Self double-stranded (ds)DNA induces IL-1β production from human monocytes by activating NLRP3 inflammasome in the presence of anti-dsDNA antibodies

Abstract

The pathogenic hallmark of systemic lupus erythematosus is the autoimmune response against self nuclear Ags, including dsDNA. The increased expression of the proinflammatory cytokine IL-1β has been found in the cutaneous lesion and PBMCs from lupus patients, suggesting a potential involvement of this cytokine in the pathogenesis of lupus. IL-1β is produced primarily by innate immune cells such as monocytes and can promote a Th17 cell response, which is increased in lupus. IL-1β production requires cleaving pro-IL-β into IL-1β by the caspase-1-associated multiprotein complex called inflammasomes. In this study we show that self dsDNA induces IL-1β production from human monocytes dependent on serum or purified IgG containing anti-dsDNA Abs by activating the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome. Reactive oxygen species (ROS) and K(+) efflux were involved in this activation. Knocking down the NLRP3 or inhibiting caspase-1, ROS, and K(+) efflux decreased IL-1β production. Supernatants from monocytes treated with a combination of self dsDNA and anti-dsDNA Ab(+) serum promoted IL-17 production from CD4(+) T cells in an IL-1β-dependent manner. These findings provide new insights in lupus pathogenesis by demonstrating that self dsDNA together with its autoantibodies induces IL-1β production from human monocytes by activating the NLRP3 inflammasome through inducing ROS synthesis and K(+) efflux, leading to the increased Th17 cell response.

Figure 1. Human monocytes produce IL-1β in response to self dsDNA in the presence of anti-dsDNA antibody-positive serum

(A–C) Measuring IL-1β in cell culture supernatants of monocytes incubated for 18 hours in the following conditions by ELISA. (A–B) Monocytes purified from a single (A) or multiple healthy donors (B, symbols indicate individual donors) were incubated with human genomic dsDNA (5 μg/ml) in the presence or absence of healthy serum or anti-dsDNA antibody-positive serum (5% final concentration) from multiple donors (A, #D1–#D4; B, #D1–#D5). (C) ANA-positive sera without anti-dsDNA antibodies (donors, #A1–#A3) were added to monocytes from a single donor in the presence or absence of dsDNA. Representative data from 2 independent experiments (A and C). *P < 0.05.

Figure 2. Anti-dsDNA antibodies are required for IL-1β production from human monocytes treated with self dsDNA

(A–D) Measuring IL-1β in cell culture supernatants of monocytes incubated for 18 hours in the following conditions by ELISA. (A) Monocytes from healthy donors were incubated with dsDNA in the presence or absence of total IgG purified from anti-dsDNA antibody-positive serum or healthy serum (n = 5). (B) Monocytes were incubated with dsDNA in the presence or absence of serum depleted or un-depleted of anti-dsDNA antibodies (2 independent experiments). (C) Monocytes were treated with anti-CD32 antibodies (FcR blocker, 2.5 μg/ml) and incubated with dsDNA and anti-dsDNA antibody-positive serum (n = 5). (D) Monocytes were incubated with anti-dsDNA antibody-positive serum or dsDNA and anti-dsDNA antibody-positive serum in the presence or absence of DNase (1 μg/ml) (n = 7). Bars and error bars indicate mean and SEM, respectively. *P < 0.05.

Figure 3. Self dsDNA induces pro-IL-1β in human monocytes in the presence of anti-dsDNA antibody-positive serum

(A–B) Monocytes from healthy donors were incubated for 6 (qPCR) or 10 (ELISA) hours with dsDNA (5 μg/ml) in the presence or absence of healthy or anti-dsDNA antibody-positive serum (5% final concentration). (A) Pro-IL1B gene expression was measured by qPCR. (B) Intracellular pro-IL-1β was measured using cell lysates by ELISA. Bars and error bars indicate mean and SEM, respectively (n = 2 and 3 independent experiments for A and B, respectively).

