Divergent functional outcomes of NLRP3 blockade downstream of multi-inflammasome activation: therapeutic implications for ALS

Abstract

NOD-Like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome modulation has emerged as a potential therapeutic approach targeting inflammation amplified by pyroptotic innate immune cell death. In diseases characterized by non-cell autonomous neurodegeneration including amyotrophic lateral sclerosis (ALS), the activation of several inflammasomes has been reported. Since functional redundancy can exist among inflammasome pathways, here we investigate the effects of NLRP3 inhibition on NLRP3, NLR family CARD Domain Containing 4 (NLRC4) and non-canonical pathways to understand whether NLRP3 blockade alone can mitigate pro-inflammatory cytokine release and pyroptotic cell death in contexts where single or multiple inflammasome pathways independent of NLRP3 are activated. In this study we do not limit our insights into inflammasome biology by solely relying on the THP-1 monocytic line under the LPS/nigericin-mediated NLRP3 pathway activation paradigm. We assess therapeutic potential and limitations of NLRP3 inhibition in multi-inflammasome activation contexts utilizing various human cellular systems including cell lines expressing gain of function (GoF) mutations for several inflammasomes, primary human monocytes, macrophages, healthy and Amyotrophic Lateral Sclerosis (ALS) patient induced pluripotent stem cells (iPSC)-derived microglia (iMGL) stimulated for canonical and non-canonical inflammasome pathways. We demonstrate that NLRP3 inhibition can modulate the NLRC4 and non-canonical inflammasome pathways; however, these effects differ between immortalized, human primary innate immune cells, and iMGL. We extend our investigation in more complex systems characterized by activation of multiple inflammasomes such as the SOD1^G93A mouse model. Through deep immune phenotyping by single-cell mass cytometry we demonstrate that acute NLRP3 inhibition does not ameliorate spinal cord inflammation in this model. Taken together, our data suggests that NLRP3 inhibition alone may not be sufficient to address dynamic and complex neuroinflammatory pathobiological mechanisms including dysregulation of multiple inflammasome pathways in neurodegenerative disease such as ALS.

Keywords: ALS; MCC950; MEFV; NLRC4; NLRP1; NLRP3; iPSC-derived microglia; inflammasomes.

Figure 1

NLRP3 KO or pharmacological inhibition blocks NLRP3 pathway activation in THP-1 cells. PMA-differentiated WT or NLRP3 KO THP-1 cells were pre-incubated with LPS (1 μg/ml, 3 h) followed by nigericin (10 μM at 2 h) to activate the NLRP3 inflammasome and the release of (A) IL-1β, (B) IL-1α and (C) TNF-α was measured. (D) Dose-dependent MCC950-mediated inhibition of IL-1β release from PMA-differentiated WT THP-1 cells following LPS (1 μg/ml, 3 h) and nigericin (10 μM, 2 h) stimulation. (E) Representative time-lapse microscopy images of PI uptake as a measure of pyroptosis in WT and NLRP3 KO THP-1 cells stimulated with LPS and Nigericin. (F) Time-lapse monitoring of PI uptake in WT and NLRP3 KO THP-1 cells up to 11 h post-stimulation with LPS and Nigericin. Data are shown as mean ± S.D. of four technical replicates representing three independent experiments. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. ****p < 0.0001.

Figure 2

Pharmacological inhibition with MCC950 blocks NLRP3 pathway activation in hMonocytes. (A) MCC950 dose-dependent inhibition of IL-1β release from primary human monocytes following LPS (0.1 μg/ml, 3 h) and nigericin (10 μM, 1 h) stimulation. Effects of MCC950 (0.37-10 μM) on (B) IL-1β, (C) IL-1α and (D) TNF-α release from hMonocytes following LPS (0.1 μg/ml, 3 h) and nigericin (10 μM, 1 h) stimulation. (E) Representative images of PI uptake in hMonocytes following various treatments. (F) Time-lapse imaging analysis of PI uptake up to 4 h post-treatment in hMonocytes (signal normalized to cellular confluency). (G) Dose-dependent inhibition of IL-1β release by MCC950 (0.01-50 μM) and (H) at top two concentrations in the dose response curve in hMDM following LPS (0.1 μg/ml, 3 h) and nigericin (10 μM, 1 h) stimulation. (I) Time lapse imaging of PI uptake normalized to cellular confluency in hMDM exposed to various stimuli and MCC950 at 10 μM, over 8 (h) Data are shown as mean ± S.D. of four technical replicates from four healthy donors. One‐way ANOVA followed by Tukey’s post hoc test. ***p < 0.001 and ****p < 0.0001.

