NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes

Abstract

Inflammation in the brain accompanies several high-impact neurological diseases including multiple sclerosis (MS), stroke, and Alzheimer's disease. Neuroinflammation is sterile, as damage-associated molecular patterns rather than microbial pathogens elicit the response. The inflammasome, which leads to caspase-1 activation, is implicated in neuroinflammation. In this study, we reveal that lysophosphatidylcholine (LPC), a molecule associated with neurodegeneration and demyelination, elicits NLRP3 and NLRC4 inflammasome activation in microglia and astrocytes, which are central players in neuroinflammation. LPC-activated inflammasome also requires ASC (apoptotic speck containing protein with a CARD), caspase-1, cathepsin-mediated degradation, calcium mobilization, and potassium efflux but not caspase-11. To study the physiological relevance, Nlrc4^-/- and Nlrp3^-/- mice are studied in the cuprizone model of neuroinflammation and demyelination. Mice lacking both genes show the most pronounced reduction in astrogliosis and microglial accumulation accompanied by decreased expression of the LPC receptor G2A, whereas MS patient samples show increased G2A. These results reveal that NLRC4 and NLRP3, which normally form distinct inflammasomes, activate an LPC-induced inflammasome and are important in astrogliosis and microgliosis.

Figure 1.

The inflammasome mediates LPC-induced IL-1β secretion in mouse BMDMs. (A–D) WT, Nlrc4^−/−, Nlrp3^−/−, Il1b^−/− (A), Asc^−/− (B), Casp1^−/− (C), and Casp11^−/− (D) BMDMs were LPS primed (100 ng/ml) overnight before LPC stimulation at concentrations of 25, 50, and 100 µM for 1 h before IL-1β was collected from supernatants for analysis by ELISA. (E–K) BMDMs were LPS primed (200 ng/ml) for 3 h before stimulation with 50 µM LPC in the presence or absence of the caspase-1 inhibitor YVAD (10 and 50 µM; E); the ROS inhibitors NAC (5 and 25 mM), MPG (1 and 2.5 mM), and APDC (10 and 50 µM; F); the mitochondrial ROS inhibitor Mitotempo (100 and 500 µM; G); the cathepsin B inhibitor (CA-074Me; 10 and 20 µM; H); the potassium efflux inhibitors KCl (50 and 100 mM) and glyburide (100 and 200 µM; I); or the calcium inhibitors BAPTA (10 and 20 µM), 2-APB (20 and 100 µM; J), and U73122 (2 and 10 µM; K). Then, IL-1β was collected from supernatants for analysis by ELISA. Each control and experimental condition was performed in triplicate. All data are representative of at least two independent experiments. Results are displayed as the mean ± SEM. *, P < 0.05; **, P < 0.01; unpaired Student's t test.

Figure 2.

The inflammasome mediates LPC-induced IL-1β maturation in mouse BMDMs. (A–D) WT, Nlrp3^−/−, Nlrc4^−/−, and Il1b^−/− (A), Asc^−/− (B), Casp1^−/− (C), and Casp11^−/− (D) BMDMs were LPS primed (100 ng/ml) overnight before LPC stimulation (25, 50, and 100 µM). Pro–IL-1β and IL-1β in the supernatant (sn) or cell lysate (Cell) and pro–caspase-1 in the cell lysate were assessed by immunoblotting. β-Actin was used as the loading control. (E–I) BMDMs were LPS primed (200 ng/ml) for 3 h and then stimulated with 50 µM LPC in the presence or absence of the caspase-1 inhibitor YVAD (E); the ROS inhibitors NAC, MPG, and APDC and the mitochondrial ROS inhibitor Mitotempo (F); the cathepsin B inhibitor CA-074Me (G); the potassium efflux inhibitors glyburide and KCl (H); and the calcium inhibitors BAPTA, 2-APB, and U73122 (I) as used in Fig. 1. IL-1β in the supernatant was assessed by immunoblotting. The image in F is assembled from two sections of the same blot, and the break between these two sections is indicated by a dash line. The data are representative of two independent experiments. w/o, without.

