MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

Abstract

Excessive activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including cardiovascular and Alzheimer's disease. Here we show that microtubule-affinity regulating kinase 4 (MARK4) binds to NLRP3 and drives it to the microtubule-organizing centre, enabling the formation of one large inflammasome speck complex within a single cell. MARK4 knockdown or knockout, or disruption of MARK4-NLRP3 interaction, impairs NLRP3 spatial arrangement and limits inflammasome activation. Our results demonstrate how an evolutionarily conserved protein involved in the regulation of microtubule dynamics orchestrates NLRP3 inflammasome activation by controlling its transport to optimal activation sites, and identify a targetable function for MARK4 in the control of innate immunity.

Figure 1. Loss of MARK4 affects IL-1β production under NLRP3 inflammasome activation.

(a) Mark4 expression under unprimed and LPS primed conditions in mouse bone marrow-derived macrophages (BMDM). Cytoplasm proximity ligation assay (PLA) signal per cell cytoplasm is used for quantification. Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group. Experiments have been repeated three times. Scale bar, 10 μm. (b) siRNA of MARK4, NLRP3 or ASC caused reduction of ATP induced IL-1β production in THP-1 cells. (c) shRNA of MARK4 caused reduction of IL-1β induced by NLRP3 stimuli, including monosodium urate (MSU) and cholesterol crystals (CC) in THP-1 cells. Mock represents macrophages without further stimulation. (d) BMDM derived from Mark4 KO mice exhibited selectivity towards NLRP3 activating stimuli. Mean±s.e.m., three to four independent experiments combined (b–d), comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. *P<0.05, **P<0.01 and ***P<0.001 were considered as statistically significant (a–d). (e) Representative western blots of caspase-1 (Casp1) and IL-1β in the supernatant (Sup.) in Mark4^+/+ and Mark4^−/− macrophages activated with indicated stimuli. Cell lysates were used as controls to indicate equal amount of cells for analysis.

Figure 2. MARK4 interacts with NLRP3 in a microtubule-dependent manner.

(a) Upon nigericin stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. (b) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. (c) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. (d) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group (a,b,d). Comparisons of the two different groups were analysed by unpaired t-test. NS was considered as not statistically significant. *P<0.05, ***P<0.001 and ****P<0.0001 were considered as statistically significant (a,c,d). Results are representatives of three independent experiments. Scale bar, 10 μm.

Figure 3. Mapping of interacting domains between MARK4 and NLRP3.

(a) Western blots of co-immunoprecipitated full-length NLRP3 with truncated MARK4 (KD: kinase domain; CD: catalytic domain; UBA: Ubiquitin-associated domain), respectively. (b) Western blots of coimmunoprecipitated truncated NLRP3 (Pyrin Del: pyrin domain deletion; Pyrin only: pyrin domain only; NACHT: Nucleotide-binding oligomerization domain; LRR: Leucine-rich repeat) with full-length MARK4, or co-immunopreciptated MARK4 with truncated NLRP3, respectively. (c) Schematic diagram showing that MARK4 catalytic kinase domain and NLRP3 Pyrin &NACHT domain were essential for their interaction. (d) Western blots of co-immunoprecipitated NLRP3 point mutants with MARK4 respectively. Whole cell lysates were analysed as indication of transfection. (e) Purified recombinant NLRP3 (1-291aa) immobilized on the glutathione sepharose can pull down purified recombinant full-length MARK4 directly. Western blots are representatives of three independent experiments.

Figure 4. MARK4 is involved in NLRP3 positioning along microtubules.

(a) Upon nigericin stimulation (3 μM), NLRP3 (arrow) was moving along microtubules (MT) to meet mitochondria (arrowhead) in THP-1 cells stably expressing NLRP3-cherry. Scale bar, 2 μm. See also Supplementary Movie 1. (b) Orthogonal view of NLRP3 with mitochondria in THP-1 cells stably expressing shRNA of MARK4 or scrambled controls. See also Supplementary Movie 2. (c) Cherry-NLRP3 and GFP-MARK4 were moving together towards MTOC in differentiated THP-1 cells upon nigericin stimulation. Histogram mean of MARK4-GFP was calculated within the indicated square. Arrowheads indicate MTOC. See also Supplementary Movie 4. (d) Orthogonal view of NLRP3 with microtubule in THP-1 cells stably expressing NLRP3-Cherry before or after stimulation by nigericin (10 μΜ for 2 h); orthogonal view was centered around MTOC. See also Supplementary Movie 5. Experiments were repeated at least three times. Scale bar, 10 μm (b–d).

Figure 5. MARK4 deficiency affects MTOC and speck nucleation.

(a) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. (b,c) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t-test. ****P<0.0001 was considered as statistically significant (b). Mitochondria distribution was shown after speck formation (c). See also Supplementary Movie 6. (d) In differentiated THP-1 cells, upon nigericin stimulation (10 μΜ for 1.5 h), NLRP3 was translocated to MTOC, indicated by γ-tubulin. NLRP3 remained in cytoplasm distribution in MARK4 shRNA cell. Experiments were repeated at least three times. Scale bar, 10 μm.

Figure 6. CagA impairs inflammasome activation by disrupting NLRP3-MARK4 interaction.

(a) CagA peptide impaired the interaction between NLRP3 and MARK4 upon NLRP3 inflammasome activation by nigericin (3 μM for 2 h) in the differentiated THP-1 cells. A control peptide was employed. Scale bar, 10 μm. (b) ELISA of IL-1β level in the supernatants of WT BMDM cells following pretreatment of CagA peptide (with indicated concentrations) and nigericin stimulation (10 μM for 1 h). Mark4 KO cells were used as controls. (c) CagA peptide prevented formation of Nlrp3 and Asc complex under nigericin stimulation (3 μM for 2 h) in WT BMDMs. Mark4 KO cells were used as controls. Scale bar, 10 μm. Mean±s.e.m. for all the cells taken from five different views at × 40 magnification for each group (a,c). (d) CagA peptide impaired the formation of NLRP3 speck on MTOC upon NLRP3 inflammasome activation (MSU 250 μg ml⁻¹ for 6 h) in the differentiated THP-1 cells. Inset was used to display magnification of the boxed region as examples of MARK4, NLRP3, and γ-tubulin localization. Scale bar, 20 μm. Mean±s.e.m., at least three experiments (a–d). Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 were considered as statistically significant (a–d).

Figure 7. Mark4 promotes Nlrp3-dependent responses in vivo.

Representative images of neutrophil recruitment by flow cytometry are shown in mice injected intraperitoneally (i.p.) with MSU (a). Neutrophil count (b), IL-1β (c) and TNF levels (d) in peritoneal exudate of mice (Mark4^−/−, Mark4^+/+, Nlrp3^−/−) injected i.p. with MSU, with or without IL-1β blocking antibody. IL-1β below the detection level of 3.5 pg ml⁻¹ was plotted along Y axis at 3.5 pg ml⁻¹. Thioglycollate was used as an inflammasome-independent inflammatory agent. Mean±s.e.m., n=5 sex and age-matched mice (males, 8 to 10 week-old) in each group. Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. **P<0.01 and ***P<0.001 were considered as statistically significant (b).