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

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.

Type of file: MOV Title of file for HTML: Supplementary Movie 3 Description: Knock-down of MARK4 affects translocation of NLRP3 on mitochondria. PMAdifferentiated THP-1 cells stably expressing Cherry-NLRP3 (red) and shRNA (scrambled control or MARK4) were stimulated with nigericin (3 µM for 2 hrs), and then stained with mitotracker dye to label mitochondrion (green). Z section pictures were stacked to reconstitute 3D view.
Type of file: MOV Title of file for HTML: Supplementary Movie 4 Description: MARK4 and NLRP3 are moving together to MTOC. PMA-differentiated THP-1 cells stably expressing Cherry-NLRP3 (red) and GFPMARK4 (green) were stimulated with nigericin (10 µM). Arrowhead indicates MTOC where MARK4 is accumulated. Video corresponds to 198.366 seconds; width of the movie is 30.3 µm.
Type of file: MOV Title of file for HTML: Supplementary Movie 5 Description: NLRP3 is shuttling towards MTOC. PMA-differentiated THP-1 cells stably expressing Cherry-NLRP3 (red) were stained with Tubulin Tracker green to label microtubules (green) before or after nigericin stimulation (10 µM for 2 hrs). Z section pictures were stacked to reconstitute 3D view.
Type of file: MOV Title of file for HTML: Supplementary Movie 6 Description: Insufficient MARK4 causes dilated ring structure of NLRP3. HEK293T cells were cooverexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or cooverexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). One day after expression, cells were subject to imaging. Z section pictures were stacked to reconstitute 3D view.
Type of file: PDF Title of file for HTML: Peer Review File Description: Supplementary Figure 1. Microtubule-disrupting drugs reduce IL-1β production. Microtubule disrupting drugs colchicine (10 µM), nocodazole (10 µM) and vinblastine (10 µM) were used to pretreat WT BMDM 30 minutes before ATP stimulation (5 mM) for half an hour. Supernatant was collected, and IL-1β was measured using ELISA. Mean ± SEM, three experiments. Comparisons of the two different groups were analysed by unpaired t test. P < 0.01 (**) were considered as statistically significant (a, b, c, d). , and macrophage differentiation markers were compared between wild-type and MARK4 deficient BMDMs. Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant.

Supplementary Figure 5. Phagocytosis ability of MARK4 knockout cells. (a)
Wild-type or MARK4 deficient BMDM were incubated with MSU crystals. Phagocytosed MSU was taken by reflection microscopy and plasma membrane was highlighted by cholera toxin staining. Scale bar= 40 µm. (b) Phagocytosis ability of MARK4 knockout cells was also analyzed by Vybrant phagocytosis assay following manufacturer's instruction. Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant.

Supplementary Figure 6. The effect of stimulation on MARK4 knock-out cells.
(a, b, c,d ) BMDM derived from MARK4 WT or KO mice were treated with various stimuli indicated. TNF (a), IL-1α (b), and IL-18(c) were measured using ELISA, and cell death was measured by using LDH cytotoxicity assay (d). (e) Cell death induced by ATP (5 mM) over a time course was measured by LDH cytotoxicity assay. Mean ± SEM, three to four experiments. Comparisons of the two different groups were analysed by unpaired t test. P < 0.05 (*), P < 0.01 (**), P < 0.001(***), and P < 0.0001(****) were considered as statistically significant. Yellow arrowheads show reversal of movement direction by an individual particle, while white arrowhead depicts 'pause' event before the particle continues to travel in the same direction. Scale bars for X (displacement in µm, 'd') and Y (time in s, 't') axis are shown as yellow lines in the kymograph. (b) Box and whisker plots presents dispersion of run lengths measured for the NLRP3 particle movements in Control or MARK4 shRNA background. Mean run length is illustrated by '+' sign in box and whisker plot, while horizontal line describes median. Mean run length of 2.417 ± 0.237 µm (Mean ± SEM) and 2.864 ± 0.222 µm was observed in cells with Control and MARK4 shRNA respectively. Mean velocity of 0.138 ± 0.010 µm/s and 0.142 ± 0.008 µm/s was observed in cells with Control and MARK4 shRNA respectively. See also Supplementary Movie 3. binds to NLRP3 and transports it along microtubules to ensure optimal positioning and activation. During the transport process, they can encounter mitochondria. (c) Where NLRP3 may receive additional activatory cues, NLRP3 and MARK4 are further transported along microtubules to reach MTOC, where NLRP3 forms the characteristic speck structure required for optimal inflammasome activation.