Abstract
Purpose :
Our untargeted metabolomics analysis revealed a time-dependent reduction in retinal NAD+ levels with a concomitant increase in necrotic cell death during experimental bacterial (S. aureus) endophthalmitis. We hypothesized that high levels of poly (ADP-ribose) polymerase 1 (PARP1) activity cause rapid NAD+ depletion and triggers parthanatos cell death pathway during endophthalmitis. The aim of this study is to investigate the role of parthanatos in the pathobiology of bacterial endophthalmitis and decipher underlying mechanisms.
Methods :
In vivo (C57BL/6 mouse) and in vitro (BMDMs, retinal Muller glia) studies were performed using pharmacological inhibitors of PARP1 and calpain signaling pathways. In mice, disease progression was assessed by non-invasive (ERG and fundus exam) and invasive (bacterial burden, cytokine levels) methods. PARP1 activity was measured by fluorescent NAD+ substrate (6-Fluo-10-NAD+) on unfixed bacterial infected retinal tissue. Similarly, calpain substrate t-BOC-Leu-Met-CMAC (CMAC) was used to assess the calpain activity. Live cell imaging was performed to determine the effect of inhibitors on cell viability. The expression of protein levels was detected by western blot and immunofluorescence whereas transcript levels were checked with qPCR. The expression of inflammatory mediators in retinal tissue or cell lysates were estimated with ELISA.
Results :
S. aureus infection induced the expression of PARP1 and Calpain in mouse retina. This coincided with increased PARP1 and Calpain activity in infected tissues. Both PARP1 or calpain inhibitors reduced parthanatos signaling resulting in diminished retinal inflammation and reduced retinal cell death. Calpain inhibitor reduced calcium buildup in infected mouse retinas and Muller glia. Our data showed that reducing PARP1 and Calpain overactivation restored cellular NAD+ and ATP levels. Moreover, NLRP3 inflammasome activation and IL-1β production is reduced by PARP1 and Calpain inhibitors in both in vivo and in vitro.
Conclusions :
Our study demonstrates the activation of parthanatos in bacterial endophthalmitis and emphasizes the role of NAD+ metabolism in regulating inflammatory cell death. Moreover, the inhibition of calpain activity could be a promising therapeutic target in ameliorating endophthalmitis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.