Background/Purpose: Systemic lupus erythematosus (SLE) is characterized by loss of immune tolerance to self-antigens and periods of waxing and waning disease. The clinical aspects of SLE differ significantly from individual to individual and are altered during periods of heightened disease activity. Activation of innate and adaptive signaling pathways, such as endosome expressed TLR3, TLR7 and TLR9, are linked to SLE development, yet differences in innate and adaptive immune signaling and responding cytokine production during variable disease activity remains unclear.

Methods: Peripheral whole blood samples of 10 African American healthy controls and 12 SLE patients with either high (SLEDAI³4) or low (SLEDAI<4) disease activity were stimulated with T-cell receptor (TCR), B-cell receptor (BCR), and Toll-like receptor (TLR) ligands for either 4 minutes (TLR and BCR) or 30 minutes (TCR) for phospho-protein analysis, and 24 hours for cytokine analysis of cell culture supernatants. Phospho-protein analysis was assessed by mass cytometry and analyzed using Cytobank. Plasma cytokine and soluble mediator concentrations of stimulated cell culture supernatants were determined by 37-plex assay and by ELISA. Spotfire (version 6.0.1) and GraphPad Prism 5.04 for Windows (GraphPad Software, San Diego, CA) was used for analysis and Mann-Whitney test was used to compare non-normally distributed data. All SLE patients met ACR classification criteria.

Results: SLE patients with high disease activity had a significantly increased fold change in T cell associated cytokines following overnight stimulation, namely sCD40L (p=0.045), IL-2 (p=0.041), and IL-13 (p=0.045) in response to TLR4, TLR7/8 and PMA-Ionomycin compared to patients with low disease activity. In general, most SLE patient cytokines had a decreased fold change in response to stimulation compared to healthy controls (p<0.05). CD4+ T cells and CD8+ T cells exhibited no significant differences in immune signaling following CD3/CD28 stimulation between SLE patients with high and low disease activity. TLR3, TLR4 and TLR9 stimulation drive heightened phosphorylation of STAT5 (p<0.0061) and PLCg₂ (p<0.045) in granulocytes of high SLE disease activity patients compared to low disease activity following Poly I:C, LPS, and CpG, respectively. In contrast, dendritic cells had a reduced signaling response to TLR7/8, TLR9 and BCR stimulation with lower pSTAT3 (TLR7/8) (p=0.0427), pp38 (TLR9) (p=0.0285), and Syk (p=0.0080), pSTAT1 (p=0.046) and pSTAT5 (p=0.0106) (BCR) in high disease activity patients compared to low disease activity. B cells also had reduced phosphorylation of p38 (p=0.0427) in response to TLR4 stimulation of high disease activity patients compared to low disease activity patients.

Conclusion: Our results suggest that altered signaling in response to TLRs in granulocyte and antigen presenting cells may contribute to elevated SLE disease activity by driving T cell proliferation and cytokine production.