U1-small nuclear ribonucleoprotein activates the NLRP3 inflammasome in human monocytes

Abstract

The NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome is a caspase-1-containing cytosolic protein complex that is essential for processing and secretion of IL-1β. The U1-small nuclear ribonucleoprotein (U1-snRNP) that includes U1-small nuclear RNA is a highly conserved intranuclear molecular complex involved in splicing pre-mRNA. Abs against this self nuclear molecule are characteristically found in autoimmune diseases like systemic lupus erythematosus, suggesting a potential role of U1-snRNP in autoimmunity. Although endogenous DNA and microbial nucleic acids are known to activate the inflammasomes, it is unknown whether endogenous RNA-containing U1-snRNP could activate this molecular complex. In this study, we show that U1-snRNP activates the NLRP3 inflammasome in CD14(+) human monocytes dependently of anti-U1-snRNP Abs, leading to IL-1β production. Reactive oxygen species and K(+) efflux were responsible for this activation. Knocking down the NLRP3 or inhibiting caspase-1 or TLR7/8 pathway decreased IL-1β production from monocytes treated with U1-snRNP in the presence of anti-U1-snRNP Abs. Our findings indicate that endogenous RNA-containing U1-snRNP could be a signal that activates the NLRP3 inflammasome in autoimmune diseases like systemic lupus erythematosus where anti-U1-snRNP Abs are present.

FIGURE 1. U1-snRNP induces IL-1β production from human monocytes in the presence of U1-snRNP antibody-positive serum

(A–C) IL-1β ELISA at 18 hours from cell culture supernatants of human monocytes incubated in the following conditions. (A–B) Monocytes from a single (A) or multiple donors (B, symbols indicate individual donors) were incubated with or without U1-snRNP (snRNP, 5 µg/ml) in the presence or absence of healthy serum or anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum (5% final concentration) from multiple donors (#S1–#S5). (C) ANA-positive sera without anti-U1-snRNP antibodies (donors, #A1–#A4) were added to monocytes from a single donor in the presence or absence of U1-snRNP. Representative data from 2 independent experiments (A and C). *P < 0.05.

FIGURE 2. The production of IL-1β from human monocytes in response to U1-snRNP requires anti-U1-snRNP antibodies

(A–D) IL-1β ELISA at 18 hours from cell culture supernatants of human monocytes incubated in the following conditions. (A) Monocytes were incubated with or without U1-snRNP in the presence or absence of total IgG purified from anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum or healthy serum. (B) Monocytes were incubated with or without U1-snRNP in the presence or absence of serum depleted or un-depleted of anti-U1-snRNP antibodies (Bars and error bars indicate mean and SEM, n = 2). (C) Monocytes were treated with anti-CD32 antibodies (FcR blocker) followed by incubation with U1-snRNP and anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum. (D) Monocytes were incubated with anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum or U1-snRNP and anti-U1-snRNP antibody-positive serum in the presence or absence of RNase. Bars and error bars indicate mean and SEM, respectively (n = 5–7 donors for A, C and D). *P < 0.05.

FIGURE 3. U1-snRNP induces IL-1β production from human monocytes by activating NF-κB through triggering TLR7 and 8 in the presence of anti-U1-snRNP antibody-positive serum

(A–B) Pro-IL-1β qPCR (A) and ELISA (B) of human monocytes incubated for 6 (qPCR) or 10 (ELISA) hours with or without U1-snRNP (snRNP, 5 µg/ml) in the presence or absence of healthy or anti-U1-snRNP antibody-positive serum (5% final concentration) (Bars and error bars indicate mean and SEM, respectively, n = 2 and 3 donors for A and B). (C) Flow cytometric analysis of TLR7 and 8 expression by CD14⁺ human monocytes. (D–E) Flow cytometric analysis of NF-κB activation in monocytes treated for 3 hours with anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum (D) or IgG purified from anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum (E) in the presence or absence of U1-snRNP. (F–H) IL-1β ELISA of culture supernatants from monocytes incubated for 18 hours with U1-snRNP and anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum in the presence or absence of the NF-κB inhibitors (F, Bay11-7082 and Cenostrol), chloroquine (G) or inhibitory nucleic acid sequences for TLR7/8 (H, ORN). Representative data from 2–3 independent experiments (C–E). Bars and error bars indicate mean and SEM, respectively (n = 4–8 donors for F–H). *P < 0.05.

FIGURE 4. The production of IL-1β from human monocytes in response to U1-snRNP and anti-U1-snRNP antibody-positive serum requires caspase-1 activation and NLRP3 inflammasome

(A–C) Flow cytometric analysis of active caspase-1 in human monocytes stimulated for 7 (A) or indicated hours (B) with or without U1-snRNP (5 µg/ml) in the presence or absence of anti-U1-snRNP (snRNP) antibody-positive (Ab⁽⁺⁾) serum (5% final concentration), healthy serum (A–B) or total IgG purified from anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum (C). (D) IL-1β ELISA of culture supernatants from monocytes incubated for 18 hours with U1-snRNP and anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum in the presence or absence of caspase-1 inhibitor. (E) IL-1β ELISA of culture supernatants from monocytes transfected with scrambled or NLRP3-specific siRNA followed by the incubation for 18 hours with U1-snRNP and anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum. Representative data from 3 independent experiments (A, C). Numbers in histograms indicate the frequency of cells stained positive. Symbols and error bars indicate mean + SD (n = 2) (B). Bars and error bars indicate mean and SEM, respectively (n = 4–7 donors for D–E). *P < 0.05.

FIGURE 5. The production of IL-1β and activation of caspase-1 by human monocytes in response to U1-snRNP and anti-U1-snRNP antibody-positive serum requires ROS synthesis and K+ efflux

(A–C) Flow cytometric analysis of reactive oxygen species (ROS) in monocytes stimulated for 3 (A) or indicated hours (B) with or without U1-snRNP (5 µg/ml) in the presence or absence of anti-U1-snRNP (snRNP) antibody-positive (Ab⁽⁺⁾) serum (5% final concentration), healthy serum (A–B) or total IgG purified from anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum (C). (D–E) IL-1β ELISA of culture supernatants from monocytes incubated for 18 hours with U1-snRNP and anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum in the presence or absence of the ROS inhibitor DPI (D) or KCl (E). (F) Flow cytometric analysis of active caspase-1 in monocytes treated for 7 hours with U1-snRNP and anti-U1-snRNP antibody-positive (Ab⁽⁺⁾) serum in the presence or absence of DPI, KCl or chloroquine. Representative data from 3–4 independent experiments (A, C, F). Symbols and error bars indicate mean + SD (n = 2) (B). Bars and error bars indicate mean and SEM, respectively (n = 4–7 donors for D–E). *P < 0.05.