Figure 3.

NLRC4 is expressed in mouse CNS during neuroinflammation and in human brain tissue from MS patients. (A) WT mice brains were examined for NLRC4 expression by IHC using a mouse NLRC4 antibody. NLRC4 expression (red) was detected after 4 wk of cuprizone-induced demyelination in WT (Nlrc4^+/+) but not in Nlrc4^−/− mice. (B) The corpus callosum from 4-wk cuprizone–treated WT mice was stained with anti-NLRC4 (top, green; bottom, red), and RCA (red) was used to detect microglia (top), and GFAP (green) was used to detect astrocytes (bottom) at the corpus callosum. DAPI (blue) was used to label nuclei. Overlay (yellow) indicates colocalization of NLRC4 with the cell-specific biomarkers. (C and D) Chronic MS brain tissues were obtained from the UCLA HBSFRC. The corpus callosum sections were stained for NLRC4 using a human NLRC4 antibody (αNLRC4), as this is the region studied in the cuprizone model. Astrocytes (C) and microglia (D) were detected using GFAP and RCA antibodies. The overlay was performed to detect colocalization of proteins. All data are representative of at least two independent experiments. Bars, 50 µM.

Figure 4.

Astrocytic NLRC4 and NLRP3 display the same inflammasome specificity as macrophage NLRs. (A) Immunocytochemical detection of GFAP (green)- but not Iba-1(red)–stained cells from a purified mouse astrocyte culture used in Fig. 4 (D and E). DAPI (blue) was used to label nuclei. Representative images from the experiment performed in triplicate are shown. Bar,16 µM. (B) WT and Nlrc4^−/− BMDMs were LPS primed (400 ng/ml) for 4 h and transfected with the transfecting reagent DOTAP and 25, 50, or 100 ng of S. typhimurium flagellin for 1 h before IL-1β was collected from supernatant. WT and Nlrc4^−/− LPS-primed BMDMs were also stimulated with 5 mM ATP, as a positive control for inflammasome activation. Cells were transfected with DOTAP alone or stimulated with flagellin alone without LPS as a negative control. Data are representative of two independent experiments. (C) WT and Nlrp3^−/− BMDMs were similarly treated as in B. Data are representative of two independent experiments. (D) WT and Nlrc4^−/− primary mouse astrocytes were LPS primed (400 ng/ml) for 4 h and transfected with 50, 100, or 200 ng flagellin for 1 h before IL-1β was collected from supernatant. LPS-primed primary mouse WT and Nlrc4^−/− astrocytes were also stimulated with 5 mM ATP, as a positive control, and transfected with DOTAP alone or stimulated with flagellin alone as a negative control. Data are representative of three independent experiments. (E) WT and Nlrp3^−/− primary mouse astrocytes were similarly treated as described in D. Data are representative of two independent experiments. (D and E) 9–12 mouse pups (0–2 d old) per genotype were used for each experiment. Each control and experimental condition was performed in triplicate. Results are displayed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; unpaired Student’s t test.

Figure 5.

NLRC4 and NLRP3 mediate LPC-induced IL-1β secretion from primary mouse astrocytes. (A) WT astrocytes were LPS primed (1 µg/ml) for 3 h and incubated with 50 and 100 µM LPC for 2 or 4 h, as indicated, before IL-1β was collected from the supernatant for analysis by ELISA. Data are representative of three independent experiments. (B) WT and Nlrc4^−/− astrocytes were treated as described in A and assayed for IL-1β secretion at 4 h. Data are representative of three independent experiments. (C and D) WT and Nlrp3^−/− astrocytes (C) or WT and Asc^−/− astrocytes (D) were treated as described in A and assayed for IL-1β secretion at 4 h. Data are representative of two independent experiments. Each experimental condition was performed in duplicate, and 9–12 mouse pups (0–2 d old) per genotype were used for each experiment. Results are displayed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student's t test.

