Monoclonal anti-dsDNA antibody 2C10 escorts DNA to intracellular DNA sensors in normal mononuclear cells and stimulates secretion of multiple cytokines implicated in lupus pathogenesis

Abstract

There have been many studies on the mechanisms of internalization of DNA-anti-DNA immune complexes by cells, including the one used for rheumatoid factor-expressing mouse B cells. In parallel, studies on the role of intracellular DNA sensors in the pathogenesis of systemic lupus erythematosus (SLE) have been conducted, including the one using a mouse model lacking one of the sensors. These and other data have established a framework for understanding the pathogenic role of anti-DNA antibodies, but studies on normal cells are limited. Here, we used the monoclonal anti-dsDNA antibody 2C10, 2-kbp dsDNA and healthy human peripheral blood mononuclear cells (PBMCs) to test whether and how 2C10 and/or DNA cause pathology in normal cells. We found that on culture with PBMCs, 2C10 preferentially entered monocytes and that DNA enhanced this internalization. In contrast, DNA alone was not significantly internalized by monocytes, but 2C10 facilitated its internalization. This was suppressed by cytochalasin D, but not by methyl-β-cyclodextrin, chloroquine or an Fc blocker, suggesting the involvement of macropinocytosis in this process. Internalization of 2C10 and DNA together resulted in production of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, IL-6, IL-10 and IL-33 by PBMCs. Cytokine production was suppressed by chloroquine and shikonin, but not by RU.521, suggesting dependence on activation of the Toll-like receptor (TLR)-9 and absent in melanoma 2 (AIM-2) pathways. These results established a simple model to demonstrate that anti-DNA antibodies can cause dysregulation of cytokine network mimicking systemic lupus erythematosus in culture of normal PBMCs, and emphasize again the importance of maintaining anti-DNA antibodies at low levels by treatment.

<p>IgGs-Abzymes from the Sera of Patients with Systemic Lupus Erythematosus Hydrolyzed miRNAs</p>

“…59,60 There is ample evidence that IgG immune complexes are transported through Fc receptors into lysosomes. [61][62][63] Therefore, it can be assumed that catalytic antibodies that hydrolyze RNA and miRNAs, along with RNase T2 and RNase 2, are involved in the activation of TLR7 and TLR8. Moreover, it is known that some antibodies can enter the cell through interaction with receptors or antigens.…”

Anti-DNA antibodies cross-reactive with β2-glycoprotein I induce monocyte tissue factor through the TLR9 pathway

“…Why WB-6 induces TF but 2C10 or H241 do not is a puzzle. Recently we reported that 2C10-DNA complex is incorporated by monocytes through macropinocytosis, and that the DNA stimulates the TLR9 and AIM-2 pathways resulting in expression of several inflammatory cytokines relevant to SLE pathogenesis [30]. We usually observe that 2C10 enters live cells more readily than WB-6 even at low concentrations as shown in Figure 2.…”

Pyroptosis and Its Role in Autoimmune Disease: A Potential Therapeutic Target

“…Similarly, the highly expressed mammalian target of rapamycin (mTOR) in lupus mice has also been shown to induce lupus nephritis (LN) through activation of mTOR/mitochondrial ROS/NLRP3 signaling ( 108 ). Another function of anti-dsDNA Abs is to help DNA in the plasma or on the cell surface enter normal monocytes through endocytosis to activate the AIM2 inflammasome, forming a vicious cycle of SLE pathogenesis ( 109 ). Naik et al.…”