The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP...

来自 : 发布时间：2025-04-04

AbstractThe NLRP3 inflammasome has an important function in inflammation by promoting the processing of pro-IL-1尾 and pro-IL-18 to their mature bioactive forms, and by inducing cell death via pyroptosis. Here we show a critical function of the E3 ubiquitin ligase Pellino2 in facilitating activation of the NLRP3 inflammasome. Pellino2-deficient mice and myeloid cells have impaired activation of NLRP3 in response to toll-like receptor priming, NLRP3 stimuli and bacterial challenge. These functions of Pellino2 in the NLRP3 pathway are dependent on Pellino2 FHA and RING-like domains, with Pellino2 promoting the ubiquitination of NLRP3 during the priming phase of activation. We also identify a negative function of IRAK1 in the NLRP3 inflammasome, and describe a counter-regulatory relationship between IRAK1 and Pellino2. Our findings reveal a Pellino2-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome. IntroductionPattern recognition receptors (PRR) are utilized by the innate immune system to recognize pathogen-associated molecular patterns (PAMP) and trigger the induction of pro-inflammatory cytokines that will promote pathogen killing and removal from the host1. Transmembrane Toll-like receptors (TLR) and cytosolic nucleotide-binding and oligomerization domain (NOD)-like receptors (NLR) are important families of PRRs2. TLRs have a cytoplasmic Toll/interleukin-1(IL-1) receptor (TIR) domain and most TLRs, upon ligand engagement, interact with the intracellular TIR domain-containing adapter myeloid differentiation primary response protein 88 (MyD88)3. The latter recruits and activates IL-1R-associated kinase 4 (IRAK4) that in turn associates with IRAK1 to promote its hyperphosphorylation4, 5. IRAK1 subsequently interacts with the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) and TAK1 kinase that activates the IkappaB (I魏B) kinase (IKK) complex to phosphorylate and promote proteasomal degradation of I魏B proteins, key inhibitors of the NF魏B transcription factor6. This allows for nuclear translocation of NF魏B. TAK1 also triggers downstream activation of the p38, ERK, and JNK MAP kinase (MAPK) pathways that co-operate with NF魏B to regulate the transcription of a plethora of pro-inflammatory genes including those encoding TNF, IL-1尾, IL-18, and IL-67. The inflammatory cytokines IL-1尾 and IL-18 are initially produced as inactive precursor proteins and require the generation of a signaling platform termed the inflammasome that processes IL-1尾 and IL-18 into their mature bioactive forms before being released from cells8,9,10. Thus, the secretion of mature IL-1尾 requires two signals. Firstly innate receptors, such as TLR4, is engaged by its ligand lipopolysaccharide (LPS) in Gram negative bacteria, to activate NF魏B and induce transcription of the gene encoding the inactive pro-IL-1尾 precursor11 with a second signal triggering inflammasome activation. Inflammasomes consist of a NLR protein such as NLRP3 that recruits the adapter protein ASC and caspase-1 into an oligomeric complex leading to auto-proteolytic processing of pro-caspase-1 into its active form that cleaves pro-IL-1尾 and pro-IL-18 precursors into their mature secreted forms12, 13. The activation of the inflammasome can also lead to a necrotic form of cell death termed pyroptosis14. The activators of NLRP3 include ATP, PAMPs, danger associated molecular patterns (DAMPs), and particulate substances. Additional NLR proteins, such as NLRC4, that is activated via NLR family apoptosis inhibitory proteins (NAIPs) and bacterial flagellin, can also form inflammasome complexes15, 16. Other inflammasome complexes include the cytosolic DNA sensor Absent in Melanoma-2 (AIM2) that associates with ASC and triggers inflammasome activation17. These canonical inflammasome pathways are also complemented by a non-canonical inflammasome in which murine caspase-11 (caspase-4 and caspase-5 in human cells) directly binds to cytosolic LPS18 and is activated to induce pyroptosis in an analogous manner to caspase-119, 20. While caspase-4, caspase-5, and caspase-11 cannot cleave pro-IL-1尾 or pro-IL-18, these caspases can promote the assembly of the NLRP3 inflammasome19, 21, 22 to indirectly induce the production and secretion of the mature forms of IL-1尾 and IL-18.There is much interest in delineating the molecular mechanisms that regulate inflammasome activation23. A number of findings have shown the significance of post-translational modification of components of the inflammasome with ubiquitination being of particular importance. TRIM31, FBXL2, and MARCH7 have been reported to promote ubiquitination and degradation of NLRP324,25,26 with NLRP3 needing to be de-ubiquitinated by the BRCC3 complex in order to facilitate NLRP3 activation27,28,29. However, there are currently no reports on E3 ubiquitin ligases directly contributing to activation of NLRP3. Here we show a critical role for the E3 ubiquitin ligase Pellino2 in regulating activation of the NLRP3 pathway.Pellino proteins constitute a three-membered family of E3 ubiquitin ligases that have various regulatory roles in innate immune signaling pathways3, 30, 31. Originally identified as IRAK-interacting proteins, each family member shares an N-terminal forkhead-associated (FHA) domain that recognizes phosphothreonine residues and mediates association with IRAK132, and a C-terminal RING-like domain that confers E3 ubiquitin ligase activity and an ability to catalyze lysine 63 (K63)-linked polyubiquitination of substrate proteins such as IRAKs33,34,35. Pellino proteins are subject to phosphorylation by kinases, such as IRAK1 and TBK1/IKK蔚, that can enhance the E3 ligase activities of the Pellino proteins35,36,37,38,39. Murine genetic models have revealed roles for Pellino1 in TLR3 and TLR4 signaling pathways40, as a suppressor of T cell activation41, 42, a driver of neuroinflammation43, and as a promoter of lymphomagenesis44 and lung carcinogenesis45. We have previously generated Pellino3-deficient mice to identify important roles for Pellino3 as a negative regulator of TLR3 signaling46 and TNF-induced cell killing47, a mediator of NOD2 signaling in the gut48 and a critical regulator of obesity-induced expression of IL-1尾 and insulin resistance49.While progress has been made in elucidating the functions of Pellino1 and Pellino3, there is a notable lack of insight into the physiological roles of Pellino2. Studies on Pellino2 have been largely confined to overexpression and knockdown approaches in cell lines that suggest a role for Pellino2 in IL-1/LPS-induced activation of ERK and JNK MAPK pathways and increased stabilization of mRNAs encoding pro-inflammatory proteins50, 51. We now generate Pellino2-deficient mice to reveal the first physiological role of Pellino2 as a mediator in the activation of the NLRP3 inflammasome pathway. We show that Pellino2 facilitates NLRP3-induced maturation and release of IL-1尾 and IL-18 in macrophages, while also promoting cell death by pyroptosis. The pathophysiological relevance of this role for Pellino2 is also shown by its contribution to the lethal effects of LPS in a murine model of endotoxemia and the in vivo induction of IL-1尾 in response to challenge with Gram negative bacteria. Pellino2 exerts its positive effects by promoting ubiquitination of NLRP3. We also identify for the first time that IRAK1 acts as an inhibitor of the NLRP3 pathway and reveal a counter-regulatory relationship between Pellino2 and IRAK1. In addition to revealing the first physiological role for Pellino2, this study also maps an entirely new regulatory pathway that controls NLRP3 activation.ResultsPellino2 mediates mature IL-1尾 production in response to LPSIn order to explore the physiological role for Pellino2 we generated Pellino2-deficient mice. Homologous recombination was used in embryonic stem cells to replace exons 2 to 6 in the Peli2 gene with an F3-flanked selection cassette (Supplementary Fig.