SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes

Shi, Chong-Shan; Nabar, Neel R.; Huang, Ning-Na; Kehrl, John H.

Intracellular protein aggregation and lysosomal stress have previously been mechanistically linked to NLRP3 activation 33, 34 . As SARS patients show increased serum IL-1β and IL-18 7,8 , we investigated whether ORF8b affects inflammasome activation in macrophages. To do so, we first attempted to generate a permanent cell line expressing GFP-8b in the human macrophage cell line THP-1 cells. While we easily established GFP expressing THP-1 cells, no cells survived the selection process when we attempted to express GFP-8b. This is likely secondary to the toxicity of the protein, as even attempts at using an inducible promoter failed to generate a useable cell line. Therefore, we switched to a transient transfection system. Evaluating inflammasome activation in macrophage cell lines following transient DNA transfection can be problematic as cytosolic DNA activates DNA sensing inflammasomes. However, we found that that by carefully titering the amount of DNA transfected, we could express the GFP vector and yet trigger minimal Il-1β production. Therefore, we compared IL-1β production by THP-1 cells transiently transfected with low amounts of the vectors producing GFP, GFP-8b, 8b-GFP, or GFP-8b V77K. We Confocal microscopy images of GFP or GFP-8b co-transfected with RFP-galectin 3 (a) or mCherry-TFEB (b). Individual and merged images are shown, and the scale bar is 5 μm. c The percentage of cells with predominantly nuclear TFEB from part b was quantified for each condition (three fields, minimum of 50 cells/field). **p < 0.01 with Student's t-test. d Immunoblot to assess LAMP1 and TFEB expression in nuclear and cytosolic fractions separated by SDS-PAGE. HeLa cells were transiently transfected with GFP or GFP-8b. e Representative confocal images from HeLa cell expressing GFP or GFP-8b and immunostained for LAMP1 expression. Individual and merged images are shown, and the scale bar is 5 μm. f Immunoblot of cytosol and nuclear extracts from GFP, GFP-8b, or GFP-8b V77K transfected HeLa cells treated overnight with or without the calcineurin inhibitor cyclosporin A (CsA-400 nM). Images and blots are representative data from experiments repeated a minimum of 3 times found that both GFP-8b and 8b-GFP increased the amount of IL-1β present in the cell supernatant compared to GFP alone (Fig. 5a) . Surprisingly, the GFP-8b V77K construct also enhanced IL-1β production suggesting that the mechanism did not solely depend upon lysosomal stress (Fig. 5a) . We also checked whether an alternatively tagged version could trigger IL-1β production by expressing Flag tagged version of ORF8b. Similar to the results with the GFP-tagged ORF8b, we found that 8b-Flag induced 3-fold more Il-1β than did the Flag vector (Fig. 5b) . A complementary approach to assess inflammasome activity is to reconstitute the requisite components in HEK293T cells. Therefore, we transfected HEK293T cells with expression vectors for NLRP3, caspase-1, ASC, and full-length IL-1β in the presence of GFP or GFP-8b. A similar experiment was performed using FLAG or 8b-FLAG. In both instances we found an increase in IL-1β in the supernatant and a decrease in fulllength IL-1β in the cell lysate in the presence of the ORF8b constructs versus the controls (Fig. 5c) . These results suggest that ORF8b activates NLRP3 inflammasomes in macrophages and likely in monocytes.