Fig 1: NLRP3, IL-1β and IL-18 are increased in the CSOM cochlea. A shows that mRNA expression of NLRP3 (p = 0.018) and IL-1β (p = 0.032) were significantly elevated in CSOM cochleae compared to control cochleae at 7d. mRNA data is shown as mean+/- SEM. Protein levels of NLRP3, IL-1β and IL-18 were determined in control and CSOM cochleae at 7d using ELISA. B shows a significant increase of NLRP3 in CSOM, whereas it was undetectable in the control group. C & D show a significant increase of IL-1β in CSOM (p < 0.001) and IL-18 in CSOM (p = 0.013). The number of mice is shown in parentheses. ELISA data is shown as mean +/- SD
Fig 2: Generation of microglia-specific NLRP3-KO or NLRP3-overexpressing mice. (A) Illustration of mice with microglia-specific depletion of NLRP3 (Tmem119p-CreERT2; NLRP3 (fx/fx)) and their control NLRP3(fx/fx) mice, as well as mice with microglia-specific persistent expression of NLRP3 (Tmem119p-CreERT2; NLRP3mut) and their control NLRP3mut mice. (B) NLRP3 staining was done in spinal discs from tamoxifen-challenged mice. (C, D) Dissociated cells from spinal discs of the mice were FAC sorted for CD68+Tmem119+ microglia, the NLRP3 levels of which were checked by ELISA. (C) The relative levels to those from NLRP3(fx/fx) (=1) were shown. (D) The presentative flow charts of FACS sorting CD68+Tmem119+ microglia. *p<0.05. ns, non-significant. Scale bars are 100µm.
Fig 3: NLRP3-depletion-induced M2 macrophage polarization reduces cytotoxic T cells in vivo. The tumor was digested and subjected to flow cytometry analysis. (A, B) FACS for CD68 and CD163, shown by quantification (A) and by representative flow charts (B). (C, D) FACS for CD3 and CD8, shown by quantification (C) and by representative flow charts (D). (E) Schematic of the study, showing that loss of NLRP3 polarizes intratumor macrophages to attenuate the immune attack on EMC both directly and indirectly through regulating cytotoxic T cells. *p <0.05. N = 5.
Fig 4: Chlorogenic acid exerts anti-inflammatory effect by reducing PKM2-depdent glycolysis of macrophage.A–E RT-qPCR analysis of Pkm2, Hk2, Pdk1, Ldha, and Glut1 mRNA levels. F, H, I ELISA of lactate production, caspase-1 and NLRP3 activity. G The Seahorse XF-96 Extracellular Flux Analyzer of ECRA. J–M Secretion of IL-1β, IL-18, IL-6 and TNF-α in RAW264.7 cells (n = 6). Data represent means ± SD of three separate experiments. Statistics was performed with unpaired two- sided Student’s t test or one-way ANOVA, followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01.
Fig 5: An active NLRP3 inflammasome leads to OHC loss in CSOM. Representative wholemount sections of the cochlea (basal, middle and apical turn) in CSOM at 14d from control (A-C) and NLRP3−/− (D-F) CSOM mice. Panel G shows the OHC survival rate in % in the cochlear basal turn of both groups, NLRP3−/− is protective for OHC loss in CSOM (p = 0.007). Red = Myosin VIIa. The number of mice per group is shown in parentheses. Data is shown as mean +/- SD. Arrows show HC loss. Scale bar = 100 μm
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