The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis

Background The innate immune system serves as the first line of host defense. Transforming growth factor-β–activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. Results In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. Conclusions Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions. Supplementary Information The online version contains supplementary material available at 10.1186/s12915-024-01901-5.


Background
The innate immune system plays a fundamental role in maintaining tissue homeostasis and establishing the first line of defense against pathogens and sterile inflammatory agents [1].Pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and homeostasis-altering molecular processes (HAMPs) are sensed by genetically encoded pattern recognition receptors (PRRs) to initiate inflammatory responses and cell death [2,3].Downstream of PRR engagement, transforming growth factor-β (TGF-β)activated kinase 1 (TAK1) is a key signaling node to drive the expression of inflammatory molecules and cell death, which are critical for host defense and cellular homeostasis [4][5][6][7].Given these critical functions, many pathogens carry TAK1 inhibitors to evade or disable host defenses.A classic example comes from Yersinia, which uses its type III secretion system to release a highly virulent Yersinia outer protein J (YopJ) that inhibits TAK1 in the host innate immune cells.To counteract pathogens, hosts sense TAK1 inhibition to induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome, which contains the NLRP3 inflammasome, caspase-8, FADD, and RIPK3 and drives cell death [8][9][10].
PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs [8,11].While the activation of PANoptosis in response to TAK1 inhibition promotes host defense, excess activation drives inflammation and pathogenesis.Therefore, the activation of TAK1 inhibitor (TAK1i)-induced PANoptosis involves a complex network of regulatory mechanisms.While it is known that regulation occurs through the TNFR1-RIPK1 signaling axis and involves the Rag-Regulator complex [5,6,[8][9][10], our understanding of the molecular mechanisms regulating TAK1i-mediated PANoptosis remains incomplete.
To advance the mechanistic understanding of TAK1iinduced PANoptosis, we evaluated the novel regulators previously identified in a whole genome cell death-based CRISPR knockout screen in immortalized bone marrowderived macrophages (iBMDMs) using TAK1i as the trigger [12].In this study, we identified the core components of the protein phosphatase 6 (PP6) holoenzyme PPP6C and PPP6R3 (also known as PP6C and PP6R3) as highly enriched CRISPR screen hits.Loss of PPP6C significantly reduced TAK1i-induced cell death and the activation of PANoptosis molecules.In contrast, combined deletion of all three PP6 regulatory subunits, PPP6R1, PPP6R2, and PPP6R3, was needed to inhibit TAK1i-induced cell death, suggesting that these molecules act redundantly in the TAK1 regulatory pathway.Additionally, we observed that the phosphatase PP6 led to a reduction in the inhibitory S321 phosphorylation of RIPK1, tipping the balance toward the pro-death S166 auto-phosphorylation of RIPK1 and triggering RIPK1-dependent PANoptosis in response to TAK1 inhibition.Overall, our findings identify a critical new regulator in the TAK1 inhibition-mediated inflammatory cell death pathway, suggesting PP6 may be a potential therapeutic target to reduce inflammation and certain forms of immunopathology.

