Received: 10 December 2019 | Revised: 16 March 2020 | Accepted: 18 March 2020 DOI: 10.1111/jcmm.15260 ORIGINAL ARTICLE Unravelling myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS): Gender-specific changes in the microRNA expression profiling in ME/CFS Amanpreet K. Cheema1,2 | Leonor Sarria1 | Mina Bekheit3 | Fanny Collado4,5 | Eloy Almenar-Pérez6 | Eva Martín-Martínez7 | Jose Alegre8 | Jesus Castro-Marrero8 | Mary A. Fletcher1,4,5 | Nancy G. Klimas1,4,5 | Elisa Oltra9 | Lubov Nathanson1,2,3 1 Institute for Neuro Immune Medicine, Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, USA 2 Department of Nutrition, Dr. Kiran C. Patel College of Osteopathic Medicine, Nova Southeastern University, Fort Lauderdale, FL, USA 3 Halmos College of Natural Sciences and Oceanography, Nova Southeastern University, Fort Lauderdale, FL, USA 4 Department of Veterans Affairs, Miami VA Healthcare System, Research Service, Miami, FL, USA 5 South Florida Veterans Affairs Foundation for Research and Education Inc, Fort Lauderdale, FL, USA 6 Escuela de Doctorado, Universidad Católica de Valencia San Vicente Mártir, Valencia, Spain 7 National Health Service, Manises Hospital, Valencia, Spain 8 Vall d'Hebron University Hospital, Vall d'Hebron Research Institute, Universitat Autónoma de Barcelona, Barcelona, Spain 9 School of Medicine, Universidad Católica de Valencia San Vicente Mártir, Valencia, Spain Correspondence Lubov Nathanson, Institute for Neuro Abstract Immune Medicine, Dr. Kiran C. Patel Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystem ill- College of Osteopathic Medicine, Nova Southeastern University, 3321 College Ave., ness characterized by medically unexplained debilitating fatigue with suggested al- Fort Lauderdale, FL 33314, USA. tered immunological state. Our study aimed to explore peripheral blood mononuclear Email: LNathanson@nova.edu cells (PBMCs) for microRNAs (miRNAs) expression in ME/CFS subjects under an ex- Funding information ercise challenge. The findings highlight the immune response and inflammation links This study was supported by NIH awards 1R15NS087604-01A1, 1R21AI124187-01 to differential miRNA expression in ME/CFS. The present study is particularly impor- and Presidential Faculty Research tant in being the first to uncover the differences that exist in miRNA expression pat- and Development Grant from Nova Southeastern University (all awarded to LN). terns in males and females with ME/CFS in response to exercise. This provides new We are grateful to all the participants who evidence for the understanding of differential miRNA expression patterns and post- took part in the studies. The study used PBMC samples collected under CDMRP exertional malaise in ME/CFS. We also report miRNA expression pattern differences award W81XWH-09-2-0071 (PI: NK), NIH associating with the nutritional status in individuals with ME/CFS, highlighting the awards 5R01NS090200-02 (PI: MAF) and 5R01AR057853-04 (PI: NK), UCV-2019- effect of subjects' metabolic state on molecular changes to be considered in clinical 270-001 (PI: EO) and Catalonia Association research within the NINDS/CDC ME/CFS Common Data Elements. The identification for Fibromyalgia, Chronic Fatigue Syndrome/Myalgic Encephalomyelitis of gender-based miRNAs importantly provides new insights into gender-specific ME/ and Environmental Sensitivities/Multiple CFS susceptibility and demands exploration of sex-suited ME/CFS therapeutics. Chemical Sensitivity (ACAF, www.fibro​mialg​ ia.cat). The sponsors had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Opinions, interpretations, conclusions and This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd J Cell Mol Med. 2020;24:5865–5877.  wileyonlinelibrary.com/journal/jcmm | 5865 5866 | CHEEMA et al. recommendations are those of the author KEYWORDS and are not necessarily endorsed by either by the Department of Defense or National exercise, gender, immune function, inflammation, microRNAs, myalgic encephalomyelitis/ Institute of Health. chronic fatigue syndrome, peripheral blood mononuclear cell 1 | I NTRO D U C TI O N 2 | M E TH O DS The underlying mechanisms associated with the onset and progres- 2.1 | Participants and samples sion of myalgic encephalomyelitis/chronic fatigue syndrome (ME/ CFS), a condition characterized by an abrupt or delayed onset of per- A community-based cross-sectional study included 63 clinically sistent or relapsing symptomatology, including memory problems, diagnosed ME/CFS subjects and 55 healthy controls matched for muscle and joint pain, gastrointestinal issues, neurological problems, age (±5 years) and BMI (±2). All individuals with ME/CFS and HCs hormonal imbalance and debilitating fatigue or easy fatigability, re- were recruited from three different geographical areas: Miami/ mains unclear. Still, a decade of research