聽1a). The selection cassette was subsequently removed by crossing mice with this targeted allele to mice expressing a Flp transgene thus generating mice with deletions of exons 2 to 6 of the Peli2 gene, as confirmed by PCR analysis (Supplementary Fig.聽1b). Semi-quantitative (Supplementary Fig.聽1c) and real-time (Supplementary Fig.聽1d) RT-PCR analysis of mRNA, isolated from bone marrow-derived macrophages (BMDMs), confirmed complete lack of expression of the Peli2 gene in cells from Peli2鈭?鈭?/sup> mice. Pellino2-deficient mice are viable and develop normally. Given the previously described roles for Pellino1 and Pellino3 in TLR signaling pathways, we initially compared the responsiveness of BMDMs from wild type (WT) and Pellino2-deficient mice to 24鈥塰 challenge with a wide array of TLR ligands, as measured by induction of the TLR-responsive proteins IL-6 (Fig.聽1a), CXCL1 (Fig.聽1b), TNF (Fig.聽1c), RANTES (Fig.1d), and IFN-尾 (Fig.聽1e). Each TLR ligand retained its ability to induce all of these cytokines in BMDMs lacking Pellino2 indicating its lack of physiological participation in these TLR signaling pathways. Since we have previously identified Pellino3 as an inhibitor of IL-1尾 expression, we next explored the role of Pellino2 in controlling the production of IL-1尾. The levels of secreted IL-1尾 protein, in response to LPS, were strongly reduced in Pellino2-deficient BMDMs (Fig.聽1f), suggesting a selective role for Pellino2 in the pathway controlling the production of IL-1尾 but not other pro-inflammatory proteins. However, Pellino2 is not involved in the transcriptional regulation of Il1b gene since LPS-induced expression of Il1b mRNA (Supplementary Fig.聽2a) and pro-IL-1尾 protein (Supplementary Fig.聽2b) is fully intact in Pellino2-deficient BMDMs. Indeed Pellino2 is dispensable for the early intracellular signaling pathways triggered by LPS that drive expression of the Il1b gene, since WT and Peli2鈭?鈭?/sup> BMDMs displayed the same patterns of time-dependent activation of NF魏B and MAPK pathways as measured by phosphorylation of I魏B伪, p38 MAPK, JNK, and ERK (Supplementary Fig.聽2c).Fig. 1Pellino2 is required for production of IL-1尾 in response to LPS. a-e Enzyme-linked immunosorbent assay (ELISA) of a IL-6, b CXCL1, c TNF, d RANTES, and e IFN-尾 expression in medium from BMDMs isolated from wild-type (WT) and Pellino2-deficient (Peli2鈭?鈭?/sup>) mice and treated with 10鈥塶g/ml Pam2CSK, 10鈥塶g/ml Pam3CSK, 1鈥壩糶/ml Zymosan, 25鈥壩糶/ml poly(I:C), 100鈥塶g/ml lipopolysaccharide (LPS), 1鈥壩糶/ml flagellin, 1鈥壩糶/ml Clo75, 1鈥壩糶/ml Clo97, and 2鈥壩糶/ml CpG for 24鈥塰. UT, untreated. f ELISA of IL-1尾 in medium from WT and Peli2鈭?鈭?/sup> BMDMs stimulated with the indicated concentrations of LPS for 24鈥塰. *p鈥?lt;鈥?.05 (paired, two-tailed Student鈥檚 t-test). Data are presented as the mean of three independent experiments (a-f). Error bars, s.e.m.Full size imagePellino2 mediates LPS-induced activation of NLRP3Given that Pellino2 is not required for induction of pro-IL-1尾 we next probed the role of Pellino2 in the activation of inflammasomes. We initially employed the widely used two-signal model of LPS and ATP to evaluate the involvement of Pellino2 in the NLRP3 pathway. The priming of BMDMs from WT mice with LPS followed by stimulation with ATP resulted in strong expression and secretion of mature IL-1尾, as measured by ELISA. However, this response was considerably reduced in cells from Peli2鈭?鈭?/sup> mice (Fig.聽2a). Pellino2-deficient cells also exhibited reduced levels of IL-18 (Fig.聽2b) and pyroptosis (Fig.聽2c) relative to WT BMDMs, suggesting that Pellino2 plays a vital role in the NLRP3 pathway. This was further supported with immunoblotting analysis that demonstrated Peli2鈭?鈭?/sup> BMDMs to be less responsive than WT cells to LPS/ATP-induced processing of pro-IL-1尾 into its mature p17 IL-1尾 form and pro-caspase-1 into its active p20 form (Fig.聽2d). 尾-Actin was used as a loading control in all immunoblotting analysis since its electrophoretic mobility is mid-range of the various proteins subjected to immunoblotting. Levels of processed IL-1尾 and caspase-1 were comparable between WT and Peli2鈭?鈭?/sup> BMDMs in response to double stranded DNA (poly (dA:dT), a trigger of the AIM2 inflammasome, suggesting some specificity for the NLRP3 pathway. To further confirm the role of Pellino2 in the NLRP3 pathway, another two-signal model, involving LPS as a prime stimulus followed by the potassium ionophore nigericin, was employed as an alternative approach to activate the NLRP3 pathway. Again Peli2鈭?鈭?/sup> BMDMs were much less responsive than WT cells with respect to LPS/nigericin-induced production of mature IL-1尾 (Fig.聽2e), IL-18 (Fig.聽2f), pyroptosis (Fig.聽2g), and processing of pro-IL-1尾 and pro-caspase-1 (Fig.聽2h). While ATP and nigericin promotes NLRP3 activation by facilitating potassium efflux, particulate material like alum can also activate NLRP3. We next demonstrated that Pellino2 is also required for alum-mediated secretion of IL-1尾 (Fig.聽2i). These data are all consistent with a role for Pellino2 in activation of the NLRP3 pathway. In keeping with its potential targeting of NLRP3, the role of Pellino2 is independent on the priming stimulus. Using the TLR2 ligand zymosan, as an alternative first signal with ATP, resulted in comparable levels of zymosan-induced expression of Il1b mRNA (Supplementary Fig.聽3a) and pro-IL-1尾 protein (Supplementary Fig.聽3b) in WT and Peli2鈭?鈭?/sup> cells. However, processing of pro-IL-1尾 (Supplementary Fig.聽3c) and secretion of mature IL-1尾 (Supplementary Fig.聽3d) in Peli2鈭?鈭?/sup> BMDMs was severely impaired.Fig. 2Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1尾 and b IL-18, and c LDH assay of medium from WT and Peli2鈭?鈭?/i> BMDMs treated with 100鈥塶g/ml LPS for 3鈥塰 with or without further stimulation with 2.5鈥塵M ATP for 1鈥塰.聽UT, untreated. d Immunoblot analysis of IL-1尾 and Caspase-1 in medium (Sup) and lysates from WT and Peli2鈭?鈭?/i> BMDMs stimulated with 100鈥塶g/ml LPS for 3鈥塰 with or without further stimulation with 2.5鈥塵M ATP for 1鈥塰 or transfection of Poly (dA:dT) (1鈥壩糶/ml) for 6鈥塰. ELISA of e IL-1尾 and f IL-18 and g LDH assay of medium from WT and Peli2鈭?鈭?/sup> BMDMs treated with 100鈥塶g/ml LPS for 3鈥塰 with or without further stimulation with 5鈥塵M Nigericin for 1鈥塰. h Immunoblot analysis of IL-1尾 and Caspase-1 in medium (Sup) and lysates from WT and Peli2鈭?鈭?/sup> BMDMs stimulated with 100鈥塶g/ml LPS for 3鈥塰 with or without further stimulation with 5鈥塵M Nigericin for 1鈥塰 or transfection of Poly (dA:dT) (1鈥壩糶/ml) for 6鈥塰. i ELISA of IL-1尾 of medium from WT and Peli2鈭?鈭?/sup> BMDMs treated with 100鈥塶g/ml LPS for 3鈥塰 with or without further stimulation with the indicated concentrations of Alum for 6鈥塰. j ELISA of IL-1尾 in medium from peritoneal-resident macrophages isolated from WT and Peli2鈭?鈭?/sup> mice. Cells were treated with 100鈥塶g/ml of LPS for 3鈥塰 with or without further stimulation with 2.5鈥塵M ATP for 1鈥塰. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1尾 in medium from transfected THP1 cells stimulated with 100鈥塶g/ml LPS for 6鈥塰 with or without further treatment with 2.5鈥塵M ATP or 5鈥塵M Nigericin for 1鈥塰. m Immunoblot analysis of IL-1尾 and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6鈥塰 with or without further stimulation with 2.5鈥塵M ATP or 5聽mM Nigericin for 1鈥塰. 