A whole genome CRISPR screen identified the phosphatase PP6 as a critical regulator of TAK1 inhibition-induced cell death
Given the established role of TAK1 as a central hub for cellular homeostasis and cell death [4][5][6][7][8][9], understanding the complex regulation of TAK1i-mediated PANoptosis is central to health and disease.A previous study to identify regulators of TAK1i-induced cell death used a whole genome CRISPR screen in iBMDMs and identified several gRNAs that were enriched in cells that survived following 24 h of TAK1i treatment [12].In this cell death screen, the genes corresponding to the enriched gRNAs represent the positive regulators required for the induction of TAK1i-induced cell death.To understand more about these novel regulators of TAK1i-induced cell death, we evaluated the hits from the previously generated CRISPR screen data and found that TNFR1 (TNFRSF1A) and RIPK1, two well-known regulators of TAK1i-mediated cell death, were the top hits from the screen (Fig. 1A).Furthermore, we also identified the enrichment of two of the phosphatase PP6 holoenzyme components, Ppp6c and Ppp6r3 (Fig. 1A).Additional analyses of the individual gRNA counts demonstrated that all four gRNAs corresponding to the genes Ppp6c and Ppp6r3 were detected in the TAK1i-treated cell pools, with the majority of them showing increased mean total gRNA counts compared to the control samples (Fig. 1B).These results suggest that deletion of Ppp6c or Ppp6r3 with any of their gRNAs was sufficient to protect the cells from TAK1i-induced cell death.Together, Fig. 1 Whole genome CRISPR screen identifies PPP6C and PPP6R3 as key regulators of TAK1 inhibition-induced cell death.A Volcano plot presenting the log 2 mean fold change for Ripk1, Tnfrsf1a, Ppp6r3, and Ppp6c gRNAs, along with all other tested gRNAs, in the TAK1 inhibitor (TAK1i) CRISPR screen, following the treatment of immortalized bone marrow-derived macrophages (iBMDMs) carrying gRNAs with TAK1i for 24 h.B Scatter plot showing the enrichment of all four gRNAs targeting Ripk1, Tnfrsf1a, Ppp6r3, and Ppp6c in the pool of iBMDMs carrying gRNAs from the whole genome CRISPR screen.C Percent cell death as measured by lactate dehydrogenase (LDH) release in primary BMDMs treated with siRNA against Ppp6c, Ppp6r3, or the combination of Ppp6r1, Ppp6r2, and Ppp6r3 in response to TAK1i treatment.D Relative expression of Ppp6c and Ppp6r3 in BMDMs treated with siRNA against Ppp6c and Ppp6r3, respectively.E Relative expression of Ppp6r1, Ppp6r2, and Ppp6r3 in the BMDMs treated with siRNAs to silence the three PP6 regulatory subunits together.The knockdown efficiency is expressed as a percentage of the respective gene expression normalized to Actb expression.The data presented are representative of three or more independent experiments (C-E).The data are shown as mean ± SEM (C-E).Statistical analysis was performed using Student's t-test and two-way ANOVA.ns, not significant; ****P < 0.0001.NT: non-targeting siRNA; Ppp6r1+r2+r3: BMDMs treated with siRNAs against Ppp6r1, Ppp6r2, and Ppp6r3 the CRISPR MageCK analysis suggests that Ppp6c and Ppp6r3 were key molecules involved in driving TAK1iinduced cell death.
The phosphatase PPP6C and its regulatory subunits are essential for TAK1i-induced cell death The phosphatase PP6 belongs to the PPP superfamily of serine/threonine phosphatases [13,14].PPP6C is the catalytic subunit, and it interacts with its regulatory subunits, the PPP6Rs (PPP6R1, PPP6R2, and PPP6R3), to form the fully functional PP6 protein phosphatase holoenzyme complex.PP6 plays key roles in cell cycle progression [15,16], and it has been implicated in restricting innate immune hyperactivation.Specifically, PP6 inhibits (i) TAK1 activation by dephosphorylating T187 in the TAK1 activation loop [17], (ii) the cGAS-STING pathway by dephosphorylating cGAS and STING at S366 [18,19], and (iii) NF-κB signaling [14].However, PP6-mediated dephosphorylation also promotes the activation of RIG-I and can therefore positively regulate protective innate immune signaling and host defense against viral infections [20].PP6 has also been linked to the modulation of LUBAC-mediated M1-ubiquitination of RIPK1 to enable RIPK1 activation and the induction of cell death [21,22].Based on these connections between PP6 and innate immune activation, combined with our finding that PP6 components are potential regulators of TAK1iinduced cell death from the CRISPR screen (Fig. 1A-B), we sought to validate the function of these components using primary murine macrophages.We transfected primary bone marrow-derived macrophages (BMDMs) with control (NT, non-targeting) or gene-specific siR-NAs to knockdown the expression of Ppp6c and Ppp6r3.Consistent with the findings from the TAK1i CRISPR screen in iBMDMs (Fig. 1A-B), the primary BMDMs transfected with NT siRNA showed robust cell death in response to TAK1i treatment, while the BMDMs transfected with siRNA targeting Ppp6c exhibited a significant reduction in cell death (Fig. 1C), as well as reductions in Ppp6c transcript and PPP6C protein expression (Fig. 1D, Additional File 1: Fig. S1A, Additional File 2: Uncropped blots).However, the siRNA-based knockdown of Ppp6r3 failed to protect the cells from TAK1i-induced cell death (Fig. 1C), despite a reduction in Ppp6r3 expression and PPP6R3 protein level in the siRNA-treated BMDMs (Fig. 1D, Additional File 1: Fig. S1B, Additional File 2: Uncropped blots).Previous studies have demonstrated that the phosphatase PPP6C can interact with other regulatory subunits [23], potentially bypassing the need for PPP6R3 in primary BMDMs.Therefore, we next assessed the potential roles of other regulatory subunits.However, similar to our results with PPP6R3, siRNA-mediated silencing of the other PP6 regulatory components, Ppp6r1 and Ppp6r2, had no effect on cell death, despite a reduction in their transcript and protein expression levels (Additional File 1: Fig. S1B-C, Additional File 2: Uncropped blots).To determine whether there was functional redundancy between the regulatory subunits that allowed the overall regulatory functions to be maintained despite the loss of an individual subunit, we then performed siRNA-mediated knockdown of the three regulatory subunits of phosphatase PP6 together.We found that the combined knockdown of Ppp6r1, Ppp6r2, and Ppp6r3 resulted in a significant reduction in each component's expression (Fig. 1E) and reduced TAK1i-induced cell death, comparable to the reduction observed in Ppp6c siRNA-treated cells (Fig. 1C).Together, these data suggest that the catalytic subunit PPP6C was required for TAK1i-mediated cell death, while its regulatory subunits PPP6R1, PPP6R2, and PPP6R3 acted redundantly to form a fully functional PP6 phosphatase enzyme that drives TAK1i-mediated cell death.

