Exercise - induced changes in cerebrospinal fluid miRNAs in Gulf War Illness, Chronic Fatigue Syndrome and sedentary control subjects

Abstract

Gulf War Illness (GWI) and Chronic Fatigue Syndrome (CFS) have similar profiles of pain, fatigue, cognitive dysfunction and exertional exhaustion. Post-exertional malaise suggests exercise alters central nervous system functions. Lumbar punctures were performed in GWI, CFS and control subjects after (i) overnight rest (nonexercise) or (ii) submaximal bicycle exercise. Exercise induced postural tachycardia in one third of GWI subjects (Stress Test Activated Reversible Tachycardia, START). The remainder were Stress Test Originated Phantom Perception (STOPP) subjects. MicroRNAs (miRNA) in cerebrospinal fluid were amplified by quantitative PCR. Levels were equivalent between nonexercise GWI (n = 22), CFS (n = 43) and control (n = 22) groups. After exercise, START (n = 22) had significantly lower miR-22-3p than control (n = 15) and STOPP (n = 42), but higher miR-9-3p than STOPP. All post-exercise groups had significantly reduced miR-328 and miR-608 compared to nonexercise groups; these may be markers of exercise effects on the brain. Six miRNAs were significantly elevated and 12 diminished in post-exercise START, STOPP and control compared to nonexercise groups. CFS had 12 diminished miRNAs after exercise. Despite symptom overlap of CFS, GWI and other illnesses in their differential diagnosis, exercise-induced miRNA patterns in cerebrospinal fluid indicated distinct mechanisms for post-exertional malaise in CFS and START and STOPP phenotypes of GWI.

Figure 1

Overlap of diagnostic criteria for CFS, GWI and major depressive disorder. Diagnostic protocols for CFS, GWI and depression selected different sets of primary and ancillary symptoms. CFS requires fatigue, then confirmation with ≥4 of 8 ancillary criteria. Active depression and other psychiatric diseases are exclusionary diagnoses for CFS. GWI requires 3 of 7 categories of symptoms. Depression requires depressed affect and anhedonia, then sufficient supporting complaints.

Figure 2

Kansas Criteria for Gulf War Illness scoring form based on Steele. ©JNBaraniukMD_17g13. Used with permission of the copyright holder.

Figure 3

Chronic Fatigue Syndrome Symptom Severity Questionnaire. ©JNBaraniukMD_17g13 Used with permission of the copyright holder.

Figure 4

Normalizer miRNAs. Data are shown as Ct for each miRNA (N0 normalizer). The N2 normalizer used (a) miR-489 and (b) miR-490-3p. N3 added (c) miR-127-3p. The N6 normalizer added (d) miR-433, (e) miR-124-3p, and (f) miR-183-3p. Each miRNA had ΔΔCt < 1.0 between groups, ANOVA > 0.05, FDR > 0.10, and were detectable in at least 180 of the 182 subjects. The blue line indicates Ct = 35. Nonexercise groups were control (sc0, grey circles), Chronic Fatigue Syndrome (cfs0, grey triangles), and Gulf War Illness (gwi0, grey diamonds). Post-exercise groups were control (SC, yellow circles), Chronic Fatigue Syndrome (CFS, blue triangles), and the Gulf War Illness START (green squares) and STOPP (red diamonds) phenotypes. Mean ± SD.

Figure 5

Intersection of miRNAs from each normalizer. The intersection of N0, N2, N3, and N6 identified 16 miRNAs that had at least 1 significant difference between groups (central yellow boxes). N0, N2 and N6 and N2, N3 and N6 added one each. Pairs of normalizers identified 6 miRNAs that were not considered significant. N0 was least selective as it identified an additional 12 miRNAs that were considered false positives. Therefore, the miRNAs selected by 3 or 4 normalizers were the set of significantly different miRNAs.

Figure 6

miRNA differences between post-exercise groups. (a) miR-22-3p was not detectable in most of the START subjects (green squares above the blue line at Ct = 35). START had significantly less miR-22-3p than SC (yellow circles) and STOPP (red diamonds) as indicated by bars over top of the groups (HSD < 0.05, FDR < 0.10). In addition, SC had significantly more miR-22-3p than sc0 (grey circles). (b) miR-9-3p was detected in START, but was found in fewer than two thirds of subjects in the other groups. START had significantly more miRNA expression than STOPP (ΔΔCt = 1.6 ± 1.4, mean ± SD, HSD < 0.05, FDR < 0.10).

Figure 7

miRNAs that were significantly elevated in post-exercise compared to appropriate nonexercise control groups. Significant differences between groups were indicated by the bars at the top of the graphs for (a) miR-99b-5p, (b) miR-425-3p, (c) miR-30d-5p, (d) miR-204-5p, and (e) miR-370 (HSD ≤ 0.05, FDR ≤ 0.10, detected with Ct ≤ 35 in more than two thirds of one group per pair). The horizontal blue line indicated Ct = 35. Mean ± SD.

Figure 8

Decreased (a) miR-328 and (b) miR-608 levels in SC, CFS, START and STOPP groups after exercise.

Figure 9

miRNAs reduced by exercise. GWI phenotypes (START and STOPP) and CFS all had reductions in (a) miR-let-7i-5p, (b) miR-200a-5p and (c) miR-93-3p. Sedentary controls had no changes.

Figure 10

Decreased miRNAs after exercise in CFS group. CFS had significant reductions in -(a) 92a-3p, (b) miR-323b-5p, (c) miR-126-5p, (d) miR-19b-3p, (e) miR-505-3p, (f) miR-532-5p, and (g) miR-186-3p, compared to its nonexercise cfs0 comparison group (bars above the groups).