Whilst the phenomenon of endotoxin tolerance has been described for over a century, the biological mechanisms underlying this critical immunological process are only now beginning to be elucidated. These include the concomitant upregulation of negative regulators of TLR signaling, induction of micro-RNAs which specifically target products of the NF-κB pathway and the selective silencing of multiple pro-inflammatory genes. Most data emanates from mouse models and there is a degree of discrepancy between genes implicated in the induction of endotoxin tolerance in animal studies and in man such that only IRAK-M has consistently been demonstrated to play a role in both mice and men (8
). The significance of a more comprehensive understanding of the basis of endotoxin tolerance are underlined by the clear role for this phenomenon in the pathogenesis of diseases such as sepsis. The tolerant state is associated with increased susceptibility to subsequent infections and indeed may reflect an underlying pro-inflammatory but paradoxically immunosupressed state (20
). Nonetheless, the highly conserved nature of tolerance and the multiplicity of mechanisms whereby it can be achieved indicate it plays a key role in the regulation of the innate immune response. Indeed it is postulated that endotoxin tolerance has evolved to act as a protective mechanism to prevent the ‘cytokine storm’ associated with onset of sepsis and strongly associated with shock (6
In this study we were interested in addressing the degree to which individuals tolerised to LPS over a short period, thus reflecting silencing of TNF release. We specifically chose a 6 h pretreatment duration because in a clinical setting the onset of sepsis can often be abrupt, with patients deteriorating rapidly over such a time period. Whilst studies of tolerance over 24 h show universal silencing of most inflammatory cytokines (11
), we find there is a large degree of inter-individual variability in the silencing of TNF over 6 h and therefore this timepoint is more applicable to an association study. It is of note that in our study we found little silencing of the IL-6 response, illustrating both the differential regulation of these genes and also the continued viability of cells.
Our interest in identifying genetic markers of endotoxin tolerance has been driven by the observations that responses to innate immune stimuli show a large degree of heritability (12
). We were curious to investigate whether there may be genetic variants associated with differential silencing of TNF release. Our initial finding that the most significantly associated SNP from a panel of over 48000 SNPs was located in the promoter region for TNFR2
, a receptor for the measured parameter (TNF release), was of high biological plausibility. We proceeded to demonstrate that this SNP, rs522807, marks a haplotype associated with increased basal expression of TNFR2 in our dataset and directly modulated allele-specific recruitment of a CRE binding protein. The observation that the basal expression of TNFR2 relates to secondary TNF release was replicated across the cohort, independently of genotype, with a highly significant correlation between expression and secondary response. By contrast there was no association with the expression of TNFR1, suggesting a specific role for TNFR2 in the regulation of LPS induced TNF release. This may reflect the differential signaling pathways of TNFR1 versus TNFR2, with TNFR2 specifically activated by membrane bound TNF and showing relative insensitivity to free TNF (27
). It is plausible that the early expression of membrane bound TNF in response to LPS and subsequent signaling via TNFR2 has later effects on tolerance. This would also explain why the basal expression levels on TNFR2 seem to be of greatest importance in influencing subsequent later responses, with individuals expressing higher levels having consequentially reduced tolerance. We substantiated this finding by using monoclonal antibodies specific for TNFR2 that enhance the affinity of the receptor for TNF (27
). Pretreatment of cells with antibody has no stimulatory effect in its own right, demonstrating that TNFR2 activity only acts to amplify other responses in this experimental model. Moreover, at 2 h cells pretreated with antibody show similar LPS induced TNF release to those pretreated with isotype control. However by 6 h, a time when endotoxin tolerance has developed (20
), there is a highly significant difference between isotype control treated cells and those treated with TNFR2 specific antibodies, indicating positive feedback of LPS induced TNF release, acting through the receptor to enhance further TNF production. Using a purified monocyte subset we were able to separately demonstrate that activity involving TNFR2 could modulate tolerance, with monocytes pretreated showing a significant increase in secondary TNF release compared to control cells.
Our data has highlighted the extent of individual variation in the endotoxin tolerance phenotype among healthy individuals and has implications for the tailored use of therapeutic interventions to modulate the inflammatory response. We have shown how expression quantitative trait mapping in primary human tissue can be used to identify functionally relevant polymorphisms for complex immunological responses. This non-biased approach allows the elucidation of pathway genes whose role may have been previously unsuspected, in this case the TNFR2 gene in endotoxin tolerance. This result has significant physiological implications for our understanding of the biological mechanisms regulating endotoxin tolerance and highlights the value of large scale genotyping in the identification of functional variation in innate immune responses in man.