Högskolan i Skövde

Formation of the NLRP3 inflammasome occurs in response to a wide range of triggers and leads to pyroptosis and release of IL-1β and IL-18. This has widely been considered an "all or nothing" response leading to similar cellular outcomes each time, once the inflammasome has formed, a view that has been challenged over the years. This assumption has largely been based on endpoint measurements at the population level, producing metrics that fail to adequately capture dynamic responses. Herein, we utilize live-cell imaging combined with an algorithmic approach to track individual ASC-GFP specks over time, formed in response to the triggers ATP, monosodium urate and nigericin. Using this approach, we report trigger-dependent differences in speck lifetime. The use of the proposed algorithm requires a relatively small dataset, while still providing insights into NLRP3 inflammasome dynamics downstream of inflammasome activation at the single-cell level.

Existing research has proven difficult to understand the interplay between upstream signaling events during NLRP3 inflammasome activation. Additionally, events downstream of inflammasome complex formation such as cytokine release and pyroptosis can exhibit variation, further complicating matters. Cell Painting has emerged as a prominent tool for unbiased evaluation of the effect of perturbations on cell morphological phenotypes. Using this technique, phenotypic fingerprints can be generated that reveal connections between phenotypes and possible modes of action. To the best of our knowledge, this was the first study that utilized Cell Painting on human THP-1 macrophages to generate phenotypic fingerprints in response to different endogenous and exogenous NLRP3 inflammasome triggers and to identify phenotypic features specific to NLRP3 inflammasome complex formation. Our results demonstrated that not only can Cell Painting generate morphological fingerprints that are NLRP3 trigger-specific but it can also identify cellular fingerprints associated with NLRP3 inflammasome activation.

The NLRP3 inflammasome is an intracellular multiprotein complex described to be involved in both an effective host response to infectious agents and various diseases. Investigation into the NLRP3 inflammasome has been extensive in the past two decades, and often revolves around the analysis of a few specific readouts, including ASC-speck formation, caspase-1 cleavage or activation, and cleavage and release of IL-1β and/or IL-18. Quantification of these readouts is commonly undertaken as an endpoint analysis, where the presence of each positive outcome is assessed independently of the others. In this study, we apply time-resolved analysis of a human macrophage model (differentiated THP-1-ASC-GFP cells) to commonly accessible methods. This approach yields the additional quantifiable metrics time-resolved absolute change and acceleration, allowing comparisons between readouts. Using this methodological approach, we reveal (potential) discrepancies between inflammasome-related readouts that otherwise might go undiscovered. The study highlights the importance of time-resolved data in general and may be further extended as well as incorporated into other areas of research. 

The NLRP3 inflammasome is an important mediator of the host inflammatory response, and downregulation of inflammation is important in cancer treatment. Here, we investigated four different pancreatic ductal adenocarcinoma (PDAC) cell lines, AsPC-1, BxPC-3, CFPAC-1 and Panc-1, with regards to NLRP3 inflammasome formation and cytokine secretion. ASC specks were observed in all the cell lines investigated, but AsPC-1 was the only cell-line with the co-localization of anti-ASC and anti-NLRP3 and spontaneously formed multiple NLRP3 inflammasomes per cell. The co-localization of NLRP3 and ASC was not accompanied by IL-1β release nor significant IL-18 release. BxPC-3 displayed relatively high expression of the inflammasome-related genes IL1B and CASP1 and had the highest levels of IL1β and IL18 secretion and the highest amount of ASC. The inflammasome-associated genes IL18 and PYCARD were up-regulated in the PDAC primary tumors compared to normal tissue, and high PDAC tumor expression of IL18, CASP1 and PYCARD correlated with low patient survival. We have shown that PDAC cell lines display significant variations in their inflammasome-related gene expression and readouts. We conclude that spontaneous ASC speck formation is possible in PDAC cells and that multiple NLRP3 inflammasomes are formed spontaneously in AsPC-1 cells but that the co-localization of NLRP3 and ASC specks does not automatically entail inflammasome function.

Purpose

Loss of expression of DLG2 has been identified in a number of cancers to contribute to the disease by resulting in increased tumor cell proliferation and poor survival. In light of the previous evidence that DLG2 alters the cell cycle and affects proliferation, combined with indications that DLG2 is involved in NLRP3 inflammasome axis we speculated that DLG2 has an immune function. So far, there is no data that clearly elucidates this role, and this study was designed to investigate DLG2 in inflammatory colon disease and in colon cancer as well as its impact on inflammasome induction.

Methods

The DLG2 expression levels were established in publicly available inflammation, colon cancer and mouse model datasets. The overexpression and silencing of DLG2 in colon cancer cells were used to determine the effect of DLG2 expression on the activation of the inflammasome and subsequent cytokine release.

Results

The expression of DLG2 is repressed in inflammatory colon diseases IBD and Ulcerative colitis as well as colorectal cancer tissue compared to healthy individuals. We subsequently show that induction with inflammatory agents in cell and animal models results in a biphasic alteration of DLG2 with an initial increase followed by an ensuing decrease. DLG2 overexpression leads to a significant increase in expression of IL1B, IκBζ and BAX, components that result in inflammasome formation. DLG2 silencing in THP1 cells resulted in increased release of IL-6 into the microenvironment which once used to treat bystander COLO205 cells resulted in an increase in STAT3 phosphorylation and an increase proliferating cells and more cells in the G2/M phase. Restoration of DLG2 to the colon resulted in reduced AKT and S6 signaling.

Conclusion

DLG2 expression is altered in response to inflammation in the gut as well as colon cancer, resulting in altered ability to form inflammasomes.

Trial registration

NCT03072641.