Figure 6.

The inflammasome mediates LPC-induced IL-1β secretion in microglia. (A and B) ICC of Iba-1 and GFAP staining of mixed glial cell cultures (A) and CD11b-positive selected cells (B) isolated from postnatal day-3–5 WT mouse pups indicates the degree of microglial purity. Bars, 50 µM. (C–G) WT, Nlrc4^−/−, Nlrp3^−/−, Il1b^−/− (C), Asc^−/− (D), Casp-1^−/− (E), Casp-11^−/− (F), and Il18^−/− (G) microglia were LPS primed (100 ng/ml) for 3 h and stimulated with LPC (20, 25, and 50 µM) for 1 h. IL-1β in the cell culture supernatant was assessed by ELISA. Data are representative of two independent experiments. Each experimental condition was performed in triplicate. Results are displayed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; unpaired Student's t test.

Figure 7.

NLRP3 and NLRC4 promote neuroinflammation. (A) Representative images of mouse brain sections stained for the microglial marker, RCA. Samples were obtained from cuprizone-treated WT, Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/−Nlrp3^−/− mice. RCA⁺ (red) microglial cells were measured at the corpus callosum in brains from untreated mice (0 wk) or after 3 wk of cuprizone treatment. DAPI (blue) was used to label nuclei. Bar, 60 µM. (B) Representative images of brain sections stained for the astrocytic marker, GFAP. Samples were obtained as described in A. DAPI (blue) was used to detect nuclei. Bar, 60 µM. (C and D) Quantification of microglial accumulation (C) and astrogliosis (D) at the midline corpus callosum in the indicated strains. Cell counts are averages of six mice per time point. Results are displayed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; unpaired Student’s t test. ND, not detectable; sq, square.

Figure 8.

The inflammasome promotes demyelination. (A) Representative images of brain sections of untreated (0 wk) and 3-wk cuprizone–treated WT, Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/−Nlrp3^−/− mice stained for the presence of myelin at the corpus callosum (purple dashed lines) with LFB (blue). Bar, 50 µM. (B) Quantification of LFB-stained WT, Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/−Nlrp3^−/− mice brain sections. Each circle represents the mean observed LFB score for one mouse. WT, Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/−Nlrp3^−/− mice were scored by a blinded reader on a scale of 0 (no demyelination) to 3 (complete demyelination). The mean value of each dataset is depicted by a red line. All scoring is restricted to the midline corpus callosum (purple dashed lines in A). LFB scores are means of four to six mice per time point. Results are displayed as the mean ± SEM. **, P < 0.01; unpaired Student’s t test.

Figure 9.

The inflammasome elevates expression of the LPC receptor G2A. (A) Brain sections from 4-wk cuprizone–treated WT mice were stained for the presence of G2A at the corpus callosum of the brain, as indicated by black arrowheads, with a hematoxylin counter stain. (Left) The negative control (no G2A antibody was used), which shows no brown staining. (Right) G2A-positive cells (brown staining as indicated by black arrowheads). Bar, 20 µm. (B) Representative images of 0-wk and 4-wk cuprizone–treated brains from WT, Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/−Nlrp3^−/− mice are shown. G2A-positive cells (as indicated by black arrowheads) are detected within the corpus callosum. Bar, 20 µM. (C) G2A-positive cells within the corpus callosum of cuprizone-treated brains of WT, Nlrc4^−/−, Nlrp3^−/−, and Nlrc4^−/−Nlrp3^−/− mice were quantified. Cell counts are means of five to six mice per time point. Results are displayed as the mean ± SEM. *, P < 0.05; unpaired Student’s t test. (D) IHC of G2A-positive cells within white matter brain tissue of normal brain and brain from an MS patient. (Top, left) G2A staining of brain section from control. (Top, right) G2A staining of brain sections from deceased MS patients. The yellow bracketed areas contain G2A-positive cells and are shown magnified to reveal the stained cells in the bottom panels. Bars, 100 µm.