Inhibition of Autophagy in Microglia and Infiltrating Monocytes/Macrophages Exacerbates Neuroinflammation and Functional Defects Following Traumatic Brain Injury

Abstract

The autophagy-lysosomal pathway serves an important role in cellular homeostasis and protection against neurodegeneration. Recently autophagy has been also implicated in regulation of immune and inflammatory responses. Specifically, high levels of autophagy flux – the progress of substrates through autophagic compartments leading to their delivery and degradation in the lysosomes – are generally associated with anti-inflammatory, and inhibition of flux with pro-inflammatory phenotypes.

To determine if autophagy may be involved in modulation of brain inflammation after traumatic brain injury (TBI), we assessed the levels of autophagy in resident microglia and infiltrating macrophages following moderate controlled cortical impact (CCI) in C57Bl/6 mice. Consistent with a potential function in neuroinflammation, our data demonstrated accumulation of autophagosomes and inhibition of autophagy flux specifically in the activated microglia and infiltrating macrophages starting by day 1 and continuing through day 28 post-CCI. Our immunofluorescence studies in transgenic Cx3Cr1-GFP microglial and Ccr2-RFP monocyte reporter mice confirmed inhibition of autophagy flux and demonstrated that while both activated resident microglia and infiltrating macrophages are affected, inhibition of autophagy is much more prominent in macrophages. We used flow cytometry analysis to demonstrate that immune cells with inhibited autophagy flux after CCI expressed increased levels of pro-inflammatory markers, including IL-1 and TNF-, as compared to corresponding immune cells with normal levels of autophagy. Furthermore, inhibition of autophagy flux correlated with impaired phagocytic function, indicating microglial/macrophage dysfunction. These trends persisted through day 28- post CCI, suggesting that autophagy flux impairment following TBI has long-term consequences. Consistent with ability of autophagy to affect inflammation, our in vitro experiments in IMG microglial and RAW 246.7 macrophage cell lines demonstrated that inhibition of autophagy can potentiate pro-inflammatory activation induced by lipopolysaccharide (LPS) treatment. In vivo, mice with microglia/macrophage-specific knockout of the autophagy gene Becn1 (Beclin1flox/flox,LysMCre/Cre) subjected to CCI showed increased expression of pro-inflammatory genes and proteins as compared to LysMCre/Cre controls. This included a marked exacerbation of the innate immune responses, including activation of the NLRP3 inflammasome and the type-I IFN pathways, indicating the importance of autophagy in suppression of innate-immunity mediated inflammation. Finally, Beclin1flox/flox, LysMCre/Cre mice performed worse than LysMCre/Cre controls on motor and cognitive behavioral tasks after CCI. Overall, these findings indicate that inhibition of autophagy in microglia/macrophages exacerbates neuroinflammation after TBI and contributes to functional deficits.