The lncRNA Neat1 promotes activation of inflammasomes in macrophages

Abstract

The inflammasome has an essential function in innate immune, responding to a wide variety of stimuli. Here we show that the lncRNA Neat1 promotes the activation of several inflammasomes. Neat1 associates with the NLRP3, NLRC4, and AIM2 inflammasomes in mouse macrophages to enhance their assembly and subsequent pro-caspase-1 processing. Neat1 also stabilizes the mature caspase-1 to promote interleukin-1β production and pyroptosis. Upon stimulation with inflammasome-activating signals, Neat1, which normally resides in the paraspeckles, disassociates from these nuclear bodies and translocates to the cytoplasm to modulate inflammasome activation using above mechanism. Neat1 is also up-regulated under hypoxic conditions in a HIF-2α-dependent manner, mediating the effect of hypoxia on inflammasomes. Moreover, in the mouse models of peritonitis and pneumonia, Neat1 deficiency significantly reduces inflammatory responses. These results reveal a previously unrecognized role of lncRNAs in innate immunity, and suggest that Neat1 is a common mediator for inflammasome stimuli.

Fig. 1

Neat1 enhances the activation of the NLRP3 inflammasome. a–c Control iBMDMs and iBMDMs devoid of Neat1 by shRNA #1 (a, left and b) or #2 (a, right and c) were untreated, primed with LPS, or treated with nigericin after LPS priming. Whole cell lysates (WCL) and the supernatant (SN) and pellet (PE) fractions were analyzed by western blotting for indicated protein levels (a). Culture supernatant was analyzed by ELISA for IL-1β secretion (b, c). d–g iBMDMs devoid of Neat1_2 (d, e) or overexpressing Neat1_1 (f, g) and the corresponding controls were treated with or without LPS or nigericin as indicated, and analyzed for protein levels (d, f) and IL-1β secretion (e, g). h, i BMDMs from Neat1 wild-type (Neat1^+/+) or knockout (Neat1^−/−) mice were primed with LPS and then stimulated with nigericin as indicated. Protein levels (h) and IL-1β secretion (i) were analyzed. In b, c, e, g, and i, data shown are mean ± SD (n = 4). ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file

Fig. 2

Neat1 enhances the activation of NLRC4 and AIM2 inflammasomes. a–d iBMDMs devoid of Neat1 by shRNA #1 (a, b), iBMDMs overexpressing Neat1_1 (c, d), and the corresponding control iBMDMs were primed with LPS, and then stimulated with flagellin (a, c) or poly(dA:dT) (b, d). WCL and SN were analyzed by western blotting. e, f BMDMs from Neat1^+/+ or Neat1^−/− mice were primed with LPS and then stimulated with flagellin (e) or poly(dA:dT) (f). WCL and SN were analyzed by western blotting. (g, h) Control iBMDMs and iBMDMs ectopically expressing Neat1_1 were primed with LPS and then stimulated with flagellin (g) or poly(dA:dT) (h). Cell lysates were immunoprecipitated with normal rabbit IgG or ASC antibody. The samples were analyzed by western blotting as indicated. Source data are provided as a Source Data file

Fig. 3

The effect of Neat1 on the NLRP3 inflammasome activation is independent of IL-6. a BMDMs derived from IL-6 wild-type (IL-6^+/+) or knockout (IL-6^−/−) mice were primed with LPS and then treated with nigericin. Protein levels in total cell lysates and supernatant fraction were analyzed by western blotting. b–d IL-6^+/+ or IL-6^−/− BMDMs were transfected with control siRNA (b–d), Neat1 siRNA #1 (b), Neat1 siRNA #2 (c), or Neat1_2 siRNA (d). Twenty-four hours later, cells were stimulated with LPS and nigericin. WCL and SN of above cells were subjected to western blotting with indicated antibodies. Source data are provided as a Source Data file

Fig. 4

Neat1 interacts with caspase-1 p20 domain. a–c Cell lysates of normal iBMDMs, LPS-primed iBMDMs or LPS-nigericin-co-stimulated iBMDMs were incubated with normal rabbit IgG (a–c), NLRP3 antibody (a), ASC antibody (b), or caspase-1 antibody (c) for RIP. The immunoprecipitates were analyzed by real-time RT-PCR to exam enrichment efficiency of Neat1 and by Western blotting for protein levels. d Cell lysates of normal, LPS-primed, or LPS-nigericin-co-stimulated iBMDMs were incubated with biotin-labeled Neat1 sense or antisense DNA probes immobilized on beads. The precipitated samples were analyzed by real-time RT-PCR for Neat1 contents, and by western blotting for indicated protein levels. e In vitro transcribed biotin-labeled Neat1_1 was incubated with lysates extracted from 293T cells expressing Flag-tagged wild-type pro-caspase-1, CARD, p20, or p10 for 3 h. The input and biotin pull-down samples were then carried out to western blotting. f In vitro transcribed biotin-labeled Neat1_1 was incubated with lysates extracted from 293T cells expressing Flag-tagged wild-type pro-caspase-1 or the indicated deletion mutants for 3 h. The input and biotin pull-down samples were analyzed by western blotting. In a, data shown are mean ± SD (n = 5). In b and d, data shown are mean ± SD (n = 3). In c, data shown are mean ± SD (n = 6). *P < 0.05, **P < 0.01, two-tailed t-test. Source data are provided as a Source Data file

