Background/Purpose:

Thymic stromal lymphopoietin (TSLP) is a cytokine that has an important role in inducing Th2 cell differentiation and has also been implicated as a pathogenic factor in the development of inflammatory arthritis.  We have previously shown that human monocyte-derived dendritic cells (DC) secrete significant quantities of TSLP. The precise stimuli which induce TSLP secretion in DC and possible autocrine effects of TSLP production are poorly understood. Endoplasmic reticulum (ER) stress is an important signalling pathway which enables cells to adapt to increased protein synthesis. PRR and ER stress pathways are both engaged in DC following uptake of bacterial pathogens and we have previously shown that both signals synergise to enhance TSLP secretion. Our aim was to identify the key signals that induce and modulate TSLP induction.

Methods:

DC were differentiated from peripheral blood monocytes, purified using positive magnetic selection, by stimulation with IL-4 and GM-CSF. Flow cytometry analysis of MoDC phenotype showed them to be CD1c⁺ CD11b^Hi CD11c^Hi CD14^Lo CD86^Lo and HLA-DR^Hi. Transcriptional analysis was assayed by qRT-PCR following RNA extraction and quantified relative to HPRT; cytokine secretion was assessed by ELISA of cell supernatants.

Results:

Heat killed Gram-negative bacteria including S. typhi, P. aeruginosa and E. coli stimulated TSLP secretion. To further examine the PRR requirements for TSLP expression we utilised the pattern recognition receptor (PRR) agonists, Peptidoglycan (TLR2 and NOD2) ultra pure LPS (TLR4) and particulate forms of β-1,3 glucan (dectin 1). These agonists stimulated TSLP mRNA transcription and protein secretion by DC, with the most potent effects induced by β-1,3 glucan (475pg/ml +/-200 SEM).

As expected, TSLP secretion induced by β-1,3 glucan was dependent on the dectin 1 adaptor molecule Syk. NF-κB and p38/MAPK were also shown to be essential signalling molecules for this response.  Addition of IL-1 receptor antagonist substantially blocked TSLP secretion. Although TNFα was previously shown to enhance TSLP by synovial fibroblasts, neutralisation of TNFα did not reduce TSLP secretion by DC.  Dectin 1 stimulation alone was sufficient to activate ER stress, as demonstrated by eIF2α phosphorylation and XBP-1 splicing. Inhibitors for the ER stress signalling molecules IRE-1α and PERK reduced TSLP secretion, suggesting that ER stress signals contribute significantly to induction of TSLP.

Conclusion:

TLR2, TLR4 and Dectin 1, are important PRRs in the induction of TSLP secretion by human DC. In addition to fungal derived β-1,3 glucans, we show that bacteria can also induce significant quantities of TSLP. TSLP secretion by DC is induced following a complex integration of signals from PRRs, ER stress pathways and cytokine receptors. Since DC have previously been shown to respond to TSLP made by epithelial cells; possible autocrine effects of TSLP on DC, and their influence on T cell differentiation, warrant further exploration. Autocrine effects of TSLP would be especially relevant in primary and secondary lymphoid tissues where epithelial cells are not present, rather than in the periphery.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/human-dendritic-cells-produce-thymic-stromal-lymphopoietin-in-response-to-pattern-recognition-receptor-ligation-and-this-secretion-is-augmented-by-endoplasmic-reticulum-stress/