Loss of the phosphatase PPP6C or its regulatory subunits abrogates the induction of TAK1i-induced PANoptosis
As the CRISPR screen and validation analyses identified a key role for PP6 components in TAK1i-induced cell death, we next sought to examine the effect of the PP6 subunits on the biochemical activation of PANoptosis molecules.Similar to the previous findings [5,6,12], we observed that TAK1i treatment induced activation of cell death molecules including caspase-1, gasdermin (GSDM) D and E, caspase-8, caspase-3, caspase-7, and MLKL in the NT siRNA-transfected BMDMs (Fig. 2, Additional File 2: Uncropped blots).The siRNA-mediated knockdown of Ppp6c, as well as the collective knockdown of Ppp6r1, Ppp6r2, and Ppp6r3, reduced the activation of each of these molecules (Fig. 2, Additional File 2: Uncropped blots).The siRNA-mediated knockdown of Ppp6r3 resulted in a slight reduction in the activation of caspase-1 and GSDMD/GSDME; however, we consistently observed that the activation of caspase-8, caspase-3, caspase-7, and MLKL was comparable or only marginally reduced in the Ppp6r3 siRNA-treated cells compared with the NT control (Fig. 2, Additional File 2: Uncropped blots).Similarly, siRNA-mediated knockdown of Ppp6r1 and Ppp6r2 decreased the activation of caspase-1 and caspase-7 and slightly decreased the activation of MLKL, with no substantial changes in the activation of other cell death proteins (Additional File 1: Fig. S1D, Additional File 2: Uncropped blots).These observations, together with the cell death data (Fig. 1C, Additional File 1: Fig. S1C), demonstrate that the PP6 regulatory subunits have complementary roles and functional redundancy to promote activation of RIPK1-dependent PANoptosis in TAK1i-treated cells.

