Impaired immune function in Gulf War Illness

Abstract

Background: Gulf War Illness (GWI) remains a serious health consequence for at least 11,000 veterans of the first Gulf War in the early 1990s. Our understanding of the health consequences that resulted remains inadequate, and this is of great concern with another deployment to the same theater of operations occurring now. Chronic immune cell dysfunction and activation have been demonstrated in patients with GWI, although the literature is not uniform. We exposed GWI patients and matched controls to an exercise challenge to explore differences in immune cell function measured by classic immune assays and gene expression profiling.

Methods: This pilot study enrolled 9 GWI cases identified from the Department of Veterans Affairs GWI registry, and 11 sedentary control veterans who had not been deployed to the Persian Gulf and were matched to cases by sex, body mass index (BMI) and age. We measured peripheral blood cell numbers, NK cytotoxicity, cytokines and expression levels of 20,000 genes immediately before, immediately after and 4 hours following a standard bicycle ergometer exercise challenge.

Results: A repeated-measures analysis of variance revealed statistically significant differences for three NK cell subsets and NK cytotoxicity between cases and controls (p < 0.05). Linear regression analysis correlating NK cell numbers to the gene expression profiles showed high correlation of genes associated with NK cell function, serving as a biologic validation of both the in vitro assays and the microarray platform. Intracellular perforin levels in NK and CD8 T-cells trended lower and showed a flatter profile in GWI cases than controls, as did the expression levels of the perforin gene PRF1. Genes distinguishing cases from controls were associated with the glucocorticoid signaling pathway.

Conclusion: GWI patients demonstrated impaired immune function as demonstrated by decreased NK cytotoxicity and altered gene expression associated with NK cell function. Pro-inflammatory cytokines, T-cell ratios, and dysregulated mediators of the stress response (including salivary cortisol) were also altered in GWI cases compared to control subjects. An interesting and potentially important observation was that the exercise challenge augments these differences, with the most significant effects observed immediately after the stressor, possibly implicating some block in the NK and CD8 T-cells ability to respond to "stress-mediated activation". This has positive implications for the development of laboratory diagnostic tests for this syndrome and provides a paradigm for exploration of the immuno-physiological mechanisms that are operating in GWI, and similar complex syndromes. Our results do not necessarily elucidate the cause of GWI, but they do reveal a role for immune cell dysfunction in sustaining illness.

Figure 1

Heatmap depicting the expression levels of genes separating GWI cases from controls immediately after the exercise challenge. Immediately after the exercise stress challenge is when we expected differences between GWI cases and controls to be maximal so the most likely time to find the most striking differences. Hierarchical clustering of the 141 probe sets correlating with NK cell subset numbers at the T1 time point was used to show that 9 genes were effective in separating cases from controls. High and low expression levels are shown as red and green respectively. Dendogram at the top shows the clustering results and gene names are given on the right side.

Figure 2

Functional network of the 9 NK genes differentiating cases from controls. The genes were overlaid onto a global molecular network developed from information contained in the Ingenuity Pathways Knowledge Base and networks were algorithmically generated based on connectivity. The genes added to the network as connecting molecules are colored grey. The node shape denotes transmembrane receptor (vertical oval), transcription factor (horizontal oval), cytokine (square), kinase (triangle), peptidase (diamond), and a group or complex (double ringed circle). The edges stand for the gene relationship; solid lines indicate a direct interaction, a dashed line an indirect interaction. A solid arrow head between two nodes denotes gene A at arrow base "acts on" gene B at arrow head. Green node color indicates protein correlated to NK cell subset by QTA that differentiates GWI cases from controls.

Figure 3

Change in perforin levels during the exercise challenge time series adjusted for the number of NK and CD8+ cells. The top graph shows intracellular perforin molecules in both NK and CD8 T-cells and the bottom graph the gene expression data (mean signal intensity). Values are mean ± SD. Repeated measures ANOVA on the intracellular perforin levels showed time series differences bordering on the significant (p-value = 0.053), with illness class differences not significant (p-value = 0.092).