+, DR+), (CD4+, DR+): A low level of activated T lymphocytes indirectly indicates a decrease in the antigenic presentation HLA-II. This decrease in CD4+ DR+ T lymphocytes can be seen in other diseases associated with EBV, such as children with EBV-associated haemophagocytic lymphohistiocytosis.26
- To verify in the laboratory the decrease of the antigenic presentation HLA-II on the part of the antigen-presenting cells.
- Molecular typing of the HLA system: to verify the existence of certain HLA alleles predisposed to developing diseases associated with EBV.
- IgG antibodies against nuclear antigen (IgG anti-EBNA): presence of a high number, as in multiple sclerosis.
- Increased CD4+ CD25+ FOXP3+ T reg lymphocytes in infected mucosa and blood.27
- CD4+ CD25+ CCR7- These are the ones that remain in the tissues, and due to the infection there is an increase.27
- Decrease of T reg lymphocytes CD4+ CD25+ CCR7+. These are the ones that recirculate through the lymph nodes. The infection causes the expression of CCR7+27 to decrease.
- Decrease in C3 and/or C4 levels. If C4a is elevated this indicates chronic inflammation.
- Elevation of TGF-B and Il-10 by the increase of T reg.
- Elevation of specific IgA against Epstein Barr in intestinal mucosa. Especially in biopsies of terminal ileum, as it is in the small intestine where they are secreted. The most effective neutralizing antibodies against viruses that infect the host through the mucous membranes are usually of the igA isotype, since they block the virus in the mucous membrane itself, preventing it from accessing its target cell.
Tests 1,2,5,6,7,9 should be present in most CFS patients, as other pathogens also evade the immune system in this way. These pathogens manage to generate an acquired functional immunodeficiency through the expression deficit of class II molecules of the major histocompatibility complex. In other subgroups of SFC for other pathogens it would be necessary to look for specific IgA against the pathogen that we want to see in the intestinal biopsies.
- Takanobu Tagawa, Manuel Albanese, Mickaël Bouvet, Andreas Moosmann, Josef Mautner, Vigo Heissmeyer, Christina Zielinski, Dominik Lutter, Jonathan Hoser, Maximilian Hastreiter, Mitch Hayes, Bill Sugden, Wolfgang Hammerschmidt. Epstein-Barr viral miRNAs inhibit antiviral CD4+T cell responses targeting IL-12 and peptide processing. Journal of Experimental Medicine Sep 2016. Available in: http://jem.rupress.org/content/early/2016/09/07/jem.20160248
- Jiun-Han Lin, Ju-Yin Lin, Ya-Ching Chou, Mei-Ru Chen, Te-Huei Yeh, Chung-Wu Lin, Sue-Jane Lin and Ching-Hwa Tsai. Epstein-Barr virus LMP2A suppresses MHC class II expression by regulating the B-cell transcription factors E47 and PU.1. American Society of Hematology. April 2, 2015. Col. 125 no. 14 2228-2238. Available in: http://www.bloodjournal.org/content/125/14/2228/tab-figures-only?sso-checked=true
- Hatton OL, Arnold-Harris A, Schaffert S, Krams SM, Martinez OM. The Interplay Between Epstein Barr Virus and B Lymphocytes: Implications for Infection, Immunity, and Disease. Immunologic research. 2014;58(0):268-276. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199828/
- Ruth F. Jarret. Etiology of Hodgkin´s Disease. Springer Science & Business Media. Dec 6, 2012. Page 68.
- Slobedman B, Barry PA, Spencer JV, Avdic S, Abendroth A. Virus-Encoded Homologs of Cellular Interleukin-10 and Their Control of Host Immune Function. Journal of Virology. 2009;83(19):9618-9629. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2747999/
- Jochum S, Moosmann A, Lang S, Hammerschmidt W, Zeidler R. ed. The EBV Immunoevasins vIL-10 and BNLF2a Protect Newly Infected B Cells from Immune Recognition and Elimination. Stevenson PG, PLoS Pathogens. 2012;8(5):e1002704. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3355093/
- Palma Ramos Alejandro, Castrillón Rivera Laura Estela, et al. Determinación de IL-10 a partir de células mononucleares humanas estimuladas in vitro con Actinomadura madurae, Nocardia asteroides, Nocardia brasiliensis, Candida albicans y Madurella mycetomatis. Dermatología Rev Mex 2008;52(5):205-10. Available in: http://www.medigraphic.com/pdfs/derrevmex/rmd-2008/rmd085a.pdf
- Albanese M, Tagawa T, Bouvet M, et al. Epstein–Barr virus microRNAs reduce immune surveillance by virus-specific CD8+T cells. Proceedings of the National Academy of Sciences of the United States of America. 2016;113(42):E6467-E6475. Available in:
