Increasing Autophagy ameliorate all-trans-retinal-activated NLRP3 Inammasome 2 in human THP-1 macrophage cells

18 Background. Profound inflammation that mediated by innate immune sensors can be observed in retina, and is considered to play an important role 20 in the pathogenesis of all- trans -retinal (atRAL)-caused retinal degeneration. 21 However, the underlying mechanism remains elusive. 22 Methods. Cell viability was detected with Cell Counting Kit-8 (CCK-8). 23 The concentration of IL-1β was evaluated using IL-1β ELISA Kits. The levels of 24 autophagy-related proteins were measured by Western blotting. The 25 measurement of autophagic flux was performed with virus vectors packing 26 tandem monomeric mCherry-eGFP-tagged LC3B. 27 Results. We focused on studying the effects of atRAL on macrophage cell 28 and the underlying pathway through pharmacological and genetical manipulation. We first found the maturation and release of IL-1β was regulated by the activation of NLRP3 inflammasome. We secondly found that mitochondria-associated reactive oxygen species (ROS) 32 were involved in the regulation of NLRP3 inflammasome activation and 33 caspase 1 cleavage. Finally, we found that atRAL functionally activated 34 autophagy in THP-1 cells, and atRAL-caused NLRP3 inflammasome activation 35 is suppressed by autophagy. Overall, our results show atRAL simultaneously 36 activates NLRP3 inflammasome and autophagy in THP-1 cells, and increasing 37 autophagy leads to the inhibition of the excessive activation of NLRP3 38 inflammasome. Our study provides new insight into the pathogenesis of aging 39 related retina degeneration. atRAL-related autophagy suppresses NLRP3 inflammasome activation in the retina. This work provides a further insight in atRAL-associated retinopathy.


Methods.
Cell viability was detected with Cell Counting . 23 The concentration of IL-1β was evaluated using IL-1β ELISA Kits. The levels of  Results. We focused on studying the effects of atRAL on macrophage cell 28 line THP-1 and determining the underlying signal pathway through 29 pharmacological and genetical manipulation. We first found the maturation and 30 release of IL-1β was regulated by the activation of NLRP3 inflammasome. We 31 secondly found that mitochondria-associated reactive oxygen species (ROS) 32 were involved in the regulation of NLRP3 inflammasome activation and 33 caspase 1 cleavage. Finally, we found that atRAL functionally activated The eye has been considered as an immune privilege organ, whereas 43 recent findings suggested profound inflammation mediated by innate immune 44 sensors also takes place in retina (Tarallo, et al. 2012). However, 45 inflammasomes is critical innate immune components, and mounting 46 evidences have suggested that the activation of NLRP3 inflammasome is 47 involved in the occurrence and development of age-related macular 48 degeneration (AMD) (Doyle, et al. 2012, Kauppinen, et al. 2016 2019, Tarallo, et al. 2012, Tseng, et al. 2013, Wang, et al. 2016. NLRP3 50 inflammasome is a large intracellular multimeric protein complex consisted by 51 NLRP3 receptor, the adaptor molecule apoptosis-associated speck-like protein 52 containing a caspase activation and recruitment domain (ASC), and caspase-1 53 (Wu, et al. 2013). NLRP3 inflammasome formation activates caspase-1, which 54 subsequent proteolytically cleaves pro-interleukin (IL)-1β and pro-IL-18 55 (Kauppinen, et al. 2016, Wu, et al. 2013. Although researches demonstrated 56 that activation of NLRP3 inflammasome was protective in neovascular AMD, 57 its promotion role in the pathogenesis of geographic atrophy has been 58 documented (Doyle, et al. 2012, Tarallo, et al. 2012. The clinical report that up 59 to 90% of advanced AMD was atrophic (Kauppinen, et al. 2016) suggests 60 NLRP3 inflammasome activation could contribute to the pathogenesis of AMD. clearance is associated to retina degeneration (Kohno, et al. 2013, Maeda, et 67 al. 2008, Shiose, et al. 2011, Zhang, et al. 2020.