The PP6 complex negatively regulates inhibitory phosphorylation of RIPK1 to drive PANoptosis
After confirming the role of PP6 components in the phenotypic and biochemical activation of PANoptosis, we next sought to determine how PP6 regulates TAK1i-induced cell death.As PP6 is a phosphatase, and RIPK1 phosphorylation is a key regulatory step in the TAK1 inhibition-mediated cell death pathway [5,8], we first assessed whether PP6 components could modulate RIPK1 phosphorylation status to regulate PANoptosis.We observed that siRNA-mediated knockdown of Ppp6c or simultaneous knockdown of all three regulatory subunits, Ppp6r1, Ppp6r2, and Ppp6r3, resulted in a reduction in the cell death-activating S166 autophosphorylation of RIPK1 (Fig. 3A, Additional File 2: Uncropped blots).Furthermore, knockdown of the PP6 catalytic subunit Ppp6c or combined knockdown of all three of its regulatory subunits resulted in an increase in the cell death-inhibiting S321 phosphorylation of RIPK1 (Fig. 3A, Additional File 2: Uncropped blots).However, knockdown of Ppp6r3 alone was insufficient to prevent S166 phosphorylation or enhance S321 phosphorylation of RIPK1 (Fig. 3A, Additional File 2: Uncropped blots), demonstrating the complementary roles of the phosphatase PP6 regulatory subunits.Moreover, consistent with the observed role for the PP6 phosphatase components in driving TAK1i-mediated lytic PANoptosis, we observed a decrease in the release of DAMPs, including lactate dehydrogenase (LDH) and high mobility group box 1 (HMGB1), in response to TAK1i treatment in the Ppp6c and Ppp6r1, Ppp6r2, and Ppp6r3 siRNA-treated groups compared to the NT group (Fig. 3A, Additional File 2: Uncropped blots).Together, these results highlight the significance of the distinct phosphorylations of RIPK1 and the effect of the phosphatase PP6 on their respective levels in determining cell fate (Fig. 3B), thereby suggesting that the therapeutic modulation of the PP6 complex may be used to treat RIPK1-dependent inflammatory cell death and inflammation in infectious and inflammatory diseases.
Our study provides evidence that phosphatase PP6 and its regulatory subunits reduced RIPK1 survival phosphorylation at S321 and promoted RIPK1-dependent PANoptosis.PP6 may also regulate RIPK1's functions by controlling its overall expression, but this remains unclear.Furthermore, siRNA-mediated knockdown of Ppp6c or the regulatory subunits did not fully block the TAK1i-induced cell death, suggesting that other phosphatase combinations may play a potential role, and future research is warranted to identify additional regulators of this inflammatory signaling and cell death mechanism.Moreover, a recent study demonstrated that the PPP1R3G/PP1γ phosphatase holoenzyme is essential to dephosphorylate the S25 residue of RIPK1 to drive the subsequent induction of RIPK1-dependent cell death [44].Future work should determine whether PP6 and its subunits cooperate with PP1γ to regulate TAK1i-induced PANoptosis and whether the effect of PP6 on RIPK1 phosphorylation is direct or indirect.Additionally, a recent study has reported that the excessive production of mitochondrial ROS (mtROS) through reverse electron transport (RET) drives PANoptosis [45].Pre-treatment with anti-RET reagents such as 1-methoxy PMS or dimethyl fumarate blocks RET-mediated mitochondrial ROS production and subsequent induction of PANoptotic cell death in BMDMs [45].Further studies will be needed Fig. 3 The PP6 complex negatively regulates inhibitory phosphorylation of RIPK1 to drive PANoptosis.A Immunoblots for S166 and S321 phospho-RIPK1 (pRIPK1) and total RIPK1 (tRIPK1) using cell lysates and immunoblots for high mobility group box 1 (HMGB1) and lactate dehydrogenase (LDH) using culture supernatants from bone marrow-derived macrophages (BMDMs) treated with TAK1 inhibitor (TAK1i) for 8 h.Blots were re-probed for β-ACTIN to serve as the internal loading control.The blots presented are representative of three independent experiments.NT: non-targeting siRNA; Ppp6r1+r2+r3: BMDMs treated with siRNAs against Ppp6r1, Ppp6r2, and Ppp6r3.B Schematic illustrating the potential mechanism for the PP6 complex to drive TAK1i-induced, RIPK1-dependent PANoptosis in BMDMs.The phosphorylation (P) marks denote the RIPK1 S166 (red, spiked) and S321 (green, rounded) phosphorylations to determine the role of mtROS in TAK1i-induced cell death.
Our findings emphasize the necessity of additional investigations to identify novel phosphatases capable of negatively regulating inhibitory phosphorylation sites on RIPK1, which can be better targeted for therapeutic modulation of RIPK1 signaling.Beyond RIPK1 and its roles in inflammatory conditions, our findings are also critical in the context of Yersinia-induced inhibition of TAK1 function, which also drives RIPK1-dependent inflammatory cell death that may depend on PP6.The identification of the central role of PP6 in mediating TAK1i-induced PANoptosis suggests that targeting PP6 or its subunits may aid in reducing RIPK1-dependent inflammatory cell death, which is critical in sterile and infectious pathologies.

Conclusions
Overall, our findings suggest an important role for PP6 phosphatase in TAK1i-mediated, RIPK1-driven PANoptosis.Our findings show that the PP6 complex is critical to regulating TAK1i-induced cell death, which may be clinically important in the context of targeting infections and inflammatory conditions.