- Casiraghi C, Dorovini-Zis K and Horwitz MS. Epstein-Barr virus infection of human brain microvessel endotjelial cells: a novel role in multiple sclerosis. Journal of Neuroinmmunology. 2011 Jan;230(1-2):173-7. Available in: https://www.ncbi.nlm.nih.gov/pubmed/20826008/
- Iwakiri D, Zhou L, Samanta M, et al. Epstein-Barr virus (EBV)–encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3. The Journal of Experimental Medicine. 2009;206(10):2091-2099.Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757889/
- Ana Isabel Alcalde Herrero. Importancia del sistema serotoninérgico intestinal. Colegio Oficial de Farmacéuticos de Zaragoza. 2011. Available in: http://www.academiadefarmaciadearagon.es/docs/Documentos/Documento31.pdf
- D’Atri LP, Etulain J, Rivadeneyra L, et al. Expression and functionality of Toll-like receptor 3 in the megakaryocytic lineage.Journal of thrombosis and haemostasis : JTH. 2015;13(5):839-850. Available in: https://www.ncbi.nlm.nih.gov/pubmed/25594115
- Anabel AS, Eduardo PC, et al. Human platelets express Toll-like receptor 3 and respond to poly I:C. Hum. Immunol. 2014 Dec;75(12):1244-51. Available in: https://www.ncbi.nlm.nih.gov/pubmed/25315747
- Foley S, et al. Impaired uptake of serotonin by platelets from patients with irritable bowel syndrome correlates with duodenal immune activation. Gastroenterology. 2011;140:1434–1443. e1. Available in: https://www.ncbi.nlm.nih.gov/pubmed/21315720
- Milka Koupenova, Lauren Clancy, Heather A. Corkrey and Jane E. Freedman. Circulating Platelets as Mediators of Immunity, Inflammation, and Thrombosis. Circulation Research. 2018;122:337-351. Available in:
- Nathalie Cloutier, Alexandre Paré, Richard W. Farndale, et al. Platelets can enhance vascular pemeability. Blood Aug 2012, 120 (6) 1334-1343. Available in: http://www.bloodjournal.org/content/120/6/1334.long?sso-checked=true
- Jason J. Chen, Zhishan Li, Hui Pan, Dennis L. Murphy, Hadassah Tamir, Hermann Koepsell and Michael D. Gershon. Maintenance of Serotonin in the Intestinal Mucosa and Ganglia of Mice that Lack the High-Affinity Serotonin Transporter: Abnormal Intestinal Motility and the Expression of Cation Transporters. Journal of Neuroscience 15 August 2001, 21(16) 6348-6361. Available in: http://www.jneurosci.org/content/21/16/6348?ijkey=da60d13d45f91574ef35ed1a15873216044d463c&keytype2=tf_ipsecsha
- Houghton LA, Atkinson W, Whitaker RP, Whorwell PJ, Rimmer MJ. Increased platelet depleted plasma 5-hydroxytryptamine concentration following meal ingestion in symptomatic female subjects with diarrhoea predominant irritable bowel syndrome. Gut. 2003;52:663–670. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1773651/
- Dukowicz AC, Lacy BE, Levine GM. Small Intestinal Bacterial Overgrowth: A Comprehensive Review. Gastroenterology & Hepatology. 2007;3(2):112-122. Available in: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3099351/
- Iglesias Alfonso José y Estévez Báez Mario. Regulación del Sistema Cardiovascular por el Sistema Nervioso Autónomo. April 13, 2008. Available in: http://fbio.uh.cu/ginvest/mesna/vfc_docs/RegulacionAutonomicaCardiovascular.pdf
- Sorayya Kheirouri, Parinaz Kalejahi, et al. Plasma levels of serotonin, gastrointestinal symptoms and sleep problems in children with autism. Turk. J. Med. Sci. 2016 Dec 20; ¡46(6):1765-1772. Available in:http://journals.tubitak.gov.tr/medical/issues/sag-16-46-6/sag-46-6-27-1507-68.pdf
- Paiseka J.L., Longman R.S., Chambers A.J., et al. Cognitive impairment associated with carcinoid syndrome. Ann. Surf. 2014 Feb;259(2):355-9. Available in: https://www.ncbi.nlm.nih.gov/pubmed/23478527
- Takayuki Ishiwata. Role of serotonergic system in thermoregulation in rats. J. Phys. Fitness Sports Med.,2014, 3(4):445-450. Available in: https://www.jstage.jst.go.jp/article/jpfsm/3/4/3_445/_pdf
- Jorgensen H. S. Studies on the neuroendocrine role of serotonin. Dan. Med. Bull. 2007 Nov; 54(4):266-88. Available in: http://faculty.virginia.edu/brain_map/L3F/Untitled.pdf
- Regueiro González J.R., López Larrea C., González Rodriguez S. y Martínez Naves E. Inmunología: Biología y patología del sistema inmunitario. 4ª edición. Editorial Médica Panamerica, 2010.
- Cheng Yang, Xiujuan Zhu, Ting Zhang and Qing Ye. EBV-HLH children with reductions in CD4+-T cells and excessive activation of CD8+ T cells. Pediatric Research. 2017. 82. 952-957. Available in: https://www.nature.com/articles/pr2017178