into ME/CFS disease ac- Fort-Lauderdale, Florida, USA, and Barcelona and Valencia, Spain, tivity has provided evidence of immune dysfunction described by as part of larger biomarker-oriented studies. For inclusion, all ME/ persistent immune activation associated with the onset and progres- CFS subjects met the 1994 CDC/Fukuda and 2003 Canadian Case sion of the illness.1-10 definitions for ME/CFS. 6,23-25 Major exclusion criteria included: During the last decade, miRNAs have been emerging as biomarkers active smoking or alcohol history, diabetes, immunodeficiency dis- in ME/CFS due to their regulatory role in the development, maturation orders, cardiovascular diseases, stroke, autoimmune conditions, and proliferation of immune cells.11-17 Brenu et al reported miR-127-3p, malignancy or systemic infection for at least 2 weeks before blood miR-142-5p and miR-143-3p as potential plasma and miR-146a, miR-223 sample collection. All female subjects were asked to complete the and miR-21 as cytotoxic lymphocyte biomarkers for ME/CFS diagno- Gynecologic Questionnaire to assess routine gynecologic param- sis,15,16 and due to this illnesses’ similarity with fibromyalgia, a complex eters and were asked to come for the assessment and collection neuroimmune condition characterized by similar symptomatology, of blood during the first two weeks of their menstrual cycle, if miR-223-3p, miR-451a, miR-338-3p, miR-143-3p and miR-145-5p have pre-menopausal. The details on health-related quality of life and 17,18 19 come into focus as well. Petty et al reported up-regulated miR- exercise challenge are provided in Appendix S1 under measures. 99b, miR-330, miR-126 and miR-30c as potential diagnostic markers for individuals with ME/CFS among the 34 miRNA which were found to be differentially expressed in individuals with ME/CFS. A recent study 2.2 | Isolation of PBMCs and RNA extraction reported significantly diminished miR-let-7i-5p, miR-93-3p and miR- 200a-5p in cerebrospinal fluid of ME/CFS subjects who underwent Up to 8 mL of whole blood/sample were collected in K 2EDTA tubes exercise challenge20 which is important due to the post-exertional mal- (Becton Dickinson) and processed within 2 hours by dilution at 1:1 aise (PEM) being a unique characteristic of ME/CFS. Altogether, despite (v/v) ratio in phosphate-buffered saline solution (PBS) with layer- 21,22 of the evidence of gender-related differences in this illness, a com- ing on top of 1 volume of Ficoll-Paque Premium (GE Healthcare) prehensive analysis of the effects of an exercise testing on differential and separation by density centrifugation at 500× g for 30 minutes miRNA expression between males and females with ME/CFS has not (20°C, brakes off). PBMC layers were isolated and washed with been performed so far. PBS. Isolated PBMC pellets were resuspended in 1 volume of red The present study aimed to investigate peripheral blood mono- blood cell lysis buffer (155 mmol/L NH4Cl, 10 mmol/L NaHCO3, nuclear cells (PBMCs) miRNA expression in ME/CFS subjects in 0.1 mmol/L EDTA and pH 7.4), kept on ice for 5 minutes, and cen- comparison with sedentary healthy controls (HCs) under an exer- trifuged (20°C at 500× g for 10 minutes). Washed pellets were re- cise challenge. The differential expression of miRNAs observed in suspended in freezing medium (90% FBS, 10% DMSO) adjusting ME/CFS was further evaluated in females and males. Our results their concentration to 107 cells/ml, aliquoted and frozen in liquid ni- indicate that miRNA expression differs between female and males trogen until use. Total RNA was extracted using RNAzol (Molecular with ME/CFS both at the baseline, and in response to the exercise Research Center, Inc.) according to the manufacturer's instructions. challenge. In addition, we found that miRNA expression was dif- RNA quality was assessed using Agilent TapeStation 4200 (Agilent). ferent in PBMCs of fasting as compared to non-fasting subjects All RNA samples had RNA Integrity number (RIN) above 7. with ME/CFS, which emphasizes the importance of the National Institute of Health (NIH), National Institute of Neurological Disorders and Stroke (NINDS) and Centers for Disease Control 2.3 | miRNA expression analysis and Prevention (CDC)’s newly adopted ME/CFS Common Data Elements (CDE) principles for detailed reporting of subjects’ We used NanoString nCounter platform (NanoString Technologies) conditions. to evaluate miRNA expression with the nCounter Human miRNA CHEEMA et al. | 5867 TA B L E 1 Baseline clinical characteristics of the study sample (a) who had breakfast; (b) who fasted (a) Variables ME/CFS (n = 35) HC (n = 34) P value Age (y) 46.60 ± 10.52 47.06 ± 11.39 .837 Gender, Female, n (%) 24 (68.57) 21 (61.76) .667 BMI (kg/m2) 26.91 ± 5.63 26.58 ± 4.49 .606 Physical health Physical functioning 43.46 ± 36.74 88.63 ± 23.46 .001 