Discussion

Fifty-one serum cytokines were measured in a cross-sectional study of 186 ME/CFS patients and 388 healthy controls matched by age and sex. A single serum sample was obtained at baseline without any physical, emotional, or neurocognitive stimulation. Only two cytokines were found to be significantly different on average in ME/CFS patients when compared as a group with healthy controls: TGF-β was elevated, and resistin was lower.

TGF-β has been found to be elevated in ME/CFS patients in five of eight studies (63%), as highlighted in a meta-analysis by Blundell et al. (10). TGF-β is a 112-amino acid protein that provides cells with the pleiotropic capacity to affect cell-developmental programs and behavior, including cell proliferation, differentiation, morphogenesis, tissue homeostasis, and regeneration (19). The principal cell sources of TGF-β include monocytes, macrophages, T cells (primarily regulatory T cells), chondrocytes, and intestinal epithelial cells, involving both innate and adaptive immune responses. Given the pleiotropism of TGF-β and its wide availability, its implication in the pathogenesis of apparently dissimilar conditions such as Marfan syndrome (20), cancer (in both control and development) (21), renal fibrosis (22), chronic pulmonary diseases (23), liver disease (24), and inflammatory bowel disease (IBD) (25) is unsurprising. Because TGF-β has been implicated in the development of cancer, elevation of this cytokine in ME/CFS patients older than 65 y of age could contribute to their possibly increased risk of NHL and two defined NHL subtypes, MZL and DLBCL, following their ME/CFS diagnosis (26). Moreover, apart from ME/CFS, other studies have found and proposed a direct link between circulating levels of TGF-β and the risk of NHL (27, 28).

TGF-β, along with IL-10, is primarily viewed as an anti-inflammatory cytokine. The TGF-β elevation in ME/CFS patients may represent down-regulatory activity by these patients’ immune systems against unremitting inflammation; if so, however, one would expect TGF-β levels to correlate with ME/CFS severity, as was observed with several proinflammatory cytokines in this study. Alternatively, it appears that TGF-β does not always function to counteract inflammation. Despite the overarching view of TGF-β as an immunosuppressive (anti-inflammatory) cytokine, its net effect may depend on the local immunological milieu at target tissues and the overall levels of TGF-β (29). For instance, in patients with active IBD, TGF-β has been found at increased levels in the inflamed gut compared with mucosa unaffected by the disease (25). Thus, elevated levels of TGF-β in ME/CFS patients may actually be detrimental and may be a major factor in promoting relentless inflammation and a “fibrotic” milieu resistant to therapeutic interventions in some ME/CFS patients.

Resistin is a cytokine produced primarily by peripheral blood mononuclear cells (PBMCs) in humans and by adipocytes (i.e., as an adipocytokine) in rodents (30). Resistin in humans appears to have a significant proinflammatory role by targeting PBMCs, endothelial cells, smooth muscle cells, platelets (30), and chondrocytes (31) and by increasing the release of IL-1β, IL-6, and TNF-α via the NF-κB pathway (32). Resistin has been reported to be a marker of inflammation in systemic lupus erythematosus (SLE) and Crohn’s disease in humans (33). It is unclear at this time why resistin had this unusual behavior in our study, increasing with mild to moderate disease severity but decreasing with moderate to severe disease. A similar trend was observed in other cytokines including ICAM1 (Fig. 1). An analogous biological behavior is observed in other disease processes such as hepatitis, in which transaminases increase with the severity of inflammation in the liver but actually decline after a certain level of severity is reached as a result of exhaustion and the inability of the hepatocytes to produce these enzymes.

