Profile of circulating microRNAs in myalgic encephalomyelitis and their relation to symptom severity, and disease pathophysiology
https://www.nature.com/articles/s41598-020-76438-y
Abstract
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex chronic disease, rooted in multi-system dysfunctions characterized by unexplained debilitating fatigue. Post-exertional malaise (PEM), defined as the exacerbation of the patient's symptoms following minimal physical or mental stress, is a hallmark of ME/CFS. While multiple case definitions exist, there is currently no well-established biomarkers or laboratory tests to diagnose ME/CFS. Our study aimed to investigate circulating microRNA expression in severely ill ME/CFS patients before and after an innovative stress challenge that stimulates PEM. Our findings highlight the differential expression of eleven microRNAs associated with a physiological response to PEM. The present study uncovers specific microRNA expression signatures associated with ME/CFS in response to PEM induction and reports microRNA expression patterns associated to specific symptom severities. The identification of distinctive microRNA expression signatures for ME/CFS through a provocation challenge is essential for the elucidation of the ME/CFS pathophysiology, and lead to accurate diagnoses, prevention measures, and effective treatment options.
Discussion
ME/CFS continues to cause significant morbidity worldwide, and it is estimated that 84–91% of persons with ME/CFS symptoms remain undiagnosed because of the lack of diagnostic biomarkers19. Using a two-step strategy, we examined the expression values of miRNAs in the plasma of all enrolled participants (patients with severe ME/CFS and matched healthy controls) at baseline (T0) and after the application of a standardized post-exertional stress challenge (T90). We quantified the changes in miRNA levels between the two time-points using the ∆∆CT method. Our analysis identified eleven miRNAs associated with ME/CFS in response to a post-exertional stress challenge. We established a Random Forest Model using miRNA expression changes (∆∆CT) before and after the post-exertional stress challenge. This unique experimental design allowed the identification of ME/CFS patients versus healthy controls with high accuracy (90%), which ultimately could then be used to predict individuals having ME/CFS. We showed that the biological sex did not influence the miRNAs expression at either baseline or in response to the induction of a PEM. Our results are in sharp contrast with the recent work by Cheema et al.16 showing that men and women with ME/CFS exhibit differential miRNA expression profiles in response to exercise. These conflicting findings could be explained primarily by the use of distinct experimental designs, the use of PBMCs vs plasma as well as by the clinical heterogeneity of ME/CFS cases tested (moderate vs severe)20. The use of the K-means method allowed us to categorize ME/CFS patients into four clusters according to their miRNA expression profiles and corresponding to changes in the severity of their symptoms (Fig. 4). Indeed, the ME/CFS patients classified in clusters 2 and 3 had the most severe symptoms and a majority of their miRNAs were upregulated. Conversely, the ME/CFS patients classified into clusters 1 and 4 had moderate symptoms, and their miRNAs were downregulated.
The majority of the circulating miRNAs identified in our cohort are involved in the regulation of immunity. Most of them are novel and are for the first time associated with ME/CFS (hsa-miR-28-5p, hsa-miR-29-3p, hsa-miR-181a-5p, hsa-miR-374b-5p, hsa-miR-486-5p, hsa-miR-3620-3p, hsa-miR-4433a-5p, hsa-miR-6819-3p) while few others have been previously reported in other ME/CFS cohorts (hsa-miR-127-3p, hsa-miR-140-5p and hsa-miR-150-5p) and replicated in our study for the first time too. Indeed, hsa-miR-127-3p has been previously reported in an Australian ME/CFS cohort13. This miRNA regulates the expression of the BCL6 gene, which encodes a transcription factor called B-cell lymphoma 6 protein that inhibits the expression of Interleukin 10 (IL-10). This anti-inflammatory cytokine plays a central role in limiting host immune responses to pathogens21. It was shown that IL-10 is elevated in the cerebrospinal fluid of some patients suffering from ME/CFS22. Previous works from Almenar-Pérez E. et al. have shown an upregulation of hsa-miR-140-5p expression in PBMCs of ME/CFS patients23. This miRNA regulates the differentiation of T cells and affects CD4 + T cell metabolism24. Moreover, overexpression of hsa-miR-140-5p in some ME/CFS patients could lead to a significant decrease in UL16 protein. This glycoprotein encoded by ULBP1 gene is responsible for the activation of natural killer (NK) cells and T-lymphocytes via the natural killer group 2, member D membrane receptor NKG2D25. More recently, Cheema et al. reporteds the upregulation of hsa-miR-150-5p in PBMCs of ME/CFS patients in response to exercise16. This miRNA is known to be associated with the modulation of immunity and inflammatory response26,27,28, while this miRNA is predicted to regulate many genes that participate in the proliferation of immune cells (Fig. 5E).