尾-Actin was used as loading controls. *p鈥?lt;鈥?.05 (paired, two-tailed Student鈥檚 t-test). Data are biological replicates that are representative of three independent experiments. Error bars, s.e.m.Full size imageHaving demonstrated a role for Pellino2 in the NLRP3 pathway in macrophages derived from bone marrow we wished to confirm a similar role in naturally occurring tissue resident macrophages. Peritoneal macrophages from Peli2鈭?鈭?/sup> mice had less LPS/ATP induction of mature IL-1尾 than corresponding WT cells (Fig.聽2j). Furthermore, we also confirm a role for Pellino2 in the NLRP3 pathway in human cells by showing that knockdown of PELI2 expression in the human monocytic THP1 cell line (Fig.聽2k) reduced LPS-, LPS/ATP-, and LPS/nigericin-induced secretion of mature IL-1尾 (Fig.聽2l) and this is consistent with less processing of IL-1尾 and caspase-1 in PELI2 knockdown cells (Fig.聽2m).We further examined the specificity of the role of Pellino2 for the NLRP3 inflammasome. Pellino2 is not employed in other canonical inflammasome pathways such as NLRC4 and AIM2 since respective triggers of these inflammasomes, flagellin and poly (dA:dT), had comparable efficacy in inducing IL-1尾 in WT and Peli2鈭?鈭?/sup> BMDMs (Supplementary Fig.聽4a). Furthermore, Pellino2 was dispensable for non-canonical inflammasome activation as Pellino2 deficiency did not affect the ability of cytosolic LPS to trigger processing of mature IL-1尾 (Supplementary Fig.聽4b) or pyroptosis (Supplementary Fig.聽4c). Additionally, cholera toxin B (CTB) co-administration with LPS provides another way to activate the non-canonical inflammasome19. Again LPS/CTB-induced release of mature IL-1尾 (Supplementary Fig.聽4d) and pyroptosis (Supplementary Fig.聽4e) were unaltered in BMDMs lacking Pellino2. Together, these data suggest that Pellino2 is not involved in non-canonical inflammasome activation and instead serves a selective role in the canonical NLRP3 pathway.Pellino2 contributes to LPS-induced lethalityWe next aimed to explore the physiological relevance of the role for Pellino2 in responding to LPS by extending our studies into in vivo models. Consequently, WT and Peli2鈭?鈭?/sup> mice were subjected to intraperitoneal administration of non-lethal dose of LPS and serum levels of a range of cytokines were assayed. LPS-induced increased serum levels of IL-6 (Fig.聽3a), TNF (Fig.聽3b), CXCL1 (Fig.聽3c), RANTES (Fig.聽3d), and IL-1尾 (Fig.聽3e) in WT mice and whereas the same levels of IL-6, TNF, CXCL1, and RANTES were observed in Pellino2-deficient mice, the serum levels of IL-1尾 in Peli2鈭?鈭?/sup> mice were reduced relative to WT mice (Fig.聽3e). These data are consistent with our earlier findings above that indicated a role for Pellino2 in controlling IL-1尾 maturation. The pathophysiological relevance of this role was further evaluated by administering mice with lethal dose of LPS and survival monitored over a 72鈥塰 period post administration. All WT mice succumbed to the lethal effects of LPS after 48鈥塰 but Peli2鈭?鈭?/sup> mice had a 50% survival rate up to 72鈥塰 (Fig.聽3f) suggesting that Pellino2 plays an important in vivo role in mediating IL-1尾 production in response to LPS and in promoting LPS-induced lethality in endotoxaemia.Fig. 3Pellino2 is required for in vivo expression of IL-1尾 and lethality in response to LPS. ELISA of a IL-6, b TNF, c CXCL1, d RANTES, and e IL-1尾, from serum of WT and Peli2鈭?鈭?/sup> mice at 16鈥塰 post-intraperitoneal injection with 20鈥塵g/kg LPS or PBS (n鈥?鈥?鈥?2). f Survival rates of age- and sex-matched WT (n鈥?鈥?2) and Peli2鈭?鈭?/sup> (n鈥?鈥?2) mice after intraperitoneal injection with 50鈥塵g/kg of LPS or PBS (n鈥?鈥? for each of WT and Peli2鈭?鈭?/sup>) as a vehicle control. Mice were monitored every 6鈥塰 for 72鈥塰. **P鈥?lt;鈥?.01 (paired, two-tailed Student鈥檚 t-test (e) and log-rank Mantel-Cox test (f)). Data indicate samples from individual mice (n鈥?鈥? for all PBS groups (a鈥揻), n鈥?鈥? for LPS groups (a), n鈥?鈥? for LPS/WT, n鈥?鈥? for LPS/ Peli2鈭?鈭?/i> (b), n鈥?鈥? for LPS groups (c, d), n鈥?鈥?1 for LPS/WT, n鈥?鈥?2 for LPS/Peli2鈭?鈭?/sup> (e). Data are pooled biological replicates from two independent experiments. Error bars, s.e.m.Full size imagePellino2 mediates NLRP3 anti-bacterial responsesWe extended our studies on the physiological relevance of the role of Pellino2 in responding to LPS and in promoting inflammasome activation by comparing the responsiveness of BMDMs from WT and Peli2鈭?鈭?/sup> mice to the Gram negative bacteria Citrobacter rodentium (C. rodentium) and Escherichia coli (E. coli). Although these bacteria activate the NLRP3 inflammasome they can also induce caspase-11 expression and synergize with activated NLRP3 to process IL-1 family cytokines and instigate pyroptosis52, 53. C. rodentium induced production of mature IL-1尾 (Fig.聽4a) and caspase-11 (Fig.聽4b) in WT BMDMs and whereas the levels of IL-1尾 were reduced in Peli2鈭?鈭?/sup> cells, the expression of caspase-11 was intact. Similarly, E.coli induced IL-1尾 maturation in a Pellino2-dependent manner (Fig.聽4c) whereas caspase-11 expression was independent of Pellino2 (Fig.聽4d) suggesting a direct and specific role for Pellino2 in the NLRP3 pathway. Pellino2 also mediated the production of IL-1尾 and IL-18 but not CXCL1 (Fig.聽4e) and processing of pro-caspase-1 (Fig.聽4f) in response to another Gram negative bacteria Pseudomonas aeruginosa (P. aeruginosa). This again supports a role for Pellino2 in bacterial-induced inflammasome activation. The physiological relevance of this role was confirmed by demonstrating that P. aeruginosa-infected Peli2鈭?鈭?/sup> mice produced less IL-1尾 and IL-18 but the same levels of another pro-inflammatory cytokine IL-6 compared with infected WT mice (Fig.聽4g).Fig. 4Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2鈭?鈭?/i> BMDMs were infected with a, b C. rodentium, c, d E.coli, or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1尾, IL-18, and CXCL1 in medium from BMDMs infected for 6鈥塰. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0鈥?鈥塰. f Immunoblot analysis of IL-1尾 and Caspase-1 in lysates from cells infected with PAO1 for 3鈥塰. 尾-Actin was used as loading controls. g ELISA of IL-1尾, IL-18, and IL-6 in peritoneal lavage from WT and Peli2鈭?鈭?/i> mice previously infected for 10鈥塰 by intraperitoneal injection of PAO1 (1.5鈥壝椻€?07 CFU). *p鈥?lt;鈥?.05, **p鈥?lt;鈥?.01 (paired, two-tailed Student鈥檚 t-test). Data represent the mean鈥壜扁€塻.e.m. of three independent experiments (a, c, e), biological replicates that are representative of 3 independent experiments (b, d, f), or samples from individual mice (n鈥?鈥?鈥? per group) (g)Full size imagePellino2 FHA and RING domains facilitate NLRP3 activationHaving provided strong supporting evidence for a role for Pellino2 in the NLRP3 inflammasome, we next probed the underlying mechanism. Our initial mechanistic studies focused on Pellino2 itself and the functional relevance of its FHA and RING-like domains for NLRP3 activation. To this end, we used murine stem cell virus (MSCV) transduction to re-constitute Peli2鈭?鈭?/sup> BMDMs with WT murine Pellino2 and mutated forms with point mutations in its FHA or RING-like domain (Fig.聽5a). The re-introduction of WT murine Pellino2 into Peli2鈭?鈭?/sup> BMDMs reconstituted the ability of LPS/ATP to induce IL-1尾 and IL-18 to levels that were equivalent to those observed in WT BMDMs (Fig.聽5b). However, the FHA mutant form of Pellino2, that lacks substrate binding activity, and the RING mutant form, that lacks E3 ubiquitin ligase activity, failed to re-constitute LPS/ATP function in Peli2鈭?