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Trasncriptional response of human whole blood following TLR2 or TLR4 ligation in vitro

ABSTRACT: Use of human whole blood for transcriptomic analysis has potential advantages over the use of isolated immune cells for studying the transcriptional response to pathogens and their products. Whole blood stimulation can be carried out in a laboratory without the expertise or equipment to isolate immune cells from blood, with the added advantage of being able to undertake experiments using very small volumes of blood. Toll like receptors (TLRs) are a family of pattern recognition receptors which recognise highly conserved microbial products. Using the TLR2 ligand (Pam3CSK4) and the TLR4 ligand (LPS) human whole blood was stimulated for 0, 1, 3, 6, 12 or 24 hours at which times mRNA was isolated and a comparative microarray was undertaken. A common NFκB transcriptional programme was identified following both TLR2 and TLR4 ligation which peaked at between 3 to 6 hours including upregulation of many of the NFκB family members. In contrast an interferon transcriptional response was observed following TLR4 but not TLR2 ligation as early as 1 hour post stimulation and peaking at 6 hours. These results recapitulate the findings observed in previously published studies using isolated murine and human myeloid cells indicating that in vitro stimulated human whole blood can be used to interrogate the transcriptional kinetics response of innate cells to TLR ligands. Our study demonstrates that with a systems biology approach analysis of mRNA isolated from human whole blood can delineate both the temporal response and the key transcriptional differences following TLR2 and TLR4 ligation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE55375 | GEO | 2014/07/11

SECONDARY ACCESSION(S): PRJNA239437

REPOSITORIES: GEO

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Project description:Toll-like receptors (TLRs) are important mediators of chronic inflammation in numerous autoimmune diseases, although the role of these receptors in primary Sjögren’s syndrome (pSS) remains incompletely understood. Previous studies in our laboratory established Myd88 as a crucial mediator of disease, although the upstream signaling events that culminate in Myd88 activation have yet to be identified. To objective of this study was to identify specific Myd88-dependent TLR-related pathways that are dysregulated both locally and systemically in a mouse model of pSS (NOD.B10Sn-H2b/J (NOD.B10)). We performed RNA-sequencing on spleens derived from NOD.B10 mice. We then harvested salivary tissue and spleens from Myd88-sufficient and deficient C57BL/10 (BL/10) and NOD.B10 mice and performed flow cytometry to determine expression of Myd88-dependent TLRs. We then cultured splenocytes with TLR2 and TLR4 agonists and measured IL-6 secretion by ELISA. Next, we evaluated spontaneous and TLR4-mediated inflammatory cytokine secretion in NOD.B10 salivary tissue. Finally, we assessed spontaneous Myd88-dependent cytokine secretion by NOD.B10 salivary cells. We identified dysregulation of numerous TLR-related networks in pSS splenocytes, particularly those employed by TLR2 and TLR4. We found upregulation of TLRs in both the splenic and salivary tissue from pSS mice. In NOD.B10 splenic tissue, B cell TLR1, TLR2, and TLR6 expression was reduced to levels of BL/10 controls in the absence of Myd88. Splenocytes from NOD.B10 mice were hyper-responsive to TLR2 ligation and the endogenous molecule decorin modulated inflammation via TLR4. Finally, we found spontaneous secretion of numerous inflammatory cytokines and this was enhanced following TLR4 ligation in female NOD.B10 salivary tissue as compared to males. Moreover, we found that spontaneous production of salivary IL-6, MCP-1 and TNFa requires Myd88 in pSS salivary tissue. Thus, our data demonstrate that Myd88-dependent TLR pathways contribute to the inflammatory landscape in pSS, and inhibition of such will likely have therapeutic utility.

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Trasncriptional response of human whole blood following TLR2 or TLR4 ligation in vitro

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