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Previously reported researches demonstrated that delayed atRAL 69 clearance after light exposure caused sub-retinal infiltration of 70 microglia/macrophages (Kohno, et al. 2013), indicating that the resident or 71 infiltrating immune cells may be involved in atRAL-induced inflammation in the 72 retina. It was also found that atRAL-induced photoreceptors apoptosis results 73 in double-stranded RNA or protein releasing, which in turn activates TLR3 and 74 TLR4 receptors (Kohno, et al. 2013, Shiose, et al. 2011, respectively. These 75 findings suggest that immunoregulatory mechanisms are implicated in the 76 pathogenesis of atRAL-caused retinal degeneration. NLRP3 inflammasome 77 has been demonstrated to be involved in sensing endogenous danger signals, 78 including uric acid crystals (Martinon, et al. 2006), extracellular ATP (Cruz, et al. 79 2007), and fatty acids (Wen, et al. 2011). In the present study, we hypothesized 80 that atRAL could activate NLRP3 inflammasome in the macrophage cell line 81 THP-1. We first found the maturation and release of IL-1β was regulated via 82 caspase 1 pathway, and the activation of NLRP3 inflammasome is 83 indispensable for such an IL-1β release. We further found that      were visualized using the Clarity TM Western ECL Substrate (Bio-rad, USA).

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The ChemiDox TM XRS+ system (Bio-rad, USA) and the Image Lab TM software 164 (Bio-rad, USA) were used for all western blot results detection and analysis.

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Statistical analysis 166 All data in the present study were expressed as the means ± SEM.

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Statistical analysis were carried out by one-way analysis of variance, followed 168 by Tukey's multiple comparisons test. P<0.05 was considered as a statistically 169 significant.

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atRAL induces the release of IL-1β by THP-1 cells 172 We first examined atRAL-caused cytotoxicity in THP-1 cells. As depicted 173 in Fig. 1B, the test concentrations of 5 μM or 10 μM atRAL appeared to 174 produce no cytotoxicity in THP1 cells; whereas at the concentration of 20 μM 175 cell viability was significantly inhibited after 24 h treatment. We also found that 176 5 or 10 μM of atRAL exposed to THP-1 cells dose-dependently enhanced the 177 expression of NLRP3 and activated caspase 1 (p20) (Fig. 1E). To avoid the 178 cytotoxic effects of high dose, we used 5 μM of atRAL in the subsequently 179 experiments. We detected the expression of NLRP3 and the result-in release 180 of IL-1β were significantly increased by 5 μM atRAL treatment within 24h in 181 time-dependent manner (Fig. 1D, C). Furthermore, these findings suggest that 182 atRAL can cause NLRP3 increasing, and activation of caspase 1, and 183 consequent mature IL-1β production.  Fig. 2A), and attenuated the maturation of IL-1β (Fig. 2B), suggesting that 193 atRAL-caused IL-1β production is mediated via caspase 1 regulation. We 194 further genetically knocked down NLRP3 with a specific siRNA, and found that 195 NLRP3-targeted siRNA dramatically diminished NLRP3 protein levels (Fig. 2C) 196 and abolished atRAL-caused cleaved caspase 1 (p20) expression (Fig. 2D).

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LC3BII is a frequently used marker for autophagosomes, and is converted 216 from LC3BI during autophagy. We therefore deployed it to determine whether 217 autophagy play a role in the activation of NLPR3 inflammasome caused by 218 atRAL. We found that atRAL (5 μM) significantly increased the expression of 219 LC3BII after incubation for 6 h, while maintaining the expression level from 12 220 to 24 h (Fig. 4A). The LC3B turnover assay was employed to further examine 221 atRAL-caused autophagy in THP-1 cells. Our data showed that atRAL-caused 222 LC3BII accumulation could further enhanced by CQ treatment (Fig. 4B). in THP-1 cells (Fig. 4C, white arrow heads). We also observed that autophagy 235 inhibitors CQ or 3-MA augmented atRAL-caused NLRP3 expression (Fig. 4D). 236 Moreover, we found that the novel autophagy activator, trehalose (Tre), 237 up-regulated the expression of atRAL-induced LC3BII, and suppressed 238 NLRP3 accumulation (Fig. 4E). These data indicate that atRAL activates 239 autophagy, which may inhibit NLRP3 inflammasome activation in THP-1 cells.

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As ROS are verified to be early inducers of autophagy (Zhang, et al. 2019), we 281 found that autophagic flux was elevated following atRAL-incubation ( Fig. 4A-C).