Single cell based phosphorylation profiling identifies alterations in Toll-like receptor 7 and 9 signaling in patients with primary Sjögren?s syndrome

To gain insights into potential dysfunctional intracellular signalling profiles of pSS patients we conducted an exploratory analysis of MAPK/ERK and JAK/STAT signalling networks in peripheral blood mononuclear cells (PBMC) from 25 female pSS patients and 25 female age-matched healthy donors using phospho-specific flow cytometry. We analyzed unstimulated samples, and samples during a 4 hour time period following activation of Toll-like receptor (TLR) 7 and 9.
Conclusion:
We here report increased signaling potentials in PBMCs from pSS patients to TLR7 and -9 stimulation through STAT3 S727 and NF-κB in B cells that correlates with a type I IFN signature. Induction of these pathways could contribute to the generation of a type I IFN signature in pSS. Patients displaying elevated potentiation of aforementioned pathways could therefore benefit from therapies targeting these pathways.
Notes:
Stimulation with TLR7 and 9 ligands was conducted using a 4 hour reverse time course. Barcoded samples included a PBMC sample from a single donor (IC) to assess assay variation. Key 9x barcoding stimulus are as follows- POlow + PB low= Unstimulated, POlow + PBmid= IC (unstimulated), POlow + PBhigh= 3 hours, POmid + PBlow= 15 minutes, POmid + PBmid= 60 minutes, POmid + PBhigh = 4 hours, POhigh + PBlow = 30 minutes, POhigh + PBmid = 2 hours, and POhigh + PBhigh= IC (stimulated). An optimized multiplex flow cytometry protocol for the analysis of intracellular signaling in peripheral blood mononuclear cells