Remarkably, 17 cytokines were associated with severity in ME/CFS patients. Thirteen of these 17 cytokines are primarily proinflammatory: CCL11, CXCL1, CXCL10, IFN-γ, IL-4, IL-5, IL-7, IL-12, IL-13, IL-17, leptin, G-CSF, and GM-CSF. Interestingly, 11 (65%) of the 17 cytokines and 9 (69%) of the 13 proinflammatory cytokines are classified as “type I” by sharing a similar 3D structure, i.e., a four α-helical bundle structure. Their linear relationship with severity was statistically significant even after correction for multiple comparisons, even though these 17 cytokines did not distinguish cases from controls overall. This apparent paradox is explained by the levels of these cytokines in patients with mild disease being below or in the lower range for healthy controls and the levels in patients with severe disease being in the higher or upper range for healthy controls (Figs. 1 and 2). This dysregulation to extremes of normative range also may explain why several studies, including ours, have reported few or no cytokine levels that distinguish ME/CFS cases from controls (10). Above all, it suggests that severity may be a key variable for subgrouping ME/CFS. Furthermore, the levels of circulating cytokines in response to inflammatory triggers such as infection have been reported by various groups to be lower than in controls for some cytokines and higher for others (34⇓–36). A response with lower levels of cytokines may represent a down-regulatory effort by the immune system in an attempt to attenuate more severe immunopathology, resulting in milder or even no symptomatology. A response with higher levels may indicate that the immune system is dealing with a greater challenge that is more likely to result in immunopathology and symptoms. ME/CFS patients in the mild category (with cytokine levels in the lower range) would be protected from more severe disease through this mechanism, whereas those in the severe category would suffer on the opposite side of the spectrum. In addition to a response to an inflammatory trigger, these cytokine findings associated with severity also suggest a dose–response defect in the metabolism or excretion of cytokines. With the lack of large and long-term longitudinal studies in ME/CFS patients, it is not possible at this time to establish whether patients evolve over time as a continuum from mild, to moderate, to severe disease or if a patient is set to stay within a range of a specific category of severity for the duration of the illness.

A second apparent paradox is harder to explain: The two cytokines that did distinguish cases from controls, TGF-β and resistin, did not exhibit a linear relationship with disease severity. It may be that TGF-β and resistin contribute to ME/CFS pathogenesis independent of disease severity.

One of the challenging clinical features of ME/CFS is the capacity of the illness to persist for several years. Thus, the inflammatory state described here might persist, unabated, for decades. Adipokines have been proposed as mediators and perpetuators of chronic inflammatory diseases (37). In this study, two adipokines were identified as being important: leptin and resistin. Thus, it is biologically plausible that changes in adipocyte tissue (in the bone marrow and/or peripheral tissues) linked to increased production of these adipokines may be a factor in the propagation of an inflammatory state in ME/CFS.

In the data presented here, leptin was found to correlate with disease severity. It also was found to correlate significantly with fatigue severity in a longitudinal study led by Younger et al. (13). In addition to playing an important role in regulating body weight by promoting satiety and increasing energy consumption, leptin has been identified as a major proinflammatory cytokine involved in innate and adaptive immune responses (38). In addition, leptin has been shown to be involved in neutrophil recruitment, macrophage activation, phagocytosis, activation of NK cells, dendritic cell survival, skewing T cells toward a proinflammatory and Th1 phenotype, and acting as a negative regulator of regulatory T cells (38, 39). Two additional clinical features in ME/CFS could be explained by the leptin levels in our patients. ME/CFS occurs more frequently in women than in men (2). Leptin levels are higher in females than males, even when corrected for confounding variables such as body mass index (BMI) or level of adiposity (40, 41), and therefore some have suggested that leptin may play a role in the influence of sex on the development of diseases including multiple sclerosis and SLE, which predominantly affect females (37); ME/CFS may be another such disease. In addition, ME/CFS patients often complain of significant cognitive and neurological symptoms, and a recent study suggested that neuroinflammation could be a central feature of the disease (42, 43). Recently, adipokines have been invoked as mediators of an ongoing crosstalk between adipose tissue and the CNS that on occasion, following an unknown trigger, can result in neuroinflammation and neurodegenerative diseases (44). Leptin also has been reported recently to up-regulate the recruitment of neutrophils into the brain in a murine model of sepsis induced by systemic administration of LPS, providing another plausible mechanism for its ability to cause neuroinflammation (45). Thus, it is possible that the CNS abnormalities observed in ME/CFS patients can be explained, at least in part, by the ability of leptin and resistin to cross and/or disrupt the blood–brain barrier (46). Moreover, systemic inflammation, such as that found in our patients, has been invoked as a mechanism for neuroinflammation in other neurodegenerative disease models (47⇓–49). In animal models, systemic administration of LPS alone has been shown to result in neuroinflammation (45, 50).