Role physical 26.07 ± 33.06 80.62 ± 33.04 .005 Bodily pain 50.36 ± 28.87 82.14 ± 24.28 .005 General health 33.06 ± 19.55 71.69 ± 23.46 .002 Mental health Vitality 33.28 ± 24.49 70.30 ± 21.17 .007 Social functioning 39.61 ± 24.5 81.82 ± 24.62 <.001 Role emotional 53.33 ± 39.92 88.54 ± 24.83 .023 Mental health 55.55 ± 30.38 71.32 ± 27.61 .595 (b) Variables ME/CFS (n = 28) HC (n = 21) P value Age (y) 54.30 ± 7.97 48.72 ± 5.43 .510 Gender, Female n (%) 13 (53.6) 12 (57.14) .458 BMI (kg/m2) 25.23 ± 3.45 24.85 ± 3.17 .433 Physical health Physical functioning 26.68 ± 22.52 96.33 ± 3.12 .003 Role physical 8.96 ± 27.38 96.57 ± 7.39 <.001 Bodily pain 16.04 ± 17.92 90.14 ± 12.66 .008 General health 19.68 ± 12.97 79.67 ± 9.18 .008 Mental health Vitality 15.14 ± 16.52 79.05 ± 9.45 .003 Social role function 25.71 ± 25.42 97.41 ± 6.31 .002 Emotional function 29.12 ± 27.66 93.55 ± 22.63 <.001 Mental health 39.96 ± 26.83 83.81 ± 8.57 .333 Note: Data are shown as mean ± standard deviation (SD) for continuous values and as numbers of cases (percentages) for categorical values. ME/CFS cases vs healthy control subjects. Significant P-values < 0.05 are shown in bold. Abbreviations: BMI, body mass index; HC, healthy controls; ME/CFS, myalgic encephalomyelitis/chronic fatigue/syndrome; SF-36, 36-item Short Form Health Survey. Panel v3 (NanoString Technologies, GXA-MIR3-12). The panel in- 2.4 | Data analysis cluded unique oligonucleotide tags for over 800 highly curated human miRNAs (from miRbase v21) and five housekeeping genes Raw counts were analysed using NanoString nSolver version 4.0 for normalization of expression between samples (ACTB, B2M, software. We calculated geometric means of negative ligation con- GAPDH, RPL19 and RPLP0). Each sample was analysed by using trols plus two standard deviations for all samples. All count values 100 ng of total RNA for processing and consecutive hybridization below this threshold were excluded from normalization. After that, (21 hours at 65°C) to the probe pairs consisting of Reporter Probe, all the normalization steps were performed according to manufac- which carry the signal on its 5′ end, and Capture Probe, which turers' instructions. holds biotin on its 3′ end. After hybridization, sample cleanup Only miRNAs that were expressed in 75% or more of samples and counting were performed according to the manufacturer's in at least one group were further analysed. We then selected miR- instructions. NAs that showed statistical significance with P < .05. Differentially 5868 | CHEEMA et al. expressed miRNAs in each contrasted group were detected by one- 3.3 | MicroRNA expression stratified by gender way ANOVA using Partek Genomic Suite v7, and false discovery rate (FDR) was calculated for multiple comparisons using the q-value. 26 Only two miRNAs (miR-150-5p and miR-342-3p) were differentially MiRNAs were differentially expressed if they met the following se- expressed between ME/CFS females and healthy females (P < .05), lection criteria: FDR ≤ 0.1, and fold change (FC) of at least 1.5 in but none passed criteria FDR < 0.1 (Figure 2, Table S1b). On the either direction. For some further analyses, we also considered miR- other hand, a total of 11 miRNAs were differentially expressed be- NAs that did not pass FDR ≤ 0.1 but passed unadjusted P < .05 (as tween males with ME/CFS and HCs (P < .05) regardless of fasting indicated in Tables S1-S5 and explained in the Section 3). Data were status and eight passed criteria of FDR < 0.1 (Table S1c). The expres- plotted using GraphPad Prism 5 (GraphPad Software). Age, BMI and sion of miR-423-5p, miR-296-5p and miR-4443 was higher, whereas SF-36 values of the participants were examined with Student's t test miR-223-3p, miR-199-3p, miR-16-5p, miR-142-3p and let-7g-5p ex- for continuous and chi-squared test for categorical variables using pression was lower in ME/CFS males than HC males. Although miR- IBM Statistical Package for the Social Science version 25 (SPSS Inc). 150-5p did not reach FDR < 0.1, its expression was higher in ME/CFS The significance level for all analyses was set at P < .05. males than healthy males (P < .05, Figure 2). 