鈭?/sup> BMDMs. These data indicate that Pellino2 is dependent on both its FHA and RING-like domains in order to trigger activation of the NLRP3 pathway. This is further supported by the observation that the re-introduction of WT Pellino2 could re-constitute the LPS/ATP-induced processing of pro-caspase-1 in Peli2鈭?鈭?/sup> BMDMs to the same degree as displayed in WT cells whereas both the FHA and RING-like mutants of Pellino2 were ineffective in this regard (Figs.聽5c, d).Fig. 5The activation of the NLRP3 pathway is dependent on the FHA and RING-like domain of Pellino2. WT and Peli2鈭?鈭?/i> BMDMs were infected with GFP-expressing murine stem cell virus (MSCV) as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (Peli2-RING), or Pellino2 FHA mutant (Peli2-FHA). a GFP expression (left panels) and immunoblot analysis of myc (right panel) (scale bar鈥?鈥?0渭m). b ELISA of IL-1尾 and IL-18 in medium from virus-infected WT and Peli2鈭?鈭?/sup> BMDMs stimulated with 100鈥塶g/ml LPS for 3鈥塰 followed by 2.5鈥塵M ATP for 1鈥塰. c, d Immunoblot analysis (c) and densitometry analysis (d) of IL-1尾 and Caspase-1 in medium (Sup) and cell lysates from virus-infected WT and Peli2鈭?鈭?/sup> BMDMs stimulated with 100鈥塶g/ml LPS for 3鈥塰 followed by 2.5鈥塵M ATP for 1鈥塰. *P鈥?i> 鈥?.05; **P鈥?i> 鈥?.01; ***P鈥?i> 鈥?.001; ****P鈥?i> 鈥?.0001 (paired, two-tailed Student鈥檚 t-test (b) or two-way ANOVA (d)). Data are biological replicates that are representative of 3鈥? independent experiments (a, c) or represent the mean鈥壜扁€塻.e.m. of 3鈥? independent experiments (b, d)Full size imagePellino2 is required for NLRP3-induced ASC oligomerizationWe next aimed to explore how Pellino2 can integrate into the NLRP3 pathway. The production of mROS has been proposed as a general converging mechanism for various NLRP3 stimuli. However, WT and Peli2鈭?鈭?/sup> BMDMs produced the same levels of mitochondrial ROS under conditions of NLRP3 activation with LPS and ATP (Supplementary Fig.聽5). This suggested that Pellino2 acts independently of such a general triggering mechanism and so we focused on more specific protein targets in the NLRP3 pathway. Given the crucial role of ASC in the NLRP3 inflammasome and since the assembly of the NLRP3 inflammasome is associated with oligomerization of ASC, that manifests as a single ASC speck in cells, we next characterized the effects of Pellino2 deficiency on ASC aggregation under conditions of NLRP3 activation. ASC speck formation was detectable in WT BMDMs that were primed with LPS and then stimulated with ATP but the number of cells displaying ASC formation under these conditions was significantly reduced in Peli2鈭?鈭?/sup> BMDMs (Fig.聽6a). Since ASC speck formation reflects oligomerization of ASC, we treated cell lysates with a bifunctional chemical crosslinking agent and subjected samples to ASC immunoblotting to assess for oligomerization. LPS/ATP-induced strong oligomerization of ASC in WT BMDMs but the levels of higher molecular weight ASC oligomers were reduced in Peli2鈭?鈭?/sup> BMDMs (Fig.聽6b), in keeping with impaired ASC speck formation under the same conditions. To further confirm the role of Pellino2 in mediating NLRP3-induced ASC aggregation, we used LPS/nigericin as a second means to activate the NLRP3 inflammasome. LPS priming, followed by nigericin stimulation, promoted ASC oligomerization in WT BMDMs but this was again reduced in Peli2鈭?鈭?/sup> BMDMs (Fig.聽6c) consistent with Pellino2 facilitating ASC oligomerization in response to NLRP3 activation. The impaired NLRP3-induced oligomerization of ASC in Peli2鈭?鈭?/sup> BMDMs was overcome by re-constituting these cells with WT Pellino2 (Fig.聽6d). However, mutated forms of Pellino2 with point mutations in its FHA or RING-like domains failed to re-constitute this function of Pellino2. This correlates closely with the earlier re-constitution of NLRP3-induced IL-1尾 and IL-18 expression with WT Pellino2 but not mutated forms (Fig.聽5b) strongly indicating that Pellino2-mediated ASC oligomerization translates into downstream production of mature bioactive forms of IL-1尾 and IL-18.Fig. 6Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2鈭?鈭?/i> BMDMs that were left untreated聽(UT) or聽 treated with 100鈥塶g/ml LPS for 3鈥塰 and further stimulated with ATP for 30鈥塵in. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar鈥?鈥?00 渭m). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2鈭?鈭?/sup> BMDMs stimulated with 100鈥塶g/ml LPS for 3鈥塰 with or without further treatment with b 2.5鈥塵M ATP, or c 5鈥塵M Nigericin for 30鈥塵in. 尾-Actin was used as loading controls. d WT and Peli2鈭?鈭?/i> BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100鈥塶g/ml LPS for 3鈥塰 followed by 2.5鈥塵M ATP for 30鈥塵in. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. **p鈥?lt;鈥?.01 (paired, two-tailed Student鈥檚 t-test). Data are the mean鈥壜扁€塻.e.m. of three independent experiments (a, right panel) or biological replicates that are representative of three independent experiments (a left panel, b, c, d)Full size imagePellino2 promotes ubiquitination of NLRP3Given that Pellino2 appears to act upstream of ASC, we next investigated NLRP3 as a potential direct target for Pellino2. We were especially interested in characterizing the regulatory mechanisms controlling ubiquitination of NLRP3 since earlier studies had shown NLRP3 to be subject to ubiquitination and our data above indicated the mediatory role of Pellino2 to be dependent on the RING-like domain that underpins its E3 ubiquitin ligase activity. We thus treated WT and Peli2鈭?鈭?/sup> BMDMs with LPS for various times, immunoprecipitated NLRP3 and analyzed the ubiquitination status by Western blotting. LPS promoted time-dependent polyubiquitination of NLRP3 in WT cells and this was considerably reduced in Peli2鈭?鈭?/sup> BMDMs (Fig.聽7a) indicating that LPS induces ubiquitination of NLRP3 in a Pellino2-dependent manner. We next defined the type of linkages in the polyubiquitin chains that are attached to NLRP3. Thus, using K63-specific TUBEs we isolated K63-linked ubiquitinated proteins followed by immunoblotting for NLRP3 to show that LPS promotes time dependent K63-linked ubiquitination of NLRP3 in WT macrophages and this is reduced in Pellino2-deficient cells (Fig.聽7b). Similar approaches using K48-specific TUBEs failed to detect any K48-linked ubiquitination of NLRP3 in response to LPS (Fig.聽7c) demonstrating that LPS promotes K63-linked ubiquitination of NLRP3 in a Pellino2-dependent manner. While previous reports have described a requirement for NLRP3 to be de-ubiquitinated in order to trigger downstream effects of inflammasome activation, our data lead us to hypothesize that part of the priming for NLRP3 inflammasome activation by LPS may be related to a requirement to initially induce ubiquitination of NLRP3. We present further evidence in support of this hypothesis by demonstrating that MCC950, a highly selective inhibitor of NLRP354, strongly suppresses LPS/ATP induction of IL-1尾 in WT BMDMs (Fig.聽7d) and also inhibits LPS-induced ubiquitination of NLRP3 (Fig.聽7e). Interestingly, while MCC950 strongly suppressed the ability of LPS to promote ubiquitination of NLRP3, MCC950 also appeared to cause some basal ubiquitination of NLRP3 in unstimulated cells. In order to address these apparent contrasting roles of MCC950 we characterized the effects of MCC950 on formation of polyubiquitin chains of different linkages. Using K63-specific TUBEs to isolate K63-linked ubiquitinated proteins we revealed LPS-induced K63-linked ubiquitination of NLRP3 and this was fully abrogated in cells pre-treated with MCC950 (Fig.