Our investigation is the first study to demonstrate that NK cytotoxic activity remains consistently decreased in CFS/ME patients during the course of the disease. However, other immune parameters, especially cytokine secretions fluctuate at different time points and therefore demonstrate inconsistencies in their distribution pattern during the course of the disease. The purpose of this investigation was to identify immune markers that can be possibly used as biomarkers for CFS/ME in a longitudinal manner.
Primarily, this longitudinal study has illustrated that NK cytotoxic activity is potentially useful as a biomarker for CFS/ME, since it was the most stable in the CFS/ME patients over the 12 months period of the study. Decreases in cytotoxic activity occur in CFS/ME and in some cases this is associated with differential expression in the levels of cytotoxic molecules [20, 21, 32, 33]. These cytotoxic proteins and their genes including perforin (PRF1), granzyme A (GZMA), granzyme B (GZMB) and granzyme K (GZMK) have been shown to be either increased or decreased in CFS/ME [24, 25, 34]. For example, the perforin gene (PRF1) may increase in some cases of CFS/ME while intracellular perforin protein may be decreased in other CFS/ME patients . Such profound differences in protein and mRNA can reduce the effectiveness of NK cells to induce lysis/cytotoxic activity of viral infected cells. Hence, defective cytotoxic activity in CFS/ME patients may be due to perturbations in the expression of cytotoxic factors resulting in reductions in cytotoxic proteins required for effective lysis of viral and microbial particles. Importantly, NK cells have both activating and inhibitory receptors, successful killing of target cells occurs through integrated signalling by activating and inhibitory receptors, and co-receptors. Inhibitory receptors are activated through the recognition of MHC class I proteins and this inhibits NK cytoxic activity [35, 36]. Activating receptors are important for eliminating tumours, and other microbes through intracellular signal transduction mechanisms that connect them to immunodominant tyrosine based activation motif (ITAMS) adaptor proteins [35–37]. Certain viruses can affect NK receptor signalling thus reducing cytoxic activity. For example, the cytomegalovirus viral genes can regulate NK inhibitory receptor expression preventing the induction of activating receptors . An increase in viral load occuring during the course of CFS/ME, may trigger defective cytotoxic receptor activations hence resulting in compromises to NK cytotoxic activity. The significance of the overall decrease in cytotoxic activity at T3 is unclear.
The exact consequence of alterations in CD56brightCD16- NK cells in CFS/ME is not fully known, however, patterns of CD56brightCD16- NK cells were affected by seasonal changes which may affect NK cytokine production. Incidentally gene expression of IFNG which is an important NK cytokine was significantly decreased in the NK cells of CFS/ME patients , which may be related to the decrease in CD56brightCD16- NK cells. Decreases in CD56brightCD16- NK cells have been observed in coronary heart disease, allergic rhinitis and juvenile rheumatoid arthritis while in diseases such as Chronic Obstructive Pulmonary Disease (COPD) CD56brightCD16- NK cells have been reported to be increased [39, 40].
IL-2, a pro-inflammatory cytokine produced by Th1 cells  is required for naïve CD4+T cell differentiation into Th2 and regulatory T cells (Tregs) in the presence of IL-4 and transforming growth factor beta (TGF-β) respectively . Binding of IL-2 to its high affinity receptor IL-2R induces the proliferation of T cells and memory CD4+ and CD8+T cells [42–44]. It is also has important roles in generating effector functions for B cells, CD56brightNK and CD8+T cells . IL-2 regulates Treg cells and interestingly, CD4+CD25+Foxp3+Treg cells, have been reported to be significantly increased in the CFS/ME patients in comparison to non-fatigued controls . An increase in IL-2 may suggest a shift towards Th1/pro-inflammatory immune response in CFS/ME patients.
Anti-inflammatory IL-10 exerts inhibitory effects on cytokine secretion and impedes pro-inflammatory cytokine secretion by multiple cells including Th1 cells (IFN-γ), macrophages/monocytes (IL-1, IL-T2, IL-8, IL-12 and TNF-) and NK cells (IFN-γ and TNF-α) . A decrease in IL-10 favours an increase in pro-inflammatory responses and this may increase the prevalence of Th1 like cytokines. IL-17A is expressed by Th17 cells, it recruits and activates neutrophils, stimulates the generation of pro-inflammatory cytokines, chemokines and increases antimicrobial gene expression [47–50]. IL-17A is therefore an important immunoregulator during microbial infections as it activates immune cells to secrete pro-inflammatory factors. A decrease in IL-17A may contribute to the prevalence of infections. A possible explanation for the observed changes in the secretion of this cytokine may be related to TGF-β which at optimal levels directly promotes IL-17A generation while reducing IL-2 . Thus, in the CFS/ME patients, TGF-β may be decreased causing an increase in IL-2. Therefore, cytokine release in CFS/ME patients undergoes shifts during the course of the disease where patients may present with either an amplified or depressed anti-inflammatory or pro-inflammatory cytokine profile. These alterations in cytokine secretion may occur during the course of the disease and at different times causing either a shift towards a predominant Th1 or Th2 immune response in CFS/ME [25–27, 52, 53]. This makes it difficult to establish a unique CFS/ME-like inflammatory cytokine profile. The observed pattern of cytokine distribution among our CFS/ME patients is consistent with equivocal findings in the literature [54–58]. In adolescents with CFS/ME cytokine secretions have been observed to be correlated with seasonal variations . Therefore, CFS/ME may be associated with oscillations in pro and anti-inflammatory cytokines, supporting the heterogeneity and multifactorial nature of the disease and the diversity in symptom presentations.