Among the novel circulating miRNAs differently expressed in the present study, hsa-miR-28-5p and hsa-miR-29a-3p are significantly associated with CD4 + T cell count29. Of note, hsa-miR-28-5p is predicted to target MS4A1 (CD20) and PLEKHA2 (Pleckstrin homology domain-containing family A member 2) genes, which are known to participate in the immune cell responses and stimulate their differentiation30,31. Similarly, hsa-miR-29a-3p is predicted to target genes such as ADA (Adenosine Deaminase), ITGB1 (Integrin Subunit Beta 1) and STAT3 (Signal transducer and activator of transcription 3), which are involved in many cellular functions including the death of immune cells32,33,34. It should be noted that overexpression of hsa-miR-29a-3p in ME/CFS patients may contribute to the reduction of their ability to respond to certain viral infections by targeting RNase L (ribonuclease L), which is known among others, for its central role in innate immunity. Moreover, RNase L plays a vital role in the modulation of antiviral and anti-proliferative activities mediated by interferon26,35. Furthermore, the expression of hsa-miR-181b-5p is reduced in invariant NK T cell-deficient mice36 and interestingly, severely ill ME/CFS patients also exhibited alteration in their invariant NK T cells37,38. Altered expression of hsa-miR-4433a-5p was previously reported in serum samples of influenza H7N9 infected patients39. Since many ME/CFS patients reported that their disease onset followed a viral infection, this miRNA is of interest given that its predicted targets are involved in the regulation of viral responses (Fig. 5J). Hsa-miR-6819-3p is another miRNA predicted to participate in the abnormal immune responses and PEM occurring in ME/CFS. It is worth mentioning that this miRNA also targets genes that have previously been related to ME/CFS, KLF3 (Krüppel-like factor 3) and TLR3 (Toll-like receptor 3)40,41 (Fig. 5K). Rintatolimod, also known commercially as Ampligen, is a dsRNA that functions as an activating ligand for TLR3 and has been tested in many clinical trials as a treatment for ME/CFS40. Therefore, high expression of hsa-miR-6819-3p could reduce the efficacy of Rintatolimod and might explain the non-responsiveness toward this drug, as previously observed in some ME/CFS patients.
Circulating miRNAs are also altered by exercise and could represent useful biomarkers to characterize PEM occurring in ME/CFS and therapeutic targets to prevent or manage this condition. Among the miRNAs associated with the physiological responses to exercise and post-exertion, Makarova et al. suggested that hsa-miR-181b-5p might play several roles in adapting to physical efforts as an endurance regulator42,43. Shah et al. have reported an overexpression of hsa-miR-181b-5p in participants’ plasma after exercise44. Indeed, exposure of mice to acute exercises resulted in a significant increase in mmu-miR-181b-5p expression in skeletal muscle tissue in young (but not older) mice. Previous work revealed a strong role for hsa-miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease44. Therefore, these results suggest that hsa-miR-181b-5p may play a role in dampening inflammation in response to acute exercise (Fig. 5F). Similarly, expression of hsa-miR-486-5p in response to regular exercise resulted in significantly increased expression in sedentary old men45, contrasting with healthy adults46 showing rather a decrease in hsa-miR-486-5p expression. Of note, acute exercice generates a significant increase in hsa-miR-486-5p expression in young men47. Interestingly, upregulation of hsa-miR-486-5p expression in endurance athletes positively correlates with VO2 max values48. Among other miRNAs associated response to exercise, hsa-miR-3620-3p is the most overexpressed circulating miRNA in endurance athletes49, and its expression is increased in ME/CFS patients, whereas it is decreased in control subjects after the application of our post-exertional stress challenge. This miRNA targets genes involved in the regulation of circadian clock (Fig. 5I) like PER 2 (period circadian protein homolog 2) and PER3 (period circadian protein homolog 3), and could be involved in sleep disturbances occurring in ME/CFS patients50,51.
