Spatiotemporal Control of Inflammatory Lytic Cell Death Through Optogenetic Induction of RIPK3 Oligomerization

Abstract

Necroptosis is a programmed lytic cell death involving active cytokine production and plasma membrane rupture through distinct signaling cascades. However, it remains challenging to delineate this inflammatory cell death pathway at specific signaling nodes with spatiotemporal accuracy. To address this challenge, we developed an optogenetic system, termed Light-activatable Receptor-Interacting Protein Kinase 3 or La-RIPK3, to enable ligand-free, optical induction of RIPK3 oligomerization. La-RIPK3 activation dissects RIPK3-centric lytic cell death through the induction of RIPK3-containing necrosome, which mediates cytokine production and plasma membrane rupture. Bulk RNA-Seq analysis reveals that RIPK3 oligomerization results in partially overlapped gene expression compared to pharmacological induction of necroptosis. Additionally, La-RIPK3 activates separated groups of genes regulated by RIPK3 kinase-dependent and -independent processes. Using patterned light stimulation delivered by a spatial light modulator, we demonstrate precise spatiotemporal control of necroptosis in La-RIPK3-transduced HT-29 cells. Optogenetic control of proinflammatory lytic cell death could lead to the development of innovative experimental strategies to finetune the immune landscape for disease intervention.

Keywords: RIPK3; RNA-seq; lytic cell death; necroptosis; optogenetics.

Figure 1:. Construction of the light-activatable RIPK3 (La-RIPK3) systems.

A. Schematic of canonical and non-canonical necroptosis pathways. B. An illustration of blue-light-activated necroptosis via activation of RIPK3. Pharmacological and optogenetic activation of RIPK3 causes its oligomerization to form necrosomes, which regulate cytokine production and phosphorylates downstream executioner protein, MLKL, for plasma membrane rupture. C. Schematic illustration of light-activatable RIPK3 (La-RIPK3) constructs by fusing cryptochrome (left) and AuLOV (right) variants to RIPK3. The control modules have either optogenetic proteins only or RIPK3 only (bottom). An abbreviation for each construct is in the parenthesis. D. Time-lapse imaging of light-induced protein puncta formation of mCherry-tagged CC and RCC in live HEK293T cells. Cells were transiently transfected and cultured for 24 hours prior to imaging. One second of blue light (472 nm) was treated to the cells every 30 s. E. Quantitation of light-activated puncta formation using the normalized fluorescence intensities in 0 s and 120 s post-irradiation images in (D).

Figure 2:. Optimization of the La-RIPK3 system and temporal profile of chemokine production and plasma membrane rupture.

A. qRTPCR analysis of the Cxcl1 mRNA level in response to optogenetic activation of each optogenetic system. Transiently transfected HEK293T cells were treated with blue light (0.6 mW/cm², 15 s on/off) for 6 h from a lab-built blue LED box (465 nm). Gapdh mRNA was used for normalization. Relative mRNA fold change was calculated by the delta-delta Ct method, normalized to non-transfected (no TF) cells in the dark. Three biological replicates (each with two to three technical replicates) were used for each analysis. B. Images of transiently transfected (mCherry, TXR filter) cells and dead cells (SytoxGreen, GFP filter) were captured after 12 h of blue light treatment to the transfected cells with the same light dose described in panels A and B. Scale bar is 50 μm. Quantification of plasma membrane rupture. The percentage of ruptured cells was calculated using the equation (numbers of SytoxGreen-stained cells/numbers mCherry transfected cells) * 100%. Representative images are shown in Sup. Fig. 2D. D-E. The level of Cxcl1 (D) and Cxcl8 (E) in La-RIPK3-expressing HEK293T cells was measured at 0.5, 2, 6, and 12 h after blue light stimulation quantified by qRTPCR. F. Quantification of time-dependent plasma membrane rupture in HEK293T cells. Live cells transiently transfected with La-RIPK3 were imaged for plasma membrane rupture (SytoxGreen-stained) in a controlled imaging chamber. Cells were illuminated with an amenable light dose (< 0.5 mW/cm²) from blue LED over 16 h. Representative images for temporal analysis are shown in Sup. Fig. 3C. Data for qPCR analysis represent the mean ± SEM. Data for cell death rate represent mean ± SD. In panels (A) and (C), the dynamic range (light-over-dark ratio) and P values (two-tailed t test) are marked as blue and black numbers above each pair of bars, respectively. In panels (D-F), P values (two-tailed t test) were marked as black numbers near each pair of bars. * P < 0.05.

Figure 3:. La-RIPK3 activation induces RIPK3 kinase-dependent cytokine production and MLKL-dependent membrane rupture.

