NADPH Oxidase 2 Regulates NLRP3 Inflammasome Activation in the Brain after Traumatic Brain Injury

Figure 10

NOX2 deletion leads to a decrease in expression and activation of the NLRP3 inflammasome effectors, caspase-1 and IL-1β in the injured cortex after TBI. (a) Quantitative RT-PCR for inflammasome components and downstream interleukins caspase 1 and IL-1β. mRNA samples were collected from the injured cortex at the 4-day post-TBI in WT and NOX2 KO mice. Caspase-1 and IL-1β mRNA show increased gene expression at the 4-day post-TBI. Deletion of NOX2 significantly attenuates these changes. mice (ctrl, sham, WT, and KO, resp.). (b) Representative Western blot showing protein expression of NLRP3 inflammasome products cleaved caspase-1 p20 and cleaved IL-1β. At 4 days after TBI, WT mice show increased expression of both cleaved effectors. However, mice deficient in NOX2 show attenuated cleavage of caspase-1 and IL-1β. Inhibition of NOX using apocynin also produces similar attenuated cleavage. Quantification of blots shown below representative image (data normalized to β-actin and presented as fold change relative to WT ctrl mice). BV2 sample included as positive control. mice (WT ctrl, KO ctrl, WT, KO, and APO, resp.). (c) Representative confocal images showing immunoreactivity of cleaved caspase 1 p20 and cleaved IL-1β in the injured cortex after TBI for WT and NOX2 KO mice. All images have been quantified to the right of the representative panel (data presented as fold change relative to sham mice). TBI increased cleavage of caspase-1 and IL-1β as detected by immunoreactivity of its cleaved products in WT mice at the 4-day post-TBI. Deletion of NOX2 attenuates this cleavage of caspase-1 and IL-1β at the 4-day post-TBI. mice (sham, WT, and KO, resp.). Scale bar represents 50 μM. (, , sham versus WT TBI; ^#, ^##, and ^### WT TBI versus KO or APO TBI).

(a)

(b)

(c)