聽7f). Notably, MCC950 alone failed to induce any K63-linked ubiquitination of NLRP3 suggesting that the basal ubiquitination of NLRP3 observed in response to MCC950 (Fig.聽7e) is due to ubiquitin chains that are joined by different linkages. Thus, LPS promotes transient K63-linked ubiquitination of NLRP3, that may prime NLRP3 for subsequent activation and this is countered by the inhibitory effects of MCC950 that oppose the K63-linked ubiquitination of NLRP3. These data arising from the use of MCC950 are consistent with our hypothesis of a requirement for LPS to prime K63-linked ubiquitination of NLRP3 for its later activation and suggests that impaired ubiquitination of NLRP3 in response to LPS in Peli2鈭?鈭?/sup> BMDMs may underlie the reduced activation of NLRP3 in these cells. Indeed MCC950 does not affect the residual LPS/ATP-induced levels of IL-1尾 in Peli2鈥?鈥?/sup> BMDMs (Fig.聽7d) raising the possibility that the targeting of NLRP3 ubiquitination may contribute to the inhibitory effects of MCC950.Fig. 7 Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples (a) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates (b) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates (c) from WT and Peli2鈭?鈭?/i> BMDMs treated with a 100鈥塶g/ml LPS for the indicated times. d ELISA of IL-1尾 in medium from WT and Peli2鈭?鈭?/sup> BMDMs treated with 100鈥塶g/ml LPS for 2鈥塰 followed by sequential treatment with 1鈥壩糓 MCC950 for 1鈥塰 and 2.5鈥塵M ATP for 30鈥塵in.聽UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples (e) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates (f) from WT and Peli2鈭?鈭?/i> BMDMs pre-treated with 1鈥壩糓 MCC950 for 1鈥塰 followed by treatment with 100鈥塶g/ml LPS for the indicated times. g Peli2鈭?鈭?/i> BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100鈥塶g/ml LPS for the indicated times. 尾-Actin was used as loading controls. *p鈥?lt;鈥?.05 (paired, two-tailed Student鈥檚 t-test). NS, not significant. Data are biological replicates that are representative of three independent experiments (a-c, e-g) or mean鈥壜扁€塻.e.m. of three independent experiments (d)Full size imageWe next characterized the nature of the functional relationship between Pellino2 and NLRP3. Using Peli2鈭?鈭?/sup> BMDMs, reconstituted with a myc-tagged form of Pellino2 that we described above to restore NLRP3 activation, we demonstrated that LPS induces a time-dependent interaction of Pellino2 with NLRP3 (Fig.聽7g). We assessed the potential of Pellino2 to act as a direct E3 ligase for NLRP3. However, the co-expression of Pellino2 and NLRP3 with WT ubiquitin (Supplementary Fig.聽6a) or a mutant ubiquitin form containing a single lysine at residue 63 (K63A) (Supplementary Fig.聽6b) failed to show any Pellino2-induced ubiquitination of NLRP3. This suggested that Pellino2 may be exerting its effects on NLRP3 in a more indirect manner and possibly, by targeting a protein that directly regulates NLRP3.IRAK1 inhibits NLRP3 and is targeted by Pellino2Given that we have previously shown Pellino2 to interact with IRAK1 and the latter can associate with NLRP3 we investigated the ability of Pellino2 to target IRAK1 and regulate its binding to NLRP3. In keeping with previous studies we show that Pellino2 can interact with IRAK1 (Fig.聽8a) and promote ubiquitination of IRAK1 in a RING domain-dependent manner (Fig.聽8b) when these proteins are co-expressed in HEK293T cells. LPS also induced time-dependent ubiquitination of IRAK1 in WT BMDMs and this was suppressed in Peli2鈭?鈭?/sup> cells (Fig.聽8c) highlighting Pellino2 as an important mediator of IRAK1 ubiquitination in response to LPS. While such ubiquitination by LPS in WT cells is followed by degradation of most of the pool of IRAK1, the greatly reduced ubiquitination of IRAK1 in Peli2鈭?鈭?/sup> cells still permitted degradation of IRAK1, albeit with slightly delayed kinetics and more residual IRAK1 than in WT cells. Furthermore, using TUBEs to isolate ubiquitinated proteins we again demonstrated that LPS-induced ubiquitination of IRAK1 was reduced in Peli2鈭?鈭?/i> cells (Fig.聽8d). We next assessed the effects of the loss of IRAK1 ubiquitination in Peli2鈭?鈥?/sup> cells on the ability of IRAK1 to interact with NLRP3. LPS promoted time-dependent interaction of IRAK1 with NLRP3 in WT BMDMs but this interaction was augmented in Peli2鈭?鈭?/sup> cells (Fig.聽8e) suggesting that Pellino2-mediated ubiquitination of IRAK1 may impair the binding of the latter to NLRP3. We were thus keen to study the functional relevance of IRAK1 binding to NLRP3 and compared NLRP3 activation between WT and IRAK1-deficient BMDMs. Intriguingly the absence of IRAK1 resulted in further enhancement of the levels of mature IL-1尾 produced in response to LPS/ATP or LPS/nigericin (Fig.聽8f, left panel), two regimes to trigger the NLRP3 inflammasome. In contrast, the absence of IRAK1 leads to reduced levels of TNF (Fig.聽8f, right panel) in keeping with the early receptor proximal role of IRAK1 in the TLR4 pathway that drives induction of TNF55. 韦his suggests that IRAK1 acts to negatively regulate NLRP3 and this was further supported by augmented processing of pro-IL-1尾 and pro-caspase-1 in IRAK1-deficient BMDMs (Fig.聽8g). Similar findings were also observed in IRAK1-deficient immortalized BMDMs (Supplementary Fig.聽7a, b) with these studies also including control experiments with IRAK4-deficient cells that produced barely detectable levels of secreted IL-1尾 (Supplementary Fig.聽7a). The latter is likely due to IRAK4 being an essential mediator of the LPS priming of pro-IL-1尾 production. We next investigated if IRAK1 could regulate the ubiquitination of NLRP3 since IRAK1 is dispensable for signal 1-mediated induction of pro-IL-1尾. We initially co-expressed IRAK1 and NLRP3 and while it proved difficult to detect any ubiquitination of NLRP3 when expressed alone or with IRAK1, intense ubiquitination of NLRP3 was observed when co-expressed with a kinase dead form of IRAK1 (Fig.聽8h). Since the latter likely acts as a dominant negative inhibitor of endogenous IRAK1, the study suggested that the kinase activity of IRAK1 may act to suppress NLRP3 ubiquitination. We also compared LPS-induced ubiquitination of NLRP3 in WT and IRAK1-deficient BMDMs and observed enhanced NLRP3 ubiquitination in the absence of IRAK1 (Fig.聽8i). Intriguingly the enhanced ubiquitination of NLRP3 in IRAK1-deficient cells is also associated with increased binding of Pellino2 to NLRP3 (Fig.聽8j) again supporting a role for Pellino2-dependent ubiquitination of NLRP3 being an important part of LPS priming of the NLRP3 inflammasome. These data are consistent with a model of NLRP3 activation that is counter-regulated by IRAK1 and Pellino2 (Fig.聽9). Thus, we propose that IRAK1 binds to NLRP3 to suppress ubiquitination and priming of NLRP3. Pellino2 can counter this braking mechanism with these effects of Pellino2 being associated with facilitation of IRAK1 ubiquitination, dissociation of IRAK1 from NLRP3 and ubiquitination of NLRP3 in a Pellino2-dependent manner. While over-expression studies have previously shown Pellino2 to promote ubiquitination of IRAK135, in vitro ubiquitination assays with purified forms of Pellino2 and IRAK would be required to confirm that IRAK1 is a direct substrate for Pellino2. Overall, our data demonstrate a role for Pellino2 in the priming phase of the NLRP3 pathway that facilitates NLRP3 inflammasome assembly, ASC oligomerization and downstream caspase-1-mediated processing of pro-IL-1尾 and pro-IL-18.Fig. 8Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates (d) from WT and Peli2鈭?鈭?/sup> BMDMs treated with 100鈥塶g/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2鈭?鈭?