Figure 3

NLRP3 KO or pharmacological inhibition blocks NLRP3 pathway activation in iPSC-derived microglia. MCC950 (0.37-10 μM) dose response on (A) IL-1β and (B) TNF-α release from iMGL (line HC4) following LPS (1 μg/ml at 3 h) and nigericin (10 μM, 3 h) stimulation. (C, D) Effects of MCC950 on PI uptake in iMGL following LPS and nigericin stimulation. For measurements of MCC950 effects on PI uptake, dye was added directly after nigericin addition and iMGL were imaged over the following 3 h. (C) Representative live-cell microscopy images of PI staining of iMGL with different treatments. Data are shown as mean ± S.D. of four technical replicates from four donors. One‐way ANOVA followed by Tukey’s post hoc test. ****p < 0.0001.

Figure 4

NLRP3 pharmacological inhibition blocks bacteria-induced NLRC4 pathway activation in THP-1 cells. (A) PMA-differentiated WT THP-1 cells and (B) NLRP3 KO THP-1 cells were transfected with flagellins from Bacillus subtilis (Fla-BS), Pseudomonas aeruginosa (Fla-PA) or Salmonella typhimurium (Fla-ST; 100 ng/well) using LF2000 (0.5 μl/well) to activate the NLRC4 inflammasome and the release of IL-1β was measured after 24 h. (C) Effects of MCC950 (0.37-10 μM) on IL-1β release from PMA-differentiated WT THP-1 cells following transfection with Fla-BS, -PA or -ST. (D) Concentration-response curve for MCC950 in blocking IL-1β release from PMA-differentiated WT THP-1 cells following transfection with Fla-BS. (E) Effects of MCC950 on PI uptake in PMA-differentiated WT THP-1 cells following transfection with Fla-BS and imaging over 24 h. Data are mean ± S.D. of three technical replicates and three independent experiments. One‐way ANOVA followed by Tukey’s post hoc test. *p < 0.05 and ****p < 0.0001.

Figure 5

NLRP3 pharmacological inhibition does not block NLRC4 pathway activation in hMonocytes and hMDM. (A) hMonocytes or hMDM were transfected with flagellins from Bacillus subtilis (Fla-BS), Pseudomonas aeruginosa (Fla-PA) or Salmonella typhimurium (50 ng/well) using LF2000 (0.25 μl/well) and the release of IL-1β was measured after 24 h. Effect of MCC950 (0.37-10 μM) on (B) IL-1β and (C) IL-18 release from hMDM following transfection with Fla-ST (500 ng/well) using LF2000 (2.5 μl/well). (D) Effect of MCC950 (0.37-10 μM) on PI uptake in hMDM following transfection with Fla-ST (500 ng/well) using LF2000 (2.5 μl/well) and imaging over 24 h. Data are mean ± S.D. of three technical replicates and representative of three and four donors for hMonocytes and hMDM, respectively. One‐way ANOVA followed by Tukey’s post hoc test. ***p < 0.001 and ****p < 0.0001.

Figure 6

NLRP3 pharmacological inhibition blocks some aspects of NLRC4 pathway activation in iMGL. (A) iMGL were transfected with flagellins from Bacillus subtilis (Fla-BS), Pseudomonas aeruginosa (Fla-PA) or Salmonella typhimurium (500 ng/well) using LF2000 (0.25 μl/well) and the release of IL-1β was measured after 24 (h) Effect of MCC950 (0.37-10 μM) on IL-1β (B) and IL-18 (C) release from iMGL following transfection with Fla-BS (500 ng/well) using LF2000 (0.25 μl/well). (D) Effects of MCC950 (0.37-10 μM) on PI uptake in iMGL following transfection with Fla-BS (500 ng/well) using LF2000 (0.25 μl/well) and time-lapse imaging during 24 h. Data are mean ± S.D. of four technical replicates from four donors. One‐way ANOVA followed by Tukey’s post hoc test. ****p < 0.0001.