A. Immunofluorescence staining against phosphor-RIPK3(Thr231/Ser232) in pharmacologically treated (TNF+zVAD) mCherry-RIPK3-expressing NIH/3T3 cells (left), La-RIPK3-expressing NIH/3T3 cells in dark (middle) and 3 h light (right). Transiently transfected cells were recognized by mCherry fluorescence (red channel, top). Levels of phosphor-RIPK3 were probed with Alexa Fluor 488 labeled secondary antibody (Green channel, middle). Overlaid images of red, green, and DAPI (cell nucleus staining) channels are presented at the bottom row. B. Analysis of pRIPK3 puncta formation under various conditions. The whole panel of representative images is shown in (Sup. Fig. 4A–B). The percentage of pRIPK3 puncta-positive cells was calculated using the equation (numbers of pRIPK3 puncta containing cells/total number of transfected cells) * 100%. C. Western blot analysis of phosphor-La-RIPK3, pan-La-RIPK3, caspase8, caspase3, and GAPDH in lentivirus-transduced cells expressing La-RIPK3 under various conditions. The RIPK3 kinase inhibitor (R3i, GSK’843) was treated with 3 μM. D. Quantification of phosphor-La-RIPK3 level (top) and cleaved caspase8 (bottom) in conditions listed in (C). E. The mRNA levels of Cxcl1 (top) and Cxcl8 (bottom) were quantified via qPCR in La-RIPK3 transduced HT-29 cells in various conditions. The normalizer gene was h36B4. F. Western blot analysis of MLKL expression in wild-type and HT-29(MLKL-KO) cells. G. Kinetics of membrane rupture in HT-29 cells with MLKL depletion or intact, transiently transfected with La-RIPK3 or CC. The percentage was calculated using the equation: (numbers of SytoxGreen-stained cells/numbers mCherry transfected cells) * 100%. Cells were illuminated with continuous blue light (< 0.5 mW/cm²) in an environmentally controlled chamber for 10 h. H. The endpoint quantification of light-induced cell rupture in La-RIPK3-transfected HT-29 cells, transiently, with or without pan-caspase inhibitor (zVAD, 20 μM) treatment. Cells were illuminated with 50ms blue light (34mW) at 5 min intervals for 22 h in an environmentally controlled chamber. Cells in (A-E) were illuminated with blue LED (465 nm, 0.6 mW/cm², 15 s on/off). Data for qPCR analysis represent the mean ± SEM. Bars represent the mean ± SD in (B), (D), (G), and (H). The black number near each pair of bars indicates the P value (two-tailed t-test).

Figure 4:. Conditioned medium from La-RIPK3-activated cells induces THP1 cell migration.

A. Schematic of undifferentiated THP1 cell trans-well migration assay. B. The percentage of migrated THP1 cells was calculated using the equation: (total number of migrated cells / total number of plated cells) * 100%. HT-29 cell conditioned medium was obtained from different conditions, including the lentivirus-transduced cells expressing La-RIPK3 or CC treated with blue light (0.5 mW/cm², 15 s on/off) or kept in the dark for 12 h. C. Quantification of secreted CXCL8 in the conditioned medium of La-RIPK3-transduced HT-29 cells in panel (B). D. Western-blot analysis of HMGB1 level in the conditioned medium of HEK293T cells, transiently transfected with La-RIPK3 or CC in dark or light for 12 h. The black number near each pair of horizontal bars indicates the P value (two-tailed t-test).

Figure 5:. Spatiotemporal induction of plasma membrane rupture in La-RIPK3-transduced HT-29 cells.

A. HT-29 cells transduced with lentivirus carrying La-RIPK3 were treated with SytoxGreen and stimulated with spatially patterned light in an environmentally controlled chamber. To spatially pattern the illumination area (2×2 grid, left top and bottom right boxes are the “light-on” area), the Mightex Polygon1000-G coupled with the GFP and TXR filter was used. The grid was created using the Mightex software (PolyScan2) using the PolyScan2 sequence profiler. Before cell stimulation, cells were imaged with a wide field (left column). Cells were exposed to spatially patterned light delivered through a 10× objective in GFP (55 ms exposure time) and TXR (100 ms exposure time) channels together with a wide-field illumination from a transmitted bright-field light source every 5 minutes for 13.5 h (middle columns), then imaged with the final wide field (right column). During all imaging sessions, bright field light was not spatially patterned. B. HT-29 cells transduced with lentivirus carrying CC were treated with SytoxGreen and similarly illuminated and imaged as in (A). Scale bar: 20 μm.

Figure 6.. Transcriptomic analysis reveals distinct genes and pathways in HT-29 cells expressing La-RIPK3 under various treatments.

A. Left: Venn diagram of the upregulated genes in three pairwise comparisons: 1) La-RIPK3 oligomerization (La-RIPK3+Light vs. La-RIPK3+Dark), 2) TSZ (TSZ vs. DMSO), 3) TNFα (TNFα vs. DMSO). Right: Enrichment analysis of common upregulated genes ranked by the Log₁₀(P-value). B. Left: Heatmap of upregulated and downregulated genes treated by La-RIPK3 light (sample of interest or SOI) over La-RIPK3 dark (reference or REF). Right: Enrichment analysis of upregulated and downregulated genes ranked by the Log₁₀(P-value). C. Same as B but with SOI of La-RIPK3 (Light) and REF of La-RIPK3+R3i treatment (GSK’843, 3 μM). D. Analysis of RIPK3’s oligomerization-dependent, kinase-independent and kinase-dependent effect. Left: Venn diagram of RIPK3 oligomerization effect (La-RIPK3 Dark vs. La-RIPK3 Light) and RIPK3 kinase effect (La-RIPK3 Light+R3i vs. La-RIPK3 Light). The La-RIPK3 in light is the common reference. Right: Enrichment analysis of the kinase-independent and kinase-dependent effects of the oligomerized RIPK3. E-H. Cell-cell adhesion was one of the common processes identified in A, B, C, and D. Validation of mRNA levels of Hhla2, Ceacam6, Cd55, and Plaur by qRTPCR. The P values (two-tailed t-test) are black numbers near each horizontal bar of sample pairs. Error bars represent the mean ± SEM.