/sup> BMDMs treated with 100鈥塶g/ml LPS for the indicated times. f ELISA of IL-1尾 (left panel) and TNF (right panel) in medium from primary WT and Irak1鈭?鈭?/i> BMDMs treated with 100鈥塶g/ml LPS for 3鈥塰 and then with 2.5鈥塵M ATP or 5鈥塵M Nigericin for 1鈥塰.聽UT, untreated. g Immunoblot analysis of IL-1尾 and Caspase-1 in medium (Sup) and lysates of WT and Irak1鈭?鈭?/i> BMDMs stimulated with 100鈥塶g/ml LPS for 3鈥塰 and 2.5鈥塵M ATP for 1鈥塰. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. 尾-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1鈭?鈭?/sup> BMDMs treated with 100鈥塶g/ml LPS for the indicated times. j Immortalized WT and Irak1鈭?鈭?/i> BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100鈥塶g/ml LPS for indicated times. *p鈥?lt;鈥?.05 (paired, two-tailed Student鈥檚 t-test). Data are biological replicates that are representative of three independent experiments (a-e, g-j) or mean鈥壜扁€塻.e.m. of three independent experiments (f)Full size imageFig. 9Schematic representation of Pellino2 mediated NLRP3 inflammasome priming. In wild type macrophages LPS promotes the association of Pellino2 with NLRP3 and so facilitates ubiquitination of NLRP3. This promotes NLRP3 inflammasome assembly, ASC oligomerization and downstream caspase-1-mediated processing of pro-IL-1尾 and pro-IL-18 and pyroptosis. Pellino2 also promotes ubiquitination of IRAK1 and so limits the interaction of IRAK1 with NLRP3 and prevents the inhibitory effects of IRAK1 on NLRP3 activation. In Peli2鈭?鈭?/sup> macrophages this braking effect of Pellino2 on IRAK1 is removed, allowing for IRAK1 to interact with NLRP3 and suppress downstream activation of the NLRP3 inflammasomeFull size imageDiscussionThis study identifies the first physiological role of Pellino2 and a new regulatory network that controls the NLRP3 inflammasome. The findings highlight an important role for Pellino2 in NLRP3 activation by promoting the K63-linked ubiquitination of NLRP3 as part of the priming phase. We also propose that IRAK1 can negatively regulate ubiquitination of NLRP3 and its downstream signaling and Pellino2 can counter this effect by ubiquitinating IRAK1 thus impairing interaction of the latter with NLRP3 and relieving the braking system of IRAK1 on the NLRP3 inflammasome. The need for such complex regulatory control of the NLRP3 inflammasome is likely due to the damaging consequences of unregulated NLRP3 activity in response to infection and also in auto-inflammatory diseases56.There is an emerging appreciation of the physiological roles of Pellino proteins. Pellino1 plays important roles in TLR3 and TLR4 pathways40, T cell activation41, CNS inflammation43, and cancer generation44, 45. We have previously shown Pellino3 to negatively regulate TLR3 signaling46, suppress TNF-induced cell death47, regulate IL-1尾 expression to control obesity-induced insulin resistance49, and mediate NOD2 signaling to facilitate gut homeostasis48. However, until this present study, there have been no reports on the physiological role of Pellino2. We now propose that it plays an important mediatory role in assembly and activation of the NLRP3 inflammasome. Pellino2 is not involved in TLR-induced activation of NF魏B or induction of pro-IL-1尾 or NLRP3 but instead facilitates the K63-linked ubiquitination of NLRP3 and we propose this as a novel pathway in the priming process. The lack of role of Pellino2 in the NF魏B pathway and other early signaling pathways such as MAPK cascades is consistent with its absence of involvement in TLR-induced expression of pro-inflammatory proteins in macrophages. Interestingly previous studies using overexpression and gene knockdown approaches in cell lines had proposed a role for Pellino2 in mediating LPS-induced activation of MAPK pathways to regulate pro-inflammatory gene expression50, 51. However, we show that these pathways are intact and functional in Pellino2-deficient cells suggesting that its role, if any, is dispensable in these signal transduction pathways. This may be due to functional redundancy with other E3 ubiquitin ligases and indeed a recent report suggests some redundancy between Pellino1, Pellino2, and TRAF6, at least with respect to IL-1 signaling57. However, the impaired activation of NLRP3 activation in Pellino2-deficient cells clearly indicates a critical role for Pellino2 in inflammasome biology that cannot be fully served by other E3 ubiquitin ligases. Interestingly, Pellino2 joins Pellino3 as E3 ubiquitin ligases that play important roles in NLR signaling. Pellino3 mediates activation of NOD2 to facilitate gut homeostasis48 and we now show that Pellino2 mediates activation of NLRP3 and its inflammasome complex. Furthermore, both Pellino proteins target kinases in manifesting these effects with Pellino3 promoting ubiquitination of RIP2 to mediate NOD2 signaling and Pellino2 targeting IRAK1 to facilitate NLRP3 inflammasome activation. Pellino1 also acts as an E3 ubiquitin ligase for RIP1 in the TLR3 and TLR4 pathways40. The present study now confirms that all Pellino family members target and ubiquitinate key regulatory kinases in innate immune signaling.Our data also highlight the intriguing ability of Pellino family members to differentially regulate the same pathway. We have recently shown that Pellino3 can negatively regulate the expression of pro-IL-1尾 by destabilizing the transcription factor HIF-1伪 and so suppress the transcription of the Il1b gene49. We now demonstrate that Pellino2 can promote increased production of mature bioactive IL-1尾 by facilitating activation of the NLRP3 inflammasome. This suggests that Pellino2 may favor the generation of a pro-inflammatory environment whereas Pellino3 seems to temper the inflammatory response.The involvement of Pellino2 in inflammasome activation appears to be limited to NLRP3. Our data suggests that Pellino2 does not regulate caspase-11 expression or non-canonical inflammasome activation in cells. Interestingly Pellino2 deficiency offers some protection in mice against the lethal effects of LPS and yet such lethality has previously been attributed to caspase-11-induced pyroptosis58. While caspase-11 cannot directly cleave pro-IL-1尾 or pro-IL-18, it can promote indirect formation of the NLRP3 inflammasome by Gasdermin D-mediated membrane pore formation and pyroptosis. Thus, caspase-11 can indirectly induce processing of the precursors of both IL-1尾 and IL-1859. Such activation of NLRP3 may be compromised in Pellino2-deficient mice and underlie the reduced serum levels of IL-1尾 and increased survival in response to in vivo administered LPS. However lack of Pellino2 does not affect the ability of transfected LPS, a means of activating non-canonical inflammasome activation, to promote secretion of IL-1尾 from BMDMs. The mechanism by which caspase-11 activates the NLRP3 pathway remains to be fully delineated and the lack of effect of Pellino2 deficiency on this non-canonical pathway may reflect a different activating mechanism than the Pellino2-dependent mechanism employed in the canonical NLRP3 pathway that is triggered by LPS/ATP.The mediatory role of Pellino2 in NLRP3 activation represents a new mechanistic insight into the molecular basis by which this inflammasome is activated. While Pellino2 is not a player in the activation of NF魏B and induction of pro-IL-1尾 and NLRP3, we now propose a new regulatory pathway that involves Pellino2-dependent ubiquitination of NLRP3. Previous reports have described TRIM31, FBXL2, and MARCH7 to promote ubiquitination and degradation of NLRP324,25,26 with de-ubiquitination by the BRCC3 complex facilitating activation of NLRP327,28,29. While TRIM31, FBXL2, and