In the present study, we showed higher expression of hsa-miR-374b-5p in the plasma of ME/CFS compared to healthy controls at baseline, which contradicts the findings of a previous study showing the opposite but which could be explained by the use of PBMCs instead of plasma samples16. Hsa-miR-374b-5p targets many genes predicted to be involved in many ME/CFS symptoms, including mitochondrial dysfunctions, wheat sensitivity, fatigue and vitamin E metabolism36,37,38,44 (Fig. 5G). Moreover, high hsa-miR-374b-5p expression levels at baseline or after PEM induction could play an essential role in the regulation of red blood cells (RBCs) shape and membrane deformability by targeting the SPTB and ACTB genes, which encode β-spectrin and β-actin proteins, respectively52. This is of interest given that RBCs of ME/CFS patients are significantly larger and less deformable compared to those of healthy individuals53.
Among possible limitations, longitudinal studies must be undertaken to characterize the variability of PEM development, symptom severity and duration following the application of our post-exertional stress challenge. While severely affected persons with ME/CFS (housebound or bedridden) cannot be tested by CPET approach, it would be interesting to compare mild to moderately affected patients using both methods to establish their sensitivity and limitations using our panel of circulating microRNAs.
In conclusion, we developed a post-exertional stress challenge that provokes PEM in ME/CFS patients. Measurement of the differential expression of circulating miRNAs in severely affected ME/CFS patients led to the discovery and validation of eleven miRNAs associated with ME/CFS. Based on these different miRNA signatures, machine learning algorithm led to the classification of ME/CFS patients into four clusters associated with symptom severity. These findings may provide a foundation for the development of a new non-invasive test to diagnose ME/CFS patients. These miRNA signatures and clusters could eventually be used to predict responses to pharmacological treatments for ME/CFS, and may even allow clinicians to identify individuals to whom such treatments could be beneficial. In addition, we present possible mechanisms that still need to be validated, by which each of the miRNAs could play a role in the pathogenesis and etiology of ME/CFS.
https://www.nature.com/articles/s41598-020-76438-y
Abstract
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex chronic disease, rooted in multi-system dysfunctions characterized by unexplained debilitating fatigue. Post-exertional malaise (PEM), defined as the exacerbation of the patient's symptoms following minimal physical or mental stress, is a hallmark of ME/CFS. While multiple case definitions exist, there is currently no well-established biomarkers or laboratory tests to diagnose ME/CFS. Our study aimed to investigate circulating microRNA expression in severely ill ME/CFS patients before and after an innovative stress challenge that stimulates PEM. Our findings highlight the differential expression of eleven microRNAs associated with a physiological response to PEM. The present study uncovers specific microRNA expression signatures associated with ME/CFS in response to PEM induction and reports microRNA expression patterns associated to specific symptom severities. The identification of distinctive microRNA expression signatures for ME/CFS through a provocation challenge is essential for the elucidation of the ME/CFS pathophysiology, and lead to accurate diagnoses, prevention measures, and effective treatment options.
Discussion
ME/CFS continues to cause significant morbidity worldwide, and it is estimated that 84–91% of persons with ME/CFS symptoms remain undiagnosed because of the lack of diagnostic biomarkers19. Using a two-step strategy, we examined the expression values of miRNAs in the plasma of all enrolled participants (patients with severe ME/CFS and matched healthy controls) at baseline (T0) and after the application of a standardized post-exertional stress challenge (T90). We quantified the changes in miRNA levels between the two time-points using the ∆∆CT method. Our analysis identified eleven miRNAs associated with ME/CFS in response to a post-exertional stress challenge. We established a Random Forest Model using miRNA expression changes (∆∆CT) before and after the post-exertional stress challenge. This unique experimental design allowed the identification of ME/CFS patients versus healthy controls with high accuracy (90%), which ultimately could then be used to predict individuals having ME/CFS. We showed that the biological sex did not influence the miRNAs expression at either baseline or in response to the induction of a PEM. Our results are in sharp contrast with the recent work by Cheema et al.16 showing that men and women with ME/CFS exhibit differential miRNA expression profiles in response to exercise. These conflicting findings could be explained primarily by the use of distinct experimental designs, the use of PBMCs vs plasma as well as by the clinical heterogeneity of ME/CFS cases tested (moderate vs severe)20. The use of the K-means method allowed us to categorize ME/CFS patients into four clusters according to their miRNA expression profiles and corresponding to changes in the severity of their symptoms (Fig. 4). Indeed, the ME/CFS patients classified in clusters 2 and 3 had the most severe symptoms and a majority of their miRNAs were upregulated. Conversely, the ME/CFS patients classified into clusters 1 and 4 had moderate symptoms, and their miRNAs were downregulated.