3 | R E S U LT S 3.4 | Response to exercise without stratification by gender 3.1 | Participant characteristics For these comparisons, we only selected data from individuals who The study compared 37 female and 26 male ME/CFS subjects with completed all three time-points in exercise and did not undergo ex- 33 female and 22 male-matched HCs. A total of 24 female and 11 tended fasting before blood draw. For figures and discussion, we male ME/CFS subjects, and 21 female and 13 male-matched HCs focused on miRNAs highlighted across analyses showing P < .05 in underwent exercise challenge (Table 1a). Blood was drawn from non-stratified analysis and at least once in sex-stratified analysis these participants for PBMC isolation at T0 (baseline before the ex- (P < .05). ercise), T1 (VO2 max, peak of exercise challenge) and T2 (four hours after T1, recovery). These individuals had unified breakfast (one ba- nana and a yogurt) 30 minutes before the exercise challenges and 3.4.1 | Comparisons for miRNAs expression in the first blood draw. T0 was designated for participants at baseline response to exercise that did not undergo exercise challenge and fasted overnight before blood draw. In the fasting group, there were 13 female and 15 male A subgroup of individuals who underwent exercise challenge was ME/CFS subjects with 12 females and 9 males as HCs (Table 1b). selected to compare differences in miRNA expression between ME/ No significant difference was observed for age, gender and body CFS and HCs in response to exercise (Figure 3), as detailed in 3.1. mass index (BMI) between individuals with ME/CFS and HCs, re- At T0, a total of 25 miRNAs were identified to be differentially ex- gardless of fasting status. ME/CFS cohort had poor (self-reported, pressed in ME/CFS compared to HCs with P < .05 (Table S2a), and six P < .05) outcomes in all reported domains of SF-36 except mental of them passed criteria of FDR < 0.1 significance. The expression of health (P = .595), as compared to healthy controls, regardless of fast- miR-423-5p was higher in individuals with ME/CFS compared with ing status (Table 1). HCs, whereas miR-22-3p, miR-199-3p, miR-126a-3p, miR-340-5p, miR-22-3p and miR-130a-3p were underexpressed in ME/CFS com- pared with HCs (Figure 3). Among differentially expressed miRNAs 3.2 | MicroRNA expression profile differences with only P < .05, the expression of miR-4443 and miR-150-5p was between ME/CFS individuals and HCs without significantly elevated, whereas miR-374-5p, miR-148a-3p, miR-146a- stratification 5p and miR-223-3p expression was lower in ME/CFS than HC (Figure 3, Table S2a). We analysed the difference in miRNA expression between all in- At T1 (VO2 max), 24 miRNAs were differentially expressed dividuals with ME/CFS and HCs regardless of gender and fasting (P < .05) with seven passing FDR < 0.1 criteria between ME/CFS status (breakfast vs fasting). At T0, the expression of miR-150-5p, and HCs. Out of the miRNAs with FDR < 0.1, the expression of miR- miR-4443, miR-423-5p and miR-342-3p was higher, whereas, for 197-3p, miR-4443, miR-423 (3p and 5p), miR-1260a, miR-125a-5p, miR-199-3p, miR-126a-3p, let-7i-5p and miR-130a-3p, the expres- miR-92a-3p and miR-331-3p was higher in those with ME/CFS than sion was lower in ME/CFS than HCs. However, out of a total of eight HCs. The expression of miR-4443 and miR-22-3p was reduced in miRNAs that were differentially expressed with P < .05, only miR- individuals with ME/CFS than HC (Table S2b). Expression of miR- 150-5p passed criteria of FDR < 0.1 (Figure 1, Table S1a). 150-5p was higher, and miR-199-3p, miR-146a-5p and miR-148-3p Only miRNAs that showed P < .05 in non-stratified and at least were lower in ME/CFS than HCs but did not pass FDR < 0.1 criteria once in gender-stratified analysis were shown in figures. (P < .05). CHEEMA et al. | 5869 F I G U R E 1 miRNA expression profile differences between diagnosed ME/CFS individuals and HCs without stratification by gender or fasting status. Data are shown as normalized counts for ME/CFS (red) and healthy controls (HC; Blue) at T0 for (A) miR-150-5p, (B) miR- 4443, (C) miR-423-5p, (D) miR-199-3p and (E) miR-130a-3p, regardless of fasting status. * denotes P < .05 and # denotes FDR < 0.1 A total of 33 miRNAs were differentially expressed (P < .05) in in PBMCs of individuals with ME/CFS (Figure 3, Table S2c). Though ME/CFS comparing to HC at T2 (recovery, four hours after T1) and FDR < 0.1 was not reached, miR-150-5p expression stayed higher in twelve miRNAs passed criteria of FDR < 0.1 significance. Out of these ME/CFS (P < .05). On the other hand, miR-22-3p, miR-151a-3p, miR- twelve, miR-197-3p, miR-4443, miR-423 (3p and 5p), miR-1260a, 199-3p and miR-126a-3p showed decreased expression in ME/CFS miR-125a-5p, miR-92a-3p and miR-331-3p showed higher expression comparing to HC at T2 (Table S2c). The expression of miR-374b-5p, 5870 | CHEEMA et al. F I G U R E 2 miRNA expression profile differences between diagnosed ME/CFS individuals and HCs at T0. Data are shown as normalized counts for males (♂) and females (♀) ME/CFS (red) and healthy controls (HC; Blue) at T0 for (A) miR-150-5p, (B) miR-4443, (C) miR-423-5p, (D) miR-199-3p and (E) miR-130a-3p, regardless of fasting status. * denotes P < .05 and # denotes FDR < 0.1 miR-148a-3p and miR-223-3p also stayed lower at T2 in ME/CFS not all miRNAs could reach FDR < 0.1 (Figure 3, Tables S2a-c). (P < .05), but FDR criteria was not reached. The expression of some miRNAs was detected or fluctuated in Interesting to