Recently, Hornig et al. (17) reported a group of cytokines that inversely correlated with fatigue duration, and in our cytokine assay we investigated the same 51 cytokines. In our study, several cytokines [GRO-α (CXCL-1), IFN-β, IL-15, IL-1A, IL-1RA, IL-2, IL-8, TGF-α, and TGF-β] also inversely correlated with fatigue duration before controlling for potentially confounding variables (age, nonspecific binding, race, and sex). After correction for age (SI Appendix, Fig. S3), nonspecific binding, and race, only one cytokine, CXCL9, inversely correlated with fatigue duration. When analysis was performed for both disease severity and fatigue duration, the findings for disease severity remained statistically significant for most cytokines, whereas those for fatigue duration did not. It is possible that disease duration and severity interact in their association with cytokine expression. To assess that possibility for each cytokine separately, we fit a regression model that allows mean cytokine expression levels to vary flexibly over the 2D distribution of total scores from the Multidimensional Fatigue Inventory (MFI-20) assessment of disease severity (51) and fatigue duration in years (52). This analysis found no evidence that the relationship between mean cytokine expression and disease severity changes with duration of disease (SI Appendix, Table S4). The importance of analyzing ME/CFS data by severity is further supported by the lack of correlation between fatigue duration and disease severity (SI Appendix, Fig. S4). However, the lack of observed correlation between disease (fatigue) duration and cytokine levels may result from the lower statistical power of the current study, because the sizes of both our overall sample of cases (n = 186) and, especially, of cases with ≤3-y fatigue duration (n = 30), were smaller than those studied by Hornig et al. Thus, our failure to discover any differences between short and long duration does not allow us to rule out definitively the possibility that mean serum levels might differ between these two specific fatigue-duration categories (≤3 y vs. >3 y). A study by Landi et al. (53) measured 31 cytokines in 100 ME/CFS patients with disease of “long duration” and in 79 healthy controls and observed reductions in IL-16, IL-7, and VEGF-A levels in ME/CFS patients. Theirs is the first ME/CFS study to measure IL-16. Although the Landi et al. study did not adjust for disease severity, the authors concluded through multivariate data analysis techniques that IL-16 could be an important candidate for a biomarker profile in ME/CFS (53). Unfortunately, our cytokine assay did not include IL-16.

A nonspecific binding in the Luminex 200 IS system potentially impacting pMFI signals of some cytokines was found in our study (SI Appendix, SI Materials and Methods). This discovery was possible through analysis of residual data that usually are considered inconsequential in most studies. The adjustment for nonspecific binding was applied to the 51 cytokines of all participants and was significant for some of the cytokines (e.g., IFN-β) (SI Appendix, Fig. S2). This adjustment allowed our study to decrease noise and, we believe, to produce data that may be closer to the actual biological underpinnings of ME/CFS. Multiplex assays that allow the measurement of a significant number of analytes, in contrast to assays that measure only a few analytes, are an efficient tool for advancing our understanding of disease processes. However, pitfalls such as this one for nonspecific binding should be anticipated and, if found, corrected.

Limitations of our study include the cross-sectional design. Future longitudinal studies are necessary to address whether ME/CFS patients remain within their cytokine signature and disease severity category over time or fluctuate among them. We sampled only peripheral blood and not other compartments such as cerebrospinal fluid (CSF). Cytokine studies in the CSF of ME/CFS patients have reported abnormalities even though the sample size has been modest and simultaneous serum samples were not analyzed (54, 55). In one study, a diathesis to allergic and inflammatory responses in CSF, similar to some of our findings, were found in these patients (54, 55). In addition, because resistin and leptin are important adipokines, correction for patients’ BMI would have been ideal. However, this correction was not possible with the current dataset. Findings in this study provide further evidence that ME/CFS likely involves a systemic inflammatory process (17). These findings also support biological plausibility for the propensity of these patients to experience several major and ongoing clinical manifestations, offer a mechanism for the disease’s predilection to affect women and the increased risk for development of NHL, and support the suitability of exploring immunomodulation as a primary or adjuvant therapy (56, 57). Future cytokine research in the peripheral blood of ME/CFS patients should embrace longitudinal designs and seek correlations with neuroradiology, neuroinflammation, and CSF studies. If using multiplex array-based technologies, investigators should pay attention to residual data in regression analysis to identify and correct unrecognized confounders such as nonspecific binding.