The majority of the circulating miRNAs identified in our cohort are involved in the regulation of immunity. Most of them are novel and are for the first time associated with ME/CFS (hsa-miR-28-5p, hsa-miR-29-3p, hsa-miR-181a-5p, hsa-miR-374b-5p, hsa-miR-486-5p, hsa-miR-3620-3p, hsa-miR-4433a-5p, hsa-miR-6819-3p) while few others have been previously reported in other ME/CFS cohorts (hsa-miR-127-3p, hsa-miR-140-5p and hsa-miR-150-5p) and replicated in our study for the first time too. Indeed, hsa-miR-127-3p has been previously reported in an Australian ME/CFS cohort13. This miRNA regulates the expression of the BCL6 gene, which encodes a transcription factor called B-cell lymphoma 6 protein that inhibits the expression of Interleukin 10 (IL-10). This anti-inflammatory cytokine plays a central role in limiting host immune responses to pathogens21. It was shown that IL-10 is elevated in the cerebrospinal fluid of some patients suffering from ME/CFS22. Previous works from Almenar-Pérez E. et al. have shown an upregulation of hsa-miR-140-5p expression in PBMCs of ME/CFS patients23. This miRNA regulates the differentiation of T cells and affects CD4 + T cell metabolism24. Moreover, overexpression of hsa-miR-140-5p in some ME/CFS patients could lead to a significant decrease in UL16 protein. This glycoprotein encoded by ULBP1 gene is responsible for the activation of natural killer (NK) cells and T-lymphocytes via the natural killer group 2, member D membrane receptor NKG2D25. More recently, Cheema et al. reporteds the upregulation of hsa-miR-150-5p in PBMCs of ME/CFS patients in response to exercise16. This miRNA is known to be associated with the modulation of immunity and inflammatory response26,27,28, while this miRNA is predicted to regulate many genes that participate in the proliferation of immune cells (Fig. 5E).
Among the novel circulating miRNAs differently expressed in the present study, hsa-miR-28-5p and hsa-miR-29a-3p are significantly associated with CD4 + T cell count29. Of note, hsa-miR-28-5p is predicted to target MS4A1 (CD20) and PLEKHA2 (Pleckstrin homology domain-containing family A member 2) genes, which are known to participate in the immune cell responses and stimulate their differentiation30,31. Similarly, hsa-miR-29a-3p is predicted to target genes such as ADA (Adenosine Deaminase), ITGB1 (Integrin Subunit Beta 1) and STAT3 (Signal transducer and activator of transcription 3), which are involved in many cellular functions including the death of immune cells32,33,34. It should be noted that overexpression of hsa-miR-29a-3p in ME/CFS patients may contribute to the reduction of their ability to respond to certain viral infections by targeting RNase L (ribonuclease L), which is known among others, for its central role in innate immunity. Moreover, RNase L plays a vital role in the modulation of antiviral and anti-proliferative activities mediated by interferon26,35. Furthermore, the expression of hsa-miR-181b-5p is reduced in invariant NK T cell-deficient mice36 and interestingly, severely ill ME/CFS patients also exhibited alteration in their invariant NK T cells37,38. Altered expression of hsa-miR-4433a-5p was previously reported in serum samples of influenza H7N9 infected patients39. Since many ME/CFS patients reported that their disease onset followed a viral infection, this miRNA is of interest given that its predicted targets are involved in the regulation of viral responses (Fig. 5J). Hsa-miR-6819-3p is another miRNA predicted to participate in the abnormal immune responses and PEM occurring in ME/CFS. It is worth mentioning that this miRNA also targets genes that have previously been related to ME/CFS, KLF3 (Krüppel-like factor 3) and TLR3 (Toll-like receptor 3)40,41 (Fig. 5K). Rintatolimod, also known commercially as Ampligen, is a dsRNA that functions as an activating ligand for TLR3 and has been tested in many clinical trials as a treatment for ME/CFS40. Therefore, high expression of hsa-miR-6819-3p could reduce the efficacy of Rintatolimod and might explain the non-responsiveness toward this drug, as previously observed in some ME/CFS patients.