note is that regardless of time-points being eval- response to exercise, namely (a) miR-4443 expression fluctuated uated, individuals with ME/CFS showed higher expression of miR- in response to exercise; higher at T0, reduced at T1 and back to 150-5p and miR-423, and reduced miR-199-3p levels, although elevated at T2, (b) miR-125a-5p which was overexpressed at T1, CHEEMA et al. | 5871 F I G U R E 3 miRNA expression profile differences between diagnosed ME/CFS individuals and HCs in response to exercise challenge. Data are shown as normalized counts for ME/CFS (red) and healthy controls (HC; Blue) at T0, T1 and T2 (non-fasting) for (A) miR-150-5p, (B) miR-4443, (C) miR-423-5p, (D) miR-199-3p, (E) miR-125a-5p, (F) miR-223-3p (G) miR-130a-3p, (H) miR-181a-5p and (I) miR-126a-3p. * denotes P < .05 and # denotes FDR < 0.1 underexpressed at T2, but did not show significant differences only miR-363-3p passed criteria of FDR < 0.1 with its expression at T0 and 3) miR-374-5p and miR-148a-5p were underexpressed being lower at T2 than at T1 (Table S3c). No miRNA passed criteria both at T1 and T2 but were not significantly different at T0. It of FDR < 0.1 for T2/T1 comparison for ME/CFS, although miR-4516 shows that expression of these miRNAs is affected by exercise in was significantly underexpressed at T2 (Table S3d). Interestingly, ME/CFS (Figure 3, Tables S2a-c). miR-181a-5p did not pass criteria of FDR < 0.1, the higher expres- sion of miR-181a-5p seen in HCs and ME/CFS between T1 and T0 (P < .05), but lowered expression between T2 and T1, for HCs only 3.4.2 | Comparisons of miRNA expression between (P < .05, Figure 3). time-points in exercise The differential expression of miRNA in response to an exercise 3.5 | Response to exercise stratified by gender challenge was evaluated between ME/CFS and HCs for inter– time-point comparisons (Figure 3). Ten miRNAs reached statisti- 3.5.1 | miRNA expression in response to exercise cal significance of P < .05 for differential expression between T1 stratified by gender and T0 (T1/T0) in HCs (Table S3a), as compared to one in ME/ CFS (Table S3b). No miRNA passed criteria of FDR < 0.1; however, Females: None of the miRNAs passed criteria of FDR < 0.1 for miR-181a-5p showed increased expression at T1 regardless of dis- analyses between ME/CFS females and HC females both at T1 ease status (Figure 3, Table S3a,b). Six miRNAs were differentially and T2 (Figure 4). However, at T1, miR-125a-5p was overex- expressed between T2 and T1 (T2/T1) in HCs (P < .05); however, pressed, whereas miR-181a-5p and miR-146a-5p were among 5872 | CHEEMA et al. F I G U R E 4 miRNA expression profile differences between diagnosed ME/CFS females and healthy females in response to exercise challenge Data are shown as normalized counts for ME/CFS (red ♀) and healthy females (HC; Blue ♀) at T0, T1 and T2 (non-fasting) for (A) miR-150-5p, (B) miR-4443, (C) miR-423-5p, (D) miR-199-3p, (E) miR-125a-5p, (F) miR-223-3p, (G) miR-130a-3p, (H) miR-181a-5p and (I) miR- 126a-3p. * denotes P < .05 and # denotes FDR < 0.1 those with reduced expression in ME/CFS females with P < .05 3.5.2 | Comparisons of miRNA expression between (Figure 4, Table S4a). At T2, miR-146-5p and miR-150-5p were time-points in exercise stratified by gender among those with higher expression (P < .05), whereas miR-199 (3p and 5p) and miR-126a-3p were among the miRNAs with The patterns of miRNA expression were further analysed in ME/CFS reduced expression in ME/CFS females compared with HC fe- and HCs for differences between time-points in both females and males (P < .05; Table S4a,b). males. Eight miRNAs showed differential expression in healthy fe- Males: At T1, 36 miRNAs were differentially expressed be- males for T1/T0, and only overexpressed miR-363-3p passed criteria tween ME/CFS males and HC males and all passed criteria of of FDR < 0.1 (Table S5a). Both increased expression of miR-181a-5p FDR < 0.1. Selectively, the expression of miR-423 (5p and 3p), and the reduced expression of miR-125a-5p could not reach FDR miR-4443, miR-125a-5p and miR-150-5p was higher in ME/CFS criteria (P < .05, Table S5a). For T2/T1 comparison in HC females, males, whereas miR-374-5p (a and b), miR-22-3p, miR-223-3p and nine miRNAs were differentially expressed where miR-125a-5p was miR-146-5p (a and b) were among those having reduced expres- overexpressed and miR-181a-5p, miR-363-3p and miR-4443 were sion in ME/CFS males (Figure 5, Table S4c,). At T2, all 36 miRNAs reduced in expression. However, only miR-181a-5p, miR-363-3p that were differentially expressed between ME/CFS males and passed criteria of FDR (<0.1) significance (P < .05, Table S5b). HC males passed criteria of FDR < 0.1. Out of the 36, miR-423 (5p In ME/CFS females, none passed criteria of FDR < 0.1 for and 3p), miR-125a-5p, miR-4443, miR-150-5p and