The proteome of Toll-like receptor 3–stimulated human immortalized fibroblasts: Implications for susceptibility to herpes simplex virus encephalitis

Abstract

Background Inborn errors in the Toll like receptor 3 (TLR3)-Interferon (IFN) type I and III pathway have been implicated in susceptibility to herpes simplex virus encephalitis (HSE) in children, but most patients studied do not carry mutations in any of the genes presently associated with HSE-susceptibility. Moreover, many patients do not display any TLR3-related fibroblastic phenotype. Objective This suggests the study of other signalling pathways downstream of TLR3 and/or other independent pathways which may contribute to HSE susceptibility. Methods We have used the stable isotope labelling of amino acids in cell culture (SILAC) proteomics methodology to measure changes in the human immortalized fibroblast proteome after TLR3 activation. Results Cells from healthy controls were compared to cells from a patient with a known genetic aetiology of HSE (UNC93B−/−) and also to cells from an HSE patient with an unknown gene defect. Consistent with known variation in susceptibility of individuals to viral infections, substantial variation in the response level of different healthy controls was observed, but common functional networks could be identified, including upregulation of superoxide dismutase 2 (SOD2). The HSE patients show clear differences in functional response networks when compared to healthy patients and also when compared to each other. Conclusions The present study delineates a number of novel proteins, TLR3-related pathways and cellular phenotypes that may help elucidate the genetic basis of childhood HSE. Furthermore, our results reveal SOD2 as a potential therapeutic target for amelioration of the neurological sequelae caused by HSE.

Proteomics in immunity and herpes simplex encephalitis

“…This often has the major obstacle that sufficient numbers of patient samples are difficult to obtain. Since TLR3-dependent pathways are clearly involved in HSV-1 susceptibility, it was considered that it might be possible to use a combination of proteomics and systems biology methods to look for other networks and/or proteins[22]. The basic idea behind this strategy is that the increasing amount of available systems biology information has changed the situation.…”

“…As described in the original publication [22], common functional pathways in healthy individuals implicated in transmigration of immune cells, apoptosis and oxidative stress that were abrogated in HSE patients were discovered. Furthermore, a set of new proteins for further investigation of possible disease aetiologies was identified (Fig.3) and evidence was obtained that manipulation of one of these (SOD2) could have therapeutic benefits.…”

“…Conversely, use of Significance B < 0.05 led to exclusion of large numbers of proteins from the C1 and C2 data sets that had large abundance changes with high reliability relative to real experimental noise. As a compromise, Significance B < 0.05 and H/L cut offs was used to select approximately equal numbers of “most significant” proteins from each sample type [22]. This “equal sampling” was successful in identifying relevant functional networks using GeneGo.…”

“…For example, only 9 of described set of 152 most significant proteins by Perez et all [22] showed overlap with the 601 genes predicted (p < 0.05) to be most closely related to the TLR3 pathway with the Human Gene Connectome approach [32]. Amongst many possibilities, this might arise because systems biology data bases are still too sparse, because aggregation of data from many cell types over many functional contexts obscures relationships for specific contexts, because other information such as post-translational state and subcellular spatial distribution of proteins as well as epigenetic modifications are insufficiently represented, or because proteomics is also detecting responses more distant from the TLR3 pathway.…”

A review with comments on herpes simplex encephalitis in adults

“…New approaches, such as TLR agonists, [30] IL-1 antagonists [31] or vaccines may be helpful in the future.…”