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The Role of Microglial Inflammasome Activation in Pyroptotic Cell Death Following Penetrating Traumatic Brain Injury

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The Role of Microglial Inflammasome Activation in Pyroptotic Cell Death Following Penetrating Traumatic Brain Injury

Stephanie W Lee et al. J Neuroinflammation.

Abstract

Background: Traumatic brain injury remains a significant cause of death and disability in the USA. Currently, there are no effective therapies to mitigate disability except for surgical interventions necessitating a need for continued research into uncovering novel therapeutic targets. In a recent study, we used a rodent model of penetrating traumatic brain injury known as penetrating ballistic-like brain injury (PBBI) to examine the role of innate immunity in post-traumatic secondary injury mechanisms. We previously reported that the inflammasome, a multiprotein complex composed of apoptosis-associated speck-like protein containing card and caspase-1, plays a role in secondary cell death mechanisms after PBBI, including inflammatory cell death (pyroptosis).

Methods: In the current study, we used flow cytometry analysis to evaluate activated microglia and CD11b-positive leukocytes after PBBI and assessed inflammasome activation and pyroptosis of specific cellular populations. Sprague-Dawley male rats underwent PBBI or sham-operated procedures and ipsilateral cortical regions processed for flow cytometry and cellular analysis. Flow cytometry results were compared using one-way ANOVA followed by Tukey's multiple comparisons.

Results: At 48 h following PBBI, there was an increase in activated microglia and infiltrating leukocytes compared to sham controls that were associated with increased caspase-1 activity. Using a florescent probe to identify caspase-1 activity and a fluorescent assay to determine cell viability, evidence for pyroptosis in CD11b+ cells was also determined. Finally, while post-traumatic treatment with an anti-ASC antibody had no effect on the number of activated microglia and infiltrating leukocytes, antibody treatment decreased caspase-1 activity in both resident microglia and infiltrating leukocytes and reduced pyroptotic CD11b+ cell death.

Conclusions: These results provide evidence for inflammasome activation in microglia and infiltrating leukocytes after penetrating traumatic brain injury and a role for pyroptotic cell death in the pathophysiology. In addition to inhibiting neuronal cell death, therapeutic treatments targeting inflammasome activation may also provide beneficial effects by reducing the potentially detrimental consequences of activated microglia and infiltrating CD11b+ leukocytes following penetrating traumatic brain injury.

Keywords: ASC; Infiltrating leukocytes; Inflammasome; Microglia; PBBI; PTBI; Penetrating ballistic-brain injury; Penetrating traumatic brain injury; Pyroptosis; Traumatic brain injury.

Conflict of interest statement

Ethics approval and consent to participate

Animal procedures were approved by the University of Miami’s Institution of Animal Care and Use Committee and adhered to the ARRIVE guidelines and those established by the National Institute of Health Guide for the Care and Use of Laboratory Animals.

Consent for publication

Not applicable.