miR-181a-5p T1/T0 comparison; however, miR-181a-5p showed increased ex- were overexpressed, whereas miR-22-3p, miR-374-5p (a and b), pression (P < .05, Table S5c). For T2/T1 comparison, miR-4516 miR-199-3p and miR-223-3p were reduced in ME/CFS males than expression was reduced (FDR < 0.1). Although the miR-181a-5p HC males (Figure 5; Table S4d). and miR-363-5p did not reach FDR < 0.1, they both showed CHEEMA et al. | 5873 F I G U R E 5 miRNA expression profile differences between diagnosed ME/CFS males and healthy males in response to exercise challenge Data are shown as normalized counts for ME/CFS (red ♂) and healthy males (HC; Blue ♂) at T0, T1 and T2 (non-fasting) for (A) miR-150-5p, (B) miR-4443, (C) miR-423-5p, (D) miR-199-3p, (E) miR-125a-5p, (F) miR-223-3p (G) miR-130a-3p, (H) miR-181a-5p and (I) miR-126a-3p. * denotes P < .05 and # denotes FDR < 0.1 decreased expression at T2 than T1 in ME/CFS females (P < .05), 3.6 | Fasting induced miRNA changes whereas both a- and b-variants of miR-146-5p showed increased expression in ME/CFS females between T2 and T1 (P < .05, The effects of fasting were assessed by evaluating differential ex- Table S4d). pression of miRNAs separately in participants who ate unified break- For T1/T0 comparison for healthy males, none of the miRNAs fast after overnight fasting and those who did not eat breakfast after passed criteria of FDR significance of 0.1. However, miR-186-5p ex- overnight fasting (at baseline only). When looking at comparison of pression increased between T1 and T0 but did not reach FDR < 0.1 miRNA expression between fasting ME/CFS and HCs, only six miR- (P < .05, Table S5e). No miRNA reached FDR criteria for T2/T1 NAs showed differential expression with P < .05, out of which only comparison; expression of miR-222-3p expression was higher and three passed FDR criteria. The expression of miR-150-5p and miR- miR-16-5p was lower in HC males (P < .05, Table S5f). ME/CFS 342-3p was higher, whereas miR-548q expression was lower in ME/ males showed increased expression for miR-125a-5p and miR- CFS than HCs (Table S6a). As discussed earlier, in participants who 15b-5p between T1 and T0, although it did not reach FDR < 0.1 had unified breakfast, regardless of gender, the ME/CFS cohort (Table S5g). For T2/T1 comparison for ME/CFS males, 12 miR- had higher expression of miR-423-5p, miR-4443 and miR-150-5p, NAs were differentially expressed (P < .05) but only miR-548q whereas miR-22-3p, miR-199-3p,miR-374b-5p, miR-126a-3p and passed criteria of FDR < 0.1 significance with reduced expression miR-130a-3p expression was reduced (Table S2a, Figure 3). Both (Table S5h). Although no other miRNA passed criteria of FDR < 0.1, miR-342-3p and miR-548q were not among the 26 differentially increased expression of miR-150-5p and miR-223-3p was observed expressed miRNAs between ME/CFS and HCs who had breakfast (Table S5h). (non-fasting) before blood draw. By contrast, miR-150-5p expression 5874 | CHEEMA et al. appears to be consistently higher in ME/CFS regardless of fast- In this study, miR-150-5p, miR-130a-3p, miR-199-3p, miR-223-3p, ing status. No other differentially expressed miRNA overlapped. miR-126a-3p, miR-4443, miR-374-5p, miR-146a-5p and miR-423 Moreover, the increased expression of miR-150-5p seen in ME/ have emerged as prominent miRNAs whose expression was altered CFS regardless of fasting status, presented higher levels in fasting in ME/CFS. These miRNAs with genes involved in cellular processes as compared to non-fasting suggesting possible modulation in ME/ and immunity as their molecular targets have been detected in other CFS by dietary factors. ME/CFS studies.10-20,27 Brenu et al had detected miR-146a-5p, miR- 223-3p and miR-126a-3p in NK and CD8 + cells, and in plasma of individuals with ME/CFS, respectively.15,16 Bjersing et al detected 3.6.1 | miRNA expression in fasting and non-fasting reduced expression of both miR-223-3p and miR-374-5p in cerebro- ME/CFS compared with HCs stratified by gender spinal fluid and serum of ME/CFS subjects.17,27 The up-regulation of miR-150-5p is seen in both T-cell and B-cell Fasting ME/CFS females had higher miR-150-5p, miR-342-3p, maturation and differentiation via Notch pathway and c-Myb, re- miR-222-3p and miR-223-3p (P < .05) (Table S6b). However, only spectively, and it influences the release of pro-inflammatory cy- miR-548q passed criteria of FDR < 0.1 with reduced expression. tokines suggesting its critical involvement in the modulation of The expression of miR-191-5p, miR-150-5p, miR-223-3p and miR- immunity and inflammatory response. 