In order to explore changes in the patterns of immune activity in CFS we constructed two distinct association networks linking the expression of 16 cytokines measured in plasma for 40 female patients and 59 case-matched healthy controls (HC). Quantitative analysis of these two networks indicated that their topologies differed far beyond what would be expected by chance alone. Indeed variation separating the patterns of cytokine-cytokine association from each subject group was 10 times greater than the variability found within each group. Interestingly the average cytokine node in either network supported the same overall exchange of mutual information. This being said a typical CFS network node relied on one less connection to do so. This is an important point as it suggests that despite differences in cytokine expression between groups both networks were equally coherent overall (p=0.689, ). Even at the basal levels of cytokine expression found in the HC group the correlation linking cytokines into a network was not only significant (all edges p<0.001) but it was virtually equivalent to the overall strength of association supporting the CFS network. Instead the difference between CFS and HC networks arose from a redistribution in the routing of mutual information with the CFS network relying more strongly on a minority of highly connected hubs. Driving these changes in structure we found that cytokines IL-1b, 2, 4, IFNγ, TNFα became much better integrated into the core CFS network, so much so that these formed a distinct subnetwork. Direct connections to anti-inflammatory cytokine IL-10 also increased substantially in CFS while the reverse was true of IL-13, 17 as well as IL-5 and 6. Despite this local restructuring these very different cytokine networks still shared a similar overall granularity. Using a novel measure of modularity we dissected these cytokine networks and found that two mid-scale communities could be isolated in both the CFS and HC group: clusters I+ and II−. However a closer look at the internal structure of these communities revealed diametrically opposite designs across illness groups. In CFS cytokine nodes in cluster I+ were more sparsely connected and adopted a more hub-like architecture whereas cytokine nodes in cluster II− were more strongly and more uniformly interconnected. The exact opposite is true of these same clusters in the control network. Differences such as these reinforce the notion that CFS manifests not only as a difference in the expression level of individual cytokines but also as an important shift in the patterns of association linking these cytokines.
The emergence of a tight-knit cluster dominated by Th1 cytokines was perhaps the most significant and most visible feature of the CFS network. Consisting of cytokine nodes IL-1b, IL-4, IFN-γ and TNF-α cluster II− also saw the recruitment of cytokines IL-2 and IL-15 from their position in cluster I+ of the HC network. This group became much more tightly associated in CFS and less centered about any individual cytokine. Interestingly IL-2, 4 and 15 belong to a family of cytokines that also includes IL-7, IL-9 and IL-21. Members of this family share a receptor complex consisting of IL-2 specific IL-2 receptor alpha (CD25), IL-2 receptor beta (CD122) and a common gamma chain (γc). It is not surprising therefore to observe a strong association between these network nodes upon immune activation. IL-2 and IL-4 are both T cell growth factors though the latter is a much more effective promoter of B cell proliferation (Burke et al., 1997
). In these data, the IL-4 median concentration was increased 3-fold in CFS while IL-2, IFNγ and TNFα concentrations remained unchanged. This would support the presence of an active Th2 component in CFS and an antagonistic role for IL-4 towards Th1 cytokines such as IFNγ within cluster II−. Additionally new recruits, IL-2 and IL-15, both contribute to NK cell proliferation. Though NK cell response was not assessed directly in this work, the lower levels of IL-15 and unchanged levels of IL-2 observed here appear consistent with reports of deficient NK cell response in CFS (Maher et al., 2005
Contrary to cluster II−, cluster I+ was dominated by cytokines typically associated with innate immunity and/or Th2 adaptive response namely IL-5, 6, 10, 12 and 13. For the most part associations between cytokine nodes in cluster I+ were fewer in number and visibly weaker than those linking their counterparts in cluster II−. Despite having weaker ties the circulating levels of IL-5, IL- 6 and IL-1a were significantly elevated suggesting an established Th-2 inflammatory environment. Indeed in CFS the mean node degree within cluster I+ was 4-fold lower than that of cluster II− () and the centrality index 6-fold higher suggesting a much sparser and more centrally directed pattern of interaction. Especially recognizable in CFS cluster I+ is the relatively strong association of pro-inflammatory cytokine IL-6 with anti-inflammatory counterpart IL-10. Recall that IL-10, though not differentially expressed, shifted from having a weak association with cluster II− in the HC network to this much more central role in cluster I+ opposite IL-6 in CFS. This altered role would have gone unnoticed in a more conventional analysis. Also recognizable are elements of the IL-23/Th17/IL-17 response (Boniface et al., 2008
; Aggarwal et al., 2003
; McGeachy et al., 2007
). The direct antagonism of IL-17 response by IL-2 (Laurence et al., 2007
) observed in the HC network was absent in CFS. Instead an alternative response emerged whereby IL-17, IL-23 and IL-6 were all separated by IL-10. IL-6 typically enhances IL-1b–driven IL-17 production (Louten et al., 2009
; Perona-Wright et al., 2009
) while IL-10 is known to effectively down-regulate Th17 cytokine expression in macrophages and T cells (Gu et al., 2008
). In these data median concentrations of IL-17 and 23 were unchanged despite elevated levels of IL-1b and IL-6. Though Th17 activation was not measured directly these observations suggest that responsiveness of this subset, like that of NK cells, may be altered in CFS.