Competing interests

JPdRV, WDD, and RWK are managing members in InflamaCORE, LLC, a company dedicated to discovering novel diagnostic and therapeutic strategies targeting abnormal inflammasome activation in clinical conditions. The other authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Representative flow cytometry density plots from the ipsilateral cortex of a sham, b PBBI injured (vehicle), and c PBBI treated with anti-ASC. d Quantification of the number of surveying microglia, activated microglia, and infiltrating CD11b+ leukocytes at 48 h post-surgery. PBBI significantly increased the number of activated microglia and infiltrating CD11b+ leukocytes 48 h after injury. There was no change in either the number of infiltrating CD11b+ leukocytes or the number of microglia after treatment with anti-ASC. Data are presented as mean ± standard error of the mean. Statistical significance was determined with one-way ANOVA followed by Tukey’s post hoc test. ****p < 0.0001. ns, no significance. n = 6 per group
Fig. 2
Fig. 2
Representative flow cytometry scatter plots of infiltrating CD11b+ leukocytes and microglia expressing caspase-1 activity from the ipsilateral cortex of a sham, b PBBI injured (vehicle), and c PBBI treated with anti-ASC. d Quantification of the number of “resting” microglia, activated microglia, and infiltrating CD11b+ leukocytes 48 h after PBBI. Forty-eight hours after injury, PBBI significantly increased the number of activated microglia and infiltrating CD11b+ leukocytes expressing caspase-1 activity. Treatment with an antibody inhibiting ASC significantly decreased the amount of caspase-1 activity in both activated microglia and infiltrating CD11b+ leukocytes. Data are presented as mean ± standard error of the mean. Statistical significance was determined with one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5 per group
Fig. 3
Fig. 3
Representative flow cytometry density plots of caspase-1 activity (FLICA) versus amine reactivity (LIVE/DEAD) from the ipsilateral cortex of sham, PBBI injured (vehicle), and PBBI treated with anti-ASC (top row). CD11b+ cells were gated for pyroptotic cells (high FLICA expression and high LIVE/DEAD expression) and caspase-1 activity live cells (high FLICA expression and low LIVE/DEAD expression). Quantification of the number of CD11b+ cells that are live cells expressing caspase-1 activity or cells undergoing pyroptosis 48 h post-injury (bottom row). PBBI significantly increased the number of CD11b+ live cells expressing caspase-1 activity and the number of CD11b+ cells undergoing pyroptosis. The number of CD11b+ cells undergoing pyroptosis significantly decreased after treatment with anti-ASC. Data are presented as mean ± standard error of the mean. Statistical significance was determined with one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5 per group
Fig. 4
Fig. 4
Representative flow cytometry scatter plots of infiltrating CD11b+ leukocytes and microglia that are pyroptotic or caspase-1 activity expressing live cells from the ipsilateral cortex of a sham, b PBBI injured (vehicle), and c PBBI treated with anti-ASC. Cells previously gated for pyroptosis or live cells expressing caspase-1 activity were further gated into the three populations of cells: infiltrating CD11b+ leukocytes, activated microglia, and microglia. d Quantification of the number of infiltrating CD11b+ leukocytes that are live cells expressing caspase-1 activity and cells undergoing pyroptosis (expressing both increased amine reactivity and caspase-1 activity) 48 h post-injury. PBBI significantly increased the number of pyroptotic and caspase-1 activity-live infiltrating CD11b+ leukocytes. Treatment with an antibody against ASC significantly decreased the amount of infiltrating CD11b+ leukocytes undergoing pyroptosis. e Quantification of “all” microglia (surveying microglia + activated microglia). f Surveying microglia versus activated microglia that are live cells expressing caspase-1 activity or pyroptotic cells 48 h post-surgery. The number of pyroptotic microglia and live microglia expressing caspase-1 significantly increased after PBBI while treatment inhibiting ASC significantly decreased microglia undergoing pyroptosis. Splitting of the microglia population into surveying phenotype versus activated phenotype showed that activated microglia were the significant contributors to the number of live microglia expressing casepase-1 and overall microglial pyroptosis after PBBI. Treatment with anti-ASC significantly decreased the number of activated microglia undergoing pyroptosis. Data are presented as mean ± standard error of the mean. Statistical significance was determined with one-way ANOVA followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5 per group
Fig. 5
Fig. 5
Schematic of inflammasome activation and pyroptosis of microglia after PBBI and proposed effects of anti-ASC on the pathway. CNS injury induces the formation of ASC specks in traumatized cells that are released into the extracellular space leading to maturation of IL-1β. ASC specks are taken up by endogenous microglia or infiltrating phagocytic cells resulting in further inflammasome activation and subsequent death by pyroptosis. Anti-ASC either binds to extracellular ASC specks blocking extracellular IL-1 maturation thereby decreasing inflammasome activation and pyroptosis of microglia and infiltrating CD11b leukocytes or binds to intracellular inflammasomes thereby leading to decreased activation and pyroptosis of microglia and infiltrating CD11b leukocytes. Adapted from Broderick et al. [25]

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