28-30 342-3p also showed higher expression along with miR-181a-5p MiR-199-3p, regulated by free fatty acids and adipokines, is a and miR-423-5p (P < .05) in fasting ME/CFS males although only negative regulator of NF-κB and IL-8.31 Additionally, low miR-199-3p few passed FDR criteria < 0.1 (Table S6c). MiR-548q was consist- expression, seen in ME/CFS subjects, is linked with poor survival ently lower in fasting ME/CFS males which also passed the FDR outcome in carcinomas possibly affecting the disease-related phys- criteria (Table S6c). iological burden.32,33 The non-fasting ME/CFS females showed six miRNAs as differ- Another dysregulated miR-223 identified in this study is reported entially expressed from healthy females (P < .05), with none reaching in other inflammatory conditions, infections and carcinomas.34 It FDR < 0.1. The expression of miR-150-5p was higher, whereas ex- modulates the TLR4/TLR2/NF-κB/STAT3 signalling pathway conse- pression of miR-126a-3p, miR-130a-3p and miR-374b-5p was lower quently affecting inflammatory cytokine expression35 and controls in ME/CFS than healthy females (Figure 4, Table S6d). In non-fast- inflammatory bowel disease (IBD)-associated intestinal inflamma- ing ME/CFS males, 48 miRNAs were differentially expressed from tion by inhibiting NLRP3 inflammasome.36 healthy males and all were underexpressed (P < .05). All of them It was shown that the cytokines released in response to inflam- passed criteria of FDR < 0.1, namely miR-374-5p (a and b), miR- matory assault, particularly TNF-α, is directly suppressed by miR- 223-3p, miR-199a-3p, miR-146-5p (a and b), miR-148a-3p, miR- 130a-3p, reducing inflammation and associated oxidative stress.37 222-3p and miR-191-5p (Figure 5, Table S6e). The higher inflammatory cytokine expression seen in ME/CFS could Three miRNAs were highlighted here that showed opposite therefore be induced by diminished miR-130a-3p expression in ME/ pattern of expression in ME/CFS between fasting and non-fasting: CFS subjects, as identified in the above-mentioned study.37 (a) the fasting ME/CFS females and males showed overexpressed Another miRNA presenting consistent reduced expression both miR-223-3p opposite to consistently underexpressed pattern in at baseline and at recovery post-exercise is miR-126a-3p, which is non-fasting ME/CFS, even in response to exercise, (b) miR-191-5p linked with inflammation. However, Petty et al19 reported that indi- was overexpressed in both fasting females and males but it was un- viduals with ME/CFS had up-regulated miR-126. The cause of this derexpressed in non-fasting ME/CFS males, (c) the consistent higher conflicting finding could be attributed to the subgroup differences expression of miR-343-3p in fasting ME/CFS which was not identi- within the ME/CFS subject population, possible gender influences fied in other group and (d) underexpressed miR-548q in both fasting due to higher proportion of females in Petty’ study or lifestyle hab- ME/CFS females and males. its. On the other hand, we observed elevated expression of miR- 423 in ME/CFS which is associated with inflammation and is also reported to be overexpressed by Petty et al.19 In a study by Gharbi 4 | D I S CU S S I O N et al,38 miR-423-3p was found to be elevated in veterans exposed to sulphur mustard (SM), a vesicant agent responsible for SM-induced This is the first ME/CFS exercise challenge study which evaluated necrosis and inflammation. gender differences in ME/CFS utilizing miRNA expression profiles. Of the highlighted miRNAs in this study, miR-146a is among the The results of this study have highlighted: (a) miRNAs that are dif- first-studied miRNA for its role as key regulator of the immune and ferentially expressed in ME/CFS, (b) altered miRNAs induced by inflammatory response. Diminished miR-146a expression leads to exercise challenge in ME/CFS, (c) gender influenced miRNAs and overexpression of STAT1 and reduced IFN-γ secretion, resulting in their differential expression in response to exercise in disease loss of repressive effect of regulatory T lymphocytes (Treg).39 state and finally (d) fasting induced miRNA difference, emphasizing miR-374a-5p regulates the expression of ubiquitin ligase, mTOR the possible role they may play in the ME/CFS-associated disease signalling pathway and monocyte chemoattractant protein (MCP)-1, biology. critical in inflammatory and immune response.40,41 CHEEMA et al. | 5875 The overexpressed miR-4443 increases pro-inflammatory cyto- matched healthy controls, particularly in response to exercise. The 41 kines by activated NF-κΒ pathway via targeting TRAF4. In fact, a findings of this study should be useful not only for the understand- consistent feature of ME/CFS is significantly reduced natural killer ing of gender-dependent disease-specific ME/CFS pathology, but (NK) cell function, unique pro- and anti-inflammatory cytokines, also in the identification of therapeutic regimes suited to ME/CFS hypersensitivity responses and viral infections prior to ME/CFS- gender-specific biology demands. associated symptom onset. In relation