Figure 7

MCC950 impact on non-canonical inflammasome pathway activation in THP-1 cells and hMDM. (A) PMA-differentiated WT THP-1 cells and (B) NLRP3 KO THP-1 were pre-incubated with PAM3CSK4 (1 μg/ml, 3 h) followed by transfection with LPS (100 ng/well, 24 h) using LF2000 (0.5 μl/well) to activate the non-canonical inflammasome pathway and the release of IL-1β was measured. (C) Dose-dependent inhibition of IL-1β release from PMA-differentiated WT THP-1 cells following incubation with PAM3CSK4 and LPS transfection. Effect of MCC950 (0.37-10 μM) on (D) IL-1β and (E) IL-1α release from hMDM following incubation with PAM3CSK4 (1 μg/ml, 3 h) and transfection with LPS (1000 ng/well, 24 h) using LF2000 (0.5 μl/well). Effect of MCC950 (0.37-10 μM) on (F) IL-1β and (G) IL-1α release from iMGL following incubation with PAM3CSK4 (1 μg/ml, 3 h) and transfection with LPS (1000 ng/well, 24 h) using LF2000 (0.5 μl/well). Data are shown as mean ± S.D. of four technical replicates and representative of three independent experiments for THP-1 and four donors for hMDM. Data are shown as mean ± S.D. of three biological replicates from three donors for iMGL. One‐way ANOVA followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01 and ****p < 0.0001.

Figure 8

IL-1β secretion and pyroptosis in THP-1 cell lines carrying GoF mutations. PMA-differentiated WT, NLRC4 S171F, NLRP1 A66V, MEFV S242R and MEFV KO THP-1 cells were assessed for the release of IL-1β (A) and PI uptake (C) in unstimulated condition after 3 h and 24 h. (B) PMA-differentiated WT, NLRC4 S171F, NLRP1 A66V, MEFV S242R and MEFV KO THP-1 were pre-incubated with LPS (1 μg/ml, 3 h) and the release of IL-1β (B) and PI uptake (D) were measured after 3 h and 24 h. Data are shown as mean ± S.D. of four technical replicates from three independent experiments. One‐way ANOVA followed by Tukey’s post hoc test. **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 9

MCC950 does not block inflammasome-mediated pyroptosis in THP-1 carrying inflammasome GoF mutations. Dose response of MCC950 (0.37-10 μM) on IL-1β release in (A) NLRC4 S171F, (C) NLRP1 A66V, (E) MEFV S242R. PI uptake in (B) NLRC4 S171F, (D) NLRP1 A66V, (F) MEFV S242R. MCC950 impact on PI uptake. The dye was added following exposure to increasing concentrations of MCC950 and THP-1 cells were imaged for 25 (h) Data are shown as mean ± S.D. of four technical replicates from three independent experiments.

Figure 10

Acute systemic administration of MCC950 in mutant SOD1 mice does not ameliorate spinal cord inflammation. (A) Schematic workflow of tissue processing for CyTOF mass cytometry. (B) Frequency of resident and infiltrating immune cells in the spinal cord of vehicle-treated WT, vehicle-treated SOD1 and SOD1 mice treated with MCC950. Mono/Mθ: monocytes and macrophages; DCs: dendritic cells. (C) Representative two-dimensional projections of single-cell data generated by viSNE of an equal number of CD45⁺ immune cells from each individual animal. Each dot represents one cell. (D) Heatmap showing the normalized mean intensity of each marker in the various immune cell subsets. Mean intensity of each marker was normalized according to the maximal and minimal mean intensity value of the marker in each population among the various treatment groups. Ag. Present: antigen presentation; Phago: phagocytosis; CRs: cytokine-related receptors. (E) Mean signal intensity levels of CD86 in the different immune cell subsets in the three treatment groups. (F) Frequency of microglial subsets 1 to 8 in each sample in the three treatment groups. (G) Representative two-dimensional projections of single-cell data generated by viSNE of an equal number of CD45^low CX3CR1⁺ microglia from each individual animal. Each dot represents one cell. Pie charts show the relative abundance among microglia of the various subsets. (H) Expression levels of functional markers across identified microglial subsets. The dot plot shows the mean (Log2) signal of each marker in each microglial subset. Data in bar graphs shown as mean ± S.E.M. Values for each animal are included (N=8 male mice per treatment group). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test. Statistically significant differences between vehicle-treated WT litter mate and SOD1^G93A mice are shown in black asterisks. White asterisks represent statistically significant differences in MCC950-treated compared to vehicle-treated SOD1 mice; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.