Another key feature of the CFS network is the central role that the hub nodes LTα and IL-12 () play in linking cytokine clusters I+ and II−. In contrast this role is almost evenly shared between IL-6, IL-15 and IL-2 in the HC network. No longer a member of cluster II− in CFS, the LTα hub nonetheless maintains strong associations to IL-1b, TNFα and IFNγ. Primarily a product of activated T and B-lymphocytes, LTα shares a strong homology with TNFα and IL-1b and is a powerful inducer of both these cytokines (Kasid et al., 1990
). Moreover IFNγ has been shown to increase the number of receptors for TNFα and LTα further promoting their action (Aggarwal et al., 1985
). In opposition to this, IL-4 will inhibit IL-2 triggered production of TNFα and LTα in mixed PBMC populations (Kasid et al., 1990
). The network links identified here indicate that these known responses of IL-1b and TNFα to LTα, and to a lesser extent IFNγ, remained consistently expressed in the data. However, while the expression of IL-1b increased 2-fold in CFS, that of TNFα remained unchanged despite an almost 4-fold increase in LTα. This attenuated TNFα response in CFS could in principle be linked with the absence of IFNγ engagement and the elevated levels of IL-4 (> 3-fold) observed in these patients. In comparison to LTα, the association of IL-12 with the nodes of cluster II− is much weaker. Typically released by macrophages and dendritic cells, IL-12 is known to stimulate the production of IFNγ and TNFα from NK and T cells. This effect is enhanced by IL-2 (Wang et al., 2000
) and to a lesser extent by IL-4 (Bream et al., 2003
), a cytokine normally suppressive of IFNγ production. Though elevated 2-fold in this CFS cohort, the absence of a concordant IFNγ response further supports a dampened sensitivity of NK cells to IL-12 signaling in CFS. This may be due at least partially to inadequate IL-2 priming of IL-12 receptor expression (Wang et al., 2000
) since IL-2 concentrations remained unchanged.
Viral triggers such as EBV and human cytomegalovirus (HCMV) have long been suspected of involvement in the onset and persistence of CFS. Recent evidence of xenotropic murine leukemia virus-related virus (XMRV) involvement in CFS (Lombardi et al., 2009
) further supports this hypothesis. While other causes may underlie the cytokine expression patterns observed in this work many of these are at least consistent with some of the disruptive effects of chronic viral infection. In one potential model, infection with one or several viral agents may trigger or exploit deficient responsiveness of NK cells to IL-12 and LTα, both of which are actively produced by EBV-immortalized B cells (Airoldi et al., 2002
; Thompson et al., 2003
), leading to impaired IFNγ production and Th1 activation. In this scenario increased IL-6, also produced by EBV-infected B cells, together with depressed levels of IL-15 may interfere with LT-α and IL-12 activation of NK cells and the resulting IFN-γ production (Wilson et al., 2001
; Saghafian-Hedengren et al., 2009
). It is important to note however that while many of the patterns found here aligned with known EBV processes others did not; for example the lack of elevated IL-10 (Samanta et al., 2008
) and IL-13 (Tsai et al., 2009
). As very distinct illnesses arise from the expression of specific subsets of the 12 known EBV induced genes (Tsuge et al., 2001
) the notion that CFS may involve a form of restricted viral latency may be worthy of consideration. Finally from a methodological perspective we observed that several significant shifts in network structure involved cytokines that were not differentially expressed across subject groups. This underscores the significance of co-expression analysis in understanding complex illnesses such as CFS. In particular such an analysis makes it possible to detect low-grade immune processes that may operate consistently with relatively modest changes in marker expression.