to exercise response, we successfully identified key AC K N OW L E D G E M E N T S miRNAs which could be critical to the post-exertional recovery-as- The authors acknowledge Dr Malav Trivedi and students of Halmos sociated pathways in ME/CFS. Consistently in ME/CFS, miR-150-5p College of Natural Sciences and Oceanography Leah Orton and and miR-423 (3p and 5p) were overexpressed while miR-199a-3p, Anna Movila for their contributions in relevant literature; students miR-130a-3p, miR-146a-3p and miR-223-3p were underexpressed of Halmos College of Natural Sciences and Oceanography Kenza regardless of stage in the exercise challenge. Additionally, the ex- Schreiber and Angelica Darmenko for assistance with data input and pression of miR-4443 was transiently reduced in ME/CFS after analysis, Beth Gilbert, MS, for formal review, Dr Connie Sol and exercise. Jeffry Cournoyer for carring out exercise challenge for ME/CFS sub- Most of the miRNAs which were found to be reduced in ME/ jects and healthy controls, the Institute for Neuro Immune Medicine CFS, regardless of gender and time-point otherwise confer pro- data management, and clinical research teams for participants' char- tective effect against inflammation.42-44 Inter-time-point compari- acteristics, Dr Vicente Serra and personnel of Umivale, Valencia, sons highlighted miR-181a-5p, miR-125a-5p and miR-363-3p to be Spain, for their help in HCs recruitment, and last but not least all influenced by gender. These miRNAs are associated with TNF-α donors, investigators, students and staff involved with the respec- levels, regulation of differential activation of macrophages and tive research studies. inflammation and mast cells function, respectively, whose expres- sion is affected by exercise.44-47 In this sense, the observed miRNA C O N FL I C T O F I N T E R E S T expression pattern in ME/CFS could be interpreted as an “echo” of The authors confirm that there is no conflict of interest. the imbalance in immune molecules reported in former ME/CFS biomarker studies.1-4 AU T H O R C O N T R I B U T I O N The reduction of miR-22-3p has been reported in ME/CFS at re- LN conceived and designed the experiments; FC, EO, EAP, EMM, covery post-exercise by Baraniuk et al, who evaluated the exercise JA, JCM, MAF and NK provided clinical data; LS performed the ex- induced changes in cerebrospinal fluid miRNAs in Gulf War Illness periments; MB, AC and LN contributed to data analysis; AC, LN, EO, and ME/CFS. 20 We saw this reduction only in males, and interest- JCM, JA, NK and MF wrote and reviewed the manuscript. ingly, the group that displaced this reduction in response to exer- cise in Baraniuk study was mostly males. 20 MiR-22 directly targets E T H I C A L A P P R OVA L A N D C O N S E N T TO PA R T I C I PAT E NLRP3 (Nod-like receptor protein 3), a key protein in the NLRP3 The research was carried out according to the World Medical inflammasome.48 Association Declaration of Helsinki. All subjects signed an informed The HCs recovered from exercise induced down-regulation of consent approved by the appropriate human subject protections both miR-130-3p and miR-223 miRNAs in response to exercise chal- committees (Institutional Review Board of the Nova Southeastern lenge, but it was also only reflected in males. University (NSU) and Miami Veterans Affairs Healthcare system Few studies have evaluated the relationship of fasting with Research Development Committee for Florida, USA; Public Health differential expression of miRNAs, but the risk diet imposes on Research Ethics Committee DGSP-CSISP for Valencia, Spain, and chronic disease development is well known. In fasting ME/CFS, Clinical Ethics Committee of the Vall d'Hebron University Hospital we identified key miRNAs that may play a critical regulatory role in for Barcelona, Spain). ME/CFS metabolic state. The inconsistencies in miRNA expression profiles between studies could have been, at least partly, a result DATA AVA I L A B I L I T Y S TAT E M E N T of fasting-induced processes influencing miRNA expression. It is, The data that support the findings of this study are available in the therefore, extremely important to align assessment and report- Appendix S1 of this article. ing with Common Data Elements (CDE) in human subject research that improves data quality and allows for comparisons across mul- ORCID tiple studies. NIH has recently adopted CDE principles in detailed Jesus Castro-Marrero https://orcid.org/0000-0002-2481-3052 reporting of ME/CFS subjects’ conditions and encourages the Lubov Nathanson https://orcid.org/0000-0003-1038-9083 use of these carefully selected CDEs in ME/CFS research which should be utilized for proper registry towards generalizability of REFERENCES findings.49 1. Fletcher MA, Zeng XR, Maher K, et al. 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