Circulating miRNAs are also altered by exercise and could represent useful biomarkers to characterize PEM occurring in ME/CFS and therapeutic targets to prevent or manage this condition. Among the miRNAs associated with the physiological responses to exercise and post-exertion, Makarova et al. suggested that hsa-miR-181b-5p might play several roles in adapting to physical efforts as an endurance regulator42,43. Shah et al. have reported an overexpression of hsa-miR-181b-5p in participants’ plasma after exercise44. Indeed, exposure of mice to acute exercises resulted in a significant increase in mmu-miR-181b-5p expression in skeletal muscle tissue in young (but not older) mice. Previous work revealed a strong role for hsa-miR-181b-5p in vascular inflammation in obesity, insulin resistance, sepsis, and cardiovascular disease44. Therefore, these results suggest that hsa-miR-181b-5p may play a role in dampening inflammation in response to acute exercise (Fig. 5F). Similarly, expression of hsa-miR-486-5p in response to regular exercise resulted in significantly increased expression in sedentary old men45, contrasting with healthy adults46 showing rather a decrease in hsa-miR-486-5p expression. Of note, acute exercice generates a significant increase in hsa-miR-486-5p expression in young men47. Interestingly, upregulation of hsa-miR-486-5p expression in endurance athletes positively correlates with VO2 max values48. Among other miRNAs associated response to exercise, hsa-miR-3620-3p is the most overexpressed circulating miRNA in endurance athletes49, and its expression is increased in ME/CFS patients, whereas it is decreased in control subjects after the application of our post-exertional stress challenge. This miRNA targets genes involved in the regulation of circadian clock (Fig. 5I) like PER 2 (period circadian protein homolog 2) and PER3 (period circadian protein homolog 3), and could be involved in sleep disturbances occurring in ME/CFS patients50,51.
In the present study, we showed higher expression of hsa-miR-374b-5p in the plasma of ME/CFS compared to healthy controls at baseline, which contradicts the findings of a previous study showing the opposite but which could be explained by the use of PBMCs instead of plasma samples16. Hsa-miR-374b-5p targets many genes predicted to be involved in many ME/CFS symptoms, including mitochondrial dysfunctions, wheat sensitivity, fatigue and vitamin E metabolism36,37,38,44 (Fig. 5G). Moreover, high hsa-miR-374b-5p expression levels at baseline or after PEM induction could play an essential role in the regulation of red blood cells (RBCs) shape and membrane deformability by targeting the SPTB and ACTB genes, which encode β-spectrin and β-actin proteins, respectively52. This is of interest given that RBCs of ME/CFS patients are significantly larger and less deformable compared to those of healthy individuals53.
Among possible limitations, longitudinal studies must be undertaken to characterize the variability of PEM development, symptom severity and duration following the application of our post-exertional stress challenge. While severely affected persons with ME/CFS (housebound or bedridden) cannot be tested by CPET approach, it would be interesting to compare mild to moderately affected patients using both methods to establish their sensitivity and limitations using our panel of circulating microRNAs.
In conclusion, we developed a post-exertional stress challenge that provokes PEM in ME/CFS patients. Measurement of the differential expression of circulating miRNAs in severely affected ME/CFS patients led to the discovery and validation of eleven miRNAs associated with ME/CFS. Based on these different miRNA signatures, machine learning algorithm led to the classification of ME/CFS patients into four clusters associated with symptom severity. These findings may provide a foundation for the development of a new non-invasive test to diagnose ME/CFS patients. These miRNA signatures and clusters could eventually be used to predict responses to pharmacological treatments for ME/CFS, and may even allow clinicians to identify individuals to whom such treatments could be beneficial. In addition, we present possible mechanisms that still need to be validated, by which each of the miRNAs could play a role in the pathogenesis and etiology of ME/CFS.