Figure 4. The production of IL-1β from human monocytes by self dsDNA and anti-dsDNA antibody-positive serum is dependent on TLR9 and NF-κB activation

(A–D) Monocytes were purified from healthy donors for the following experiments. (A) Monocytes were incubated for 4 hours with dsDNA in the presence or absence of healthy or anti-dsDNA antibody-positive serum. NF-κB activation (phosphorylation) was determined by flow cytometry. Numbers in histograms indicate the frequency (%) of cells stained for phosphorylated NF-κB (pS529). (B–D) Monocytes were incubated for 18 hours with dsDNA and anti-dsDNA antibody-positive serum in the presence or absence of the NF-κB inhibitors (B, 5 μM Bay11-7082 and 5 μM Cenostrol), chloroquine (C, 5 μg/ml) or inhibitory nucleic acid sequence for TLR9 (D, 5 μM ODN). IL-1β in cell culture supernatants were measured by ELISA. Representative data from 4 independent experiments (A). Bars and error bars indicate mean and SEM, respectively (n = 4, 7 and 8 for B, C and D, respectively). *P < 0.05.

Figure 5. Caspase-1 and NLRP3 are involved in the production of IL-1β from human monocytes in response to self dsDNA and anti-dsDNA-positive serum

(A) Monocytes from a healthy donor were incubated for 7 hours with dsDNA in the presence or absence of healthy or anti-dsDNA antibody-positive serum. Active capspase-1 was measured using flow cytometry. Numbers in histograms indicate the frequency (%) of cells positive for active caspase-1. (B) IL-1β ELISA of culture supernatants from monocytes incubated for 18 hours with dsDNA and anti-dsDNA antibody-positive serum in the presence or absence of caspase-1 inhibitor (10 μM) (n = 4). (C) Measuring the NLRP3 gene expression in human monocytes incubated for 6 hours in the presence or absence of anti-dsDNA antibody-positive serum or a combination of dsDNA and anti-dsDNA antibody-positive serum by qPCR (n = 2). (D) IL-1β ELISA of culture supernatants from monocytes nucleofected with scrambled or NLRP3-specific siRNA followed by the incubation for 18 hours with dsDNA and anti-dsDNA antibody-positive serum (n = 6). Representative data from 4 independent experiments (A). Bars and error bars indicate mean and SEM, respectively. *P < 0.05.

Figure 6. The production of IL-1β from human monocytes in response to self dsDNA and anti-dsDNA antibody-positive serum requires reactive oxygen species (ROS) synthesis and K+ efflux

(A) Flow cytometric analysis of reactive oxygen species (ROS) in monocytes stimulated for 4 hours with dsDNA in the presence or absence of healthy or anti-dsDNA antibody-positive serum. (B–C) IL-1β ELISA of culture supernatants from monocytes incubated for 18 hours with dsDNA and anti-dsDNA antibody-positive serum in the presence or absence of the ROS inhibitor DPI (50 μM, B) or KCl (100 mM, C). Representative data from 3 independent experiments (A). Bars and error bars indicate mean and SEM, respectively (n = 5 and 7 for B and C, respectively). *P < 0.05.

Figure 7. Cell culture supernatant from monocytes treated with a combination of self dsDNA and anti-dsDNA antibody-positive serum promotes IL-17 production from IL-1 receptor I (IL-1RI)-positive memory CD4⁺ T cells in an IL-1β-dependent manner

ELISA of IL-17 production from sorted human IL-1RI⁺ memory CD4⁺ T cells that were stimulated for 5 days with anti-CD3 and -CD28 antibody-coated beads in 80% culture medium with 20% of supernatants from monocytes treated for 18 hours with self dsDNA in the presence or absence of anti-dsDNA antibody-positive serum. Some IL-1RI⁺ memory CD4⁺ T cells were incubated with culture medium alone or medium with anti-CD3 and anti-CD28 antibody-coated beads in the presence or absence of recombinant IL-1β (20 ng/ml). IL-1R antagonist (100 ng/ml) was added on days 0 and 2. Bars and error bars indicate mean and SEM, respectively (n = 5). The presence and absence of each treatment are indicated by + and −, respectively. *P < 0.05.