Fig. 5

Neat1 stabilizes the caspase-1 hetero-tetramers and increases caspase-1 activity. a–d 293T cells were infected with lentiviruses expressing control RNA or Neat1_1 and then co-transfected with Flag-p20 and HA-p10 (a, b) or Flag-p33 and HA-p10 (c, d). Cell lysates were incubated with anti-Flag mAb (M2) beads. Input samples and pull-down products were subjected to western blotting (a, c). Relative ratios of HA-p10 pulled down by Flag-p20 (b) or Flag-p33 (d) were shown in histogram, based on the data of three independent replication experiments. e Neat1^+/+ and Neat1^−/− BMDMs were primed with LPS and then activated with nigericin, flagellin, or poly(dA:dT). Mature caspase-1 activity was measured. f Control or Neat1_1 overexpressing iBMDMs were primed with LPS and then activated with nigericin, flagellin, or poly(dA:dT). Mature caspase-1 activity were measured. In b, d, e and f, data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file

Fig. 6

Neat1 promotes caspase-1-dependent pyroptosis. a–f Neat1_1-overexpressing iBMDMs (a, b), Neat1-knockdown iBMDMs (c, d), Neat1^−/− BMDMs (e, f), and the corresponding control cells were primed with LPS and then stimulated with nigericin. Cells were stained with PI and analyzed under microscope (a, c, and e). Percentages of pyroptotic cells were measured by LDH release into supernatants (b, d, and f). In a, c, and e, the bar graph indicates 300 μm. In b, d, and f, data shown are mean ± SD (n = 4). ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file

Fig. 7

Neat1 is exported from nucleus to cytoplasm in response to inflammasome-activating stimuli. a–c Untreated iBMDMs (a–c), LPS-primed iBMDMs (a–c), nigericin-activated iBMDMs (a), flagellin-activated iBMDMs (b), or poly(dA:dT)-activated iBMDMs (c) were subjected to fractionation into cytoplasmic and nuclear extracts. Cytoplasmic RNA was analyzed for the Neat1 expression by real-time RT-PCR. d The subcellular co-localization of Neat1 (green) and ASC (purple) in untreated, LPS-primed or LPS-nigericin-co-stimulated iBMDMs was analyzed by RNA-FISH and IF. In d, the bar graph indicates 5 μm. In a, b, and c, data shown are mean ± SD (n = 3). *P < 0.05, ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file

Fig. 8

Neat1 is released from nuclear paraspeckles into cytoplasm during inflammasome activation. a–c Cell lysates of untreated iBMDMs (a–c), LPS-primed iBMDMs (a–c), nigericin-activated iBMDMs (a), flagellin-activated iBMDMs (b), or poly(dA:dT)-activated iBMDMs (c) were analyzed by Western blotting. d Colocation of paraspeckle proteins, Sfpq (green), and Pspc1 (red) were assayed by immunostaining. The location of paraspeckles (yellow) was obtained by merging two signals of Sfpq and Pspc1. The position of the nucleus is shown in the bright-field image. e, f Interactions among paraspeckle components in untreated iBMDMs (e, f), LPS-primed iBMDMs (e, f), flagellin-activated iBMDMs (e), or poly(dA:dT)-activated iBMDMs (f) were analyzed by co-immunoprecipitation assay. g, h iBMDMs infected with lentiviruses expressing control, Pspc1, Sfpq, or Nono shRNA were analyzed for the cytoplasmic and nuclear levels of Neat1 and Neat1_2. Percentages of cytoplasmic Neat1 or Neat1_2 among total Neat1 or Neat1_2 RNA were shown in h. In d, the bar graph indicates 10 μm. In g and h, data shown are mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file

Fig. 9

Neat1 mediates the effect of HIF-2α on inflammasomes under hypoxic condition. a–c iBMDMs cultured under normoxic or hypoxic conditions were untreated (a–c), primed with LPS (a–c,or co-stimulated with LPS and nigericin (a), flagellin (b), or poly(dA:dT) (c). WCL (a–c), SN (a–c), and PE (a) were subjected to western blotting. d–i Control (d–i), HIF-1α-silenced (d, f, and h), HIF-2α-silenced (e, g and i), Neat1-silenced (d–i), HIF-1α-Neat1-double-silenced (d, f and h), or HIF-2α-Neat1-double-silenced (e, g and i) iBMDMs were cultured under hypoxic conditions and primed with LPS, followed by stimulation with nigericin (d, e), flagellin (f, g), or poly(dA:dT) (h, i). WCL and SN were analyzed by western blotting. Source data are provided as a Source Data file

Fig. 10

Loss of Neat1 impairs alum-induced peritoneal inflammation and attenuates flagellin-induced lung inflammation in vivo. a IL-1β content in the lavage fluid from Neat1^+/+ or Neat1^−/− mice 12 h after PBS or alum injection. b Absolute numbers of Neat1^+/+ or Neat1^−/− PECs recovered 12 h after PBS or alum injection. c, d Absolute numbers of neutrophils (c) and Ly6C⁺ monocytes (d) exuded to the peritoneum 12 h after PBS or alum injection. e IL-1β content in the BALF from Neat1^+/+ or Neat1^−/− mice 12 h after isotonic saline or flagellin intranasal instillation. f Absolute numbers of Neat1^+/+ or Neat1^−/− PECs recovered 12 h after isotonic saline or flagellin intranasal instillation. g, h Absolute numbers of neutrophils (g) and Ly6C⁺ monocytes (h) exuded to the lung 12 h after isotonic saline or flagellin intranasal instillation. In a–h, data shown are mean ± SD (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. Source data are provided as a Source Data file