Mice
The C57/BL6J control mice (wild type; WT) and CRISPR-Cas9 knockin mice (Rosa26-Cas9 knockin, JAX stock #024858) [46] used in the present study were maintained under specific pathogen-free conditions and bred at St. Jude Children's Research Hospital's Animal Resources Center.Mice were fed standard chow and maintained under a 12 h light-dark cycle.Animal studies were conducted using the standard protocols approved by the St. Jude Children's Research Hospital Committee on the Use and Care of Animals (protocol 482).

Isolation and stimulation of primary macrophages
To differentiate the murine bone marrow cells into macrophages, the bone marrow cells were cultured in macrophage differentiation medium (BMDM medium).BMDM medium was prepared using IMDM (12440053, Thermo Fisher Scientific), supplemented with 30% L929 cell-conditioned medium, 10% heat-inactivated fetal bovine serum (HI-FBS) (S1620, Biowest), 1% nonessential amino acids (11140-050, Thermo Fisher Scientific), and 1% penicillin-streptomycin (15070-063, Thermo Fisher Scientific).Freshly isolated murine bone marrow cells were mixed to form a single cell suspension in BMDM medium and seeded into three 15 cm 2 non-tissue culture-treated plates, each containing 20 mL of the medium.The cells in these plates were fed with an additional 5 mL of fresh BMDM medium on days 3 and 5 to support growth and differentiation.For siRNA knockdown experiments, day 6 cells were scraped, nucleofected with the respective siRNAs, and seeded at a density of 10 6 cells per well in 12-well culture plates in DMEM (11995-065, Gibco) containing 10% HI-FBS, 1% nonessential amino acids, and 1% penicillin-streptomycin for 2 h, followed by supplementing with an equal volume (1 mL) of fresh BMDM medium.The cells were then supplemented with fresh BMDM medium 24 h post-transfection and cultured for a total of 36-48 h from the time of transfection, followed by experimental stimulations.The TAK1 inhibitor (TAK1i, (5Z)-7-oxozeaenol, 100 nM final concentration; 17459, Cayman Chemical) was used to treat the cells for the inhibition of TAK1 kinase activity, as previously described [5,6,12], and the cell lysates were collected at the different time points as stated in the figure legends for the western blot analyses.

Cell death analysis
Cell death was measured and presented as a percentage of LDH release.The LDH release was quantified using the Cyto Tox 96 ® Non-Radioactive Cytotoxicity assay kit (G1782, Promega), following the manufacturer's instructions.

Transfection
The fully differentiated WT BMDMs were transfected with the respective siRNAs using the nucleofector machine (Neon ™ Transfection System, 100 μL/reaction kit, MPK10025, Thermo Fisher Scientific).Using the nucleofector settings of 1500 V and a single pulse at 20 mS, 50 μM of siGENOME SMARTpool siRNA were delivered to 10 6 BMDMs.Similar parameters were followed for the combined knockdown of Ppp6r1+r2+r3, where 50 μM of each of the siGENOME SMARTpool siRNAs for the respective genes was transfected together into the BMDMs.The transfected cells were transferred to 1 mL of warm, complete DMEM and seeded in a well of the 12-and/or 24-well tissue culture plate to conduct the experimental stimulations.Subsequently, to promote growth and recovery, the transfected cells were supplemented with an equal volume of warm BMDM medium 2 h post-transfection.The spent medium was replaced with fresh BMDM at 24 h after transfection, and the cells were allowed to rest for another 12-24 h.In total, the cells were allowed to rest for 36-48 h after transfection before proceeding to experimental stimulations.The siGENOME SMARTpool siRNAs specific for mouse Ppp6c (M-062583-01-0010, Horizon Discovery), Ppp6r1 (M-059588-00-0010, Horizon Discovery), Ppp6r2 (M-044631-02-0010, Horizon Discovery), and Ppp6r3 (M-040027-00-0010, Horizon Discovery), along with a non-targeting (NT) control siGENOME SMARTpool siRNA (D-001206-14-20, Horizon Discovery), were used in this study.

Statistical analysis
Statistical significance was determined by t tests (twotailed) for two groups and a two-way ANOVA for more than two groups.The data are represented as the mean ± SEM.The GraphPad Prism version 8.0 and 9.0 software packages were used for data analyses.Yersinia outer protein J