MARCH7 are more associated with K48-linked ubiquitination and degradation of NLRP3, we show that NLRP3 is initially modified with K63-linked polyubiquitin chains, in response to priming signals such as LPS. The loss of Pellino2-dependent ubiquitination of NLRP3 leads to impaired inflammasome activation suggesting K63-linked ubiquitination of NLRP3 to be an important part of the priming process. Indeed MCC950, a highly potent and selective inhibitor of NLRP3 strongly suppresses this priming-induced K63-linked ubiquitination of NLRP3.Having performed its role as part of the priming process, the ubiquitin chains on NLRP3 presumably need to be removed by BRCC3 to facilitate downstream inflammasome complex formation. While the molecular basis to the positive effects of Pellino2-dependent ubiquitination remains to be delineated, it may facilitate the transient recruitment of an accessory protein(s) that facilitates NLRP3 activation or alternatively impair the binding of an inhibitory protein such as IRAK1, as demonstrated in this study. While our data clearly demonstrate that Pellino2 can promote ubiquitination of NLRP3, we were unable to show, using in vitro ubiquitination assays, that NLRP3 is a direct substrate for Pellino2. This suggests that the ubiquitination is mediated by another E3 ubiquitin ligase or that the Pellino2-induced ubiquitination is dependent on some ancillary protein or process that is triggered by the priming signal. Our data also adds to a growing appreciation of the importance of ubiquitination for the NLRP3 inflammasome and its various constituents. Linear ubiquitination of ASC, by the LUBAC component HOIL-1L, is required for NLRP3 inflammasome assembly60 and polyubiquitination of pro-IL-1尾 facilitates its interaction with caspase-161, a prologue to assembly of the NLRP3 complex61, 62. In addition, the E3 ligases cIAP1 and cIAP2 promote K63-linked polyubiquitination of caspase-1, a pre-requisite for its full activation63 whereas autophagy targets ubiquitinated pro-caspase-1 and inflammasomes for degradation64.Since we were unable to show that Pellino2 directly ubiquitinates NLRP3, we explored the possibility that it could target an intermediate protein to effect such ubiquitination. We have previously shown Pellino2 to interact with IRAK165 and the latter can also associate with NLRP366, 67 and thus we explored the regulatory effects of Pellino2 on IRAK1. We now show that Pellino2 mediates LPS-induced ubiquitination of IRAK1 that is associated with impaired binding to NLRP3. Intriguingly we also show that IRAK1 acts to negatively regulate the NLRP3 pathway and this is consistent with reduced binding of Pellino2 to NLRP3 and ubiquitination of the latter. Thus, we now present a novel network for regulating NLRP3 in which Pellino2 acts to promote ubiquitination of NLRP3, an important part of the priming process, and this is counter-regulated by IRAK1 that can bind to NLRP3 to suppress its ubiquitination and activation. IRAK1 kinase activity appears to be very important for mediating these effects since a kinase inactive form of IRAK1 strongly favors ubiquitination of NLRP3. Interestingly, previous studies have suggested a positive role for IRAK1 in acute NLRP3 activation that is triggered by short co-treatment with LPS and ATP that is independent of priming and new protein synthesis66, 67. However, we now propose a negative role for IRAK1 in the priming phase of NLRP3 activation and this is consistent with an earlier study showing increased processing of caspase-1 in IRAK1 knockout cells under these conditions67. The counter-regulatory roles of Pellino2 and IRAK1 in this pathway highlights the close interplay between the IRAK and Pellino families. Previous reports have described IRAKs as being capable of phosphorylating Pellino proteins to increase their E3 ubiquitin ligase activities. However, it is highly unlikely that IRAK1-induced phosphorylation and activation of Pellino2 is upstream of NLRP3 inflammasome activation since we show enhanced interaction of Pellino2 with NLRP3 and increased ubiquitination and activation of NLRP3 in IRAK1-deficient cells. Furthermore, in the context of LPS signaling, IRAKs are not employed for this purpose and instead TBK1/IKK蔚 have been proposed to act as the activating kinases for Pellino proteins38. Instead, we speculate that IRAK1 may promote phosphorylation of NLRP3 to restrict recruitment of Pellino2. Intriguingly, NLRP3 activation is negatively controlled by phosphorylation of its pyrin domain68. While the effector kinases were not identified in the latter report, our studies now highlight the importance of IRAK1 as a negative regulator of NLRP3 and as a lead candidate kinase that underlies the inhibitory effects of NLRP3 phosphorylation. We also describe a new regulatory network, involving an E3 ubiquitin ligase and a kinase, that controls NLRP3 activation and ascribe the first physiological role to Pellino2. Such intricate control of this inflammasome likely serves a crucial role in precluding dysregulated NLRP3 activation and uncontrolled inflammatory diseases. Future studies on this pathway are warranted to provide a better understanding of associated disease and to provide important clues for novel interventive strategies.MethodsMicePeli2鈭?鈭?/sup> mice were generated by Taconic Artemis using proprietary technology. To generate constitutive Peli2鈭?鈭?/sup> mice, mice that were heterozygous for the targeted allele were bred with mice containing a Flpe transgene (C57BL/6-Tg(CAG-Flpe)2Arte). This resulted in the deletion of exon 2鈥? and loss of function of the Peli2 gene. The Flpe transgene was removed by breeding the resulting Peli2+/鈭?/sup> mice with C57BL/6 mice during colony expansion. Mice were genotyped by PCR analysis of DNA isolated from ear punches using primers 鈥渁,鈥?GCCTCTACAGGATGCTCATTT; 鈥渂,鈥?GGACAGTCATGCTAGTCTGAGG; 鈥渃,鈥?GAGACTCTGGCTACTCATCC; and 鈥渄,鈥?CCTTCAGCAAGAGCTGGGGAC. Bone marrow from Irak1鈭?鈭?/sup> mice were provided by Prof. Katherine Fitzgerald, the University of Massachusetts Medical School. Genotype of mice was confirmed by western blot for IRAK1 in cultured BMDMs55. All animal experiments were performed under licenses of the Health Products Regulatory Authority (HPRA) of Ireland and the UK Home Office with all protocols being approved by the Research Ethics committees of Maynooth University or Queens University Belfast. Sample sizes used are in line with other similar published studies. All animals were used at age 8鈥?2 weeks. Animals were allocated to experimental groups based on genotype, age, and sex. No randomization methods were used. Mice were housed in individually ventilated cages. Mice were culled using cervical dislocation in isolation from any other mice.Plasmids and reagentsThe plasmids myc-tagged Pellino2, Pellino2-C335A/338A (Peli2-RING), and Pellino2-T187A/N188A (Peli2-FHA) were generated in house; V5-NLRP3 was donated by Prof. Jae Jung, the University of Southern California. IRAK1 and FLAG-tagged IRAK4 were generated in house. HA-ubiquitin was from Addgene. All antibodies were used at a dilution of 1:1000 unless otherwise stated. Anti-p-ERK (9101), anti-ERK (9102), anti-p-I魏B伪 (9246), anti-pIKK (14938), anti-p65 (3033) anti-p-P38 (9211), anti-P38 (9212), anti-p-Jnk (9251), anti-Jnk (9252), anti-myc (2276), anti-IRAK1 (4504), anti-IRAK4 (4363), and anti-human caspase 1 p20 (4199) were from CellSignaling; anti-mouse caspase1 p20 (AG-20B-0042-C100) and anti-mouse NLRP3 (AG-20B-0014-C100) was from adipogen; anti-mouse IL1尾 (AF-401-NA) was from R anti-I魏B-伪 (C-21; sc-371), anti-Ub (P4D1; sc-8017), and anti-ASC (sc-514414) was from Santa Cruz; anti-HA (16B12; MMS-101P) was from Covance; anti-K63 ubiquitin (HWA4C4; BML-PW0600) was from Enzo life science; anti-K48 ubiquitin (Apu2; 05-1307) was from Millipore; anti-flag (F3165) and anti-尾-actin (AC-15; A 1978) were from Sigma; anti-mouse IRDyeTM 680 (926-68070) and anti-rabbit IRDyeTM 800 (926-32211) were from LI-COR Biosciences; anti-mouse-HRP and anti-rabbit-HRP were from Promega. Anti-rabbit Alexa Fluor 568 (A-11011) was from Invitrogen. Adenosine 5鈥?triphosphate disodium salt hydrate (ATP) (A1852) and Nigericin (N7143) were from Sigma. LPS (ALX-581-010-L002) was from Enzo. Pam3CSK4, Pam2CSK4, Zymosan, Poly (I:C), Flagellin, Clo75, Clo97, CpG, Alum, and Poly(dA:dT) were from Invivogen. Murine IL-1尾 was from R D. Cells were transfected using Lipfectamine 2000 (Invitrogen) according to the manufacturer鈥檚 instructions. LDH cytotoxicity assay kit (61780) was from Promega.Cell cultureHEK293T (ATCC CRL-11268) were cultured in Dulbecco鈥檚 modified Eagle鈥檚 medium supplemented with 10% (v/v) fetal bovine serum, 100鈥塙/ml penicillin and 100鈥壩糶/ml streptomycin. THP1 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 100鈥塙/ml penicillin and 100鈥壩糶/ml streptomycin. All cell lines were obtained from the ATCC and have been previously validated using the STR method by the ATCC. Cell lines were not tested for mycoplasma contamination. For isolation of BMDMs, tibias and femurs were removed from WT and Peli2鈭?鈭?/sup> mice by sterile techniques and bone marrow was flushed with fresh RPMI-1640 plus GlutaMAX-I medium using a 271/4 gage needle. Cells were plated in medium supplemented with 10% (v/v) conditioned medium of L929 mouse fibroblasts and were maintained for 6 days at 37鈥壜癈 in a humidified atmosphere of 5% CO2. Medium was replaced every 2 days. Peritoneal macrophages were harvested by injecting sterile PBS containing 10% (v/v) fetal bovine serum (5鈥塵l) into the peritoneal cavity. The peritoneal exudate was withdrawn and centrifuged at 1500鈥塯 for 5鈥塵in. Pelleted peritoneal cells were resuspended in RPMI containing 10% (v/v) fetal bovine serum and cultured at 37鈥壜癈 prior to indicated stimulations. Immortalized WT, Irak1鈭?鈭?/sup>, and Irak4鈭?鈭?/sup> BMDMs (iBMDMS) were provided by Prof. Katherine Fitzgerald, the University of Massachusetts Medical School. iBMDMs were cultured and passaged every 3 days in fresh RPMI-1640 plus GlutaMAX-I medium.ELISAPrimary BMDMs were seeded (1鈥壝椻€?06 cells per ml; 200鈥壩糽/well) in 96-well plates and allowed to rest for 24鈥塰. Cells were then stimulated with the indicated ligands. Conditioned media was collected at the indicated time points and IL-1尾, IL-18, TNF, IL-6, RANTES, and CXCL1 were quantified by sandwich ELISA (R D Systems).Cytotoxicity assayConditioned medium from treated BMDMs was assessed for LDH release using the CytoTox96 non-radioactive cytotoxicity assay (Promega) as per manufacturer鈥檚 instructions.Inflammasome activation assaysCells were primed with LPS (100鈥塶g/ml) from E. coli serotype EH100 (ra) TLRgrade for 3鈥塰 followed by stimulation with the inflammasome activators: adenosine 5鈥?triphosphate disodium salt hydrate (ATP) (5鈥塵M) for 1鈥塰, Poly (dA:dT) (1鈥壩糶/ml) transfected with Lipofectamine 2000 (Invitrogen) for 6鈥塰 or Nigericin (5鈥壩糓) for 1鈥塰. For noncanonical inflammasome activation, cells were primed with 100鈥塶g/ml Pam3CSK4 (Invivogen) for 3鈥塰, followed by transfection of LPS (2鈥壩糶/ml) using Lipofectamine 2000 for 6鈥塰 or primed with LPS for 3鈥塰 and stimulated with CTB (20鈥壩糶/ml) for 16鈥塰.Immunoblotting and immunoprecipitationPrimary BMDMs from WT and Peli2鈭?鈭?/sup> mice were cultured in 12-well plates (1鈥壝椻€?06 cells per ml; 1鈥塵l) or 10鈥塩m dishes (2鈥壝椻€?06 cells per ml; 10鈥塵l). HEK293T cells (2.5鈥壝椻€?05 cells per ml; 3鈥塵l) were grown in 6-well plates and where indicated were transfected with the appropriate expression constructs. For whole cell lysate analysis, cells were lysed in NP-40 lysis buffer (50鈥塵M Tris-HCl, pH 7.4, containing 150鈥塵M NaCl, 1% (w/v) IgePal, 50鈥塵M NaF, 1鈥塵M Na3VO4, 1鈥塵M dithiothreitol, 1鈥塵M phenylmethylsulfonyl fluoride and complete protease inhibitor mixture (Roche)). For co-immunoprecipitation, cells were treated as indicated and then collected in 200鈥壩糽 0.5鈥壝椻€塏P-40 lysis buffer, followed by incubation for 30鈥塵in at 4鈥壜癈. Cell lysates were then incubated with the appropriate antibody and an aliquot (50鈥壩糽) of protein A鈥損rotein G鈥揳garose was added to each sample, followed by incubation overnight at 4鈥壜癈. Immunoprecipitates were collected by centrifugation for 1鈥塵in at 1000鈥塯 at 4鈥壜癈 and the beads were then washed four times with 1鈥塵l of NP-40 lysis buffer (without Na3VO4, dithiothreitol, phenylmethylsulfonyl fluoride, or protease-inhibitor 鈥渃ocktail鈥?. An aliquot (40鈥壩糽) of SDS-PAGE sample buffer (62.5鈥塵M TrisHCl, pH 6.8, 10% (w/v) glycerol, 2% (w/v) SDS, 0.7鈥塎 尾-mercaptoethanol, and 0.001% (w/v) bromophenol blue) was added to the beads. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes and analyzed by immunoblot with the following antibodies as appropriate: anti-NLRP3, anti-IRAK1, anti-myc, and anti-FLAG. Immunoreactivity was visualized by the Odyssey Imaging System (LICOR Biosciences) or enhanced chemiluminescence. For experiments assessing the ubiquitination status of NLRP3 or IRAK1, cells were collected in 200鈥壩糽 RIPA buffer (25鈥塵M Tris-HCL, 150鈥塵M NaCl, 1% (w/v) IgePal, 1% (w/v) sodium deoxycholate). Cell lysates were treated with 1% (w/v) SDS and heated to 95鈥壜癈 for 5鈥塵in to dissociate NLRP3 or IRAK1 from any associated proteins. Lysates were then diluted 10 fold in RIPA buffer before immunoprecipitation. Immunoprecipitated samples were analyzed by immunoblot with anti-ubiquitin (P4D1; sc-8017; Santa Cruz). For immunoblotting of IL-1尾 and Caspase-1 in cell supernatants, conditioned medium was collected and filtered using filter spin columns to reduce salt and remove abundant serum proteins. Filtrates were added to 4鈥壝椻€塖DS-PAGE sample buffer and resolved by SDS-PAGE for immunoblot analysis.Tandem ubiquitin binding entity analysisPrimary BMDMs from WT and Peli2鈭?鈭?/sup> mice were cultured in 10鈥塩m dishes (2鈥壝椻€?06 cells per ml; 10鈥塵l). For enrichment of ubiquitinated proteins, Ubq-TUBE-Agarose (UM-401) or K63-TUBE-FLAG (UM-604) or K48-TUBE-FLAG (UM-605) (Life Sensors) were used according to manufacturer instructions. Briefly, cells were lysed in TUBE lysis buffer (100鈥塵M Tris HCL pH 7.5, 150鈥塵M NaCl, 5鈥塵M EDTA, 1% NP-40, 0.5% Triton) containing 1,10-phenanthroline (5鈥塵M), PR-619 (100鈥壩糓) N-Ethylmalamide (5鈥塵M) and 20鈥壩糽 of Ubq-TUBE agarose or 500鈥塶M of K63-TUBE-FLAG or K48-TUBE-FLAG. Cell lysates were then incubated for 1鈥塰 on ice to allow TUBE binding. For enrichment of FLAG-tagged TUBEs, M2-FLAG affinity gel (A220, Sigma) was equilibrated in TBST for 5鈥塵in then added to samples (20鈥壩糽/sample) and incubated at 4鈥壜癈 for 2鈥塰 with rotation. TUBE-enriched FLAG affinity gel was then collected by centrifugation for 5鈥塵in at 5000鈥塺pm at 4鈥壜癈 and washed three times with 1鈥塵l of wash buffer (TUBE lysis buffer without Triton). An aliquot (25鈥壩糽) of SDS-PAGE sample buffer (62.5鈥塵M TrisHCl, pH 6.8, 10% (w/v) glycerol, 2% (w/v) SDS, 0.7鈥塎 尾-mercaptoethanol and 0.001% (w/v) bromophenol blue) was added to the beads. Samples were resolved by SDS-PAGE, transferred to nitrocellulose membranes and analyzed by immunoblot with the following antibodies as appropriate: anti-NLRP3, anti-IRAK1, anti-K63-linked ubiquitin, anti-K48 linked ubiquitin, and anti-ubiquitin. Immunoreactivity was visualized by the Odyssey Imaging System (LICOR Biosciences).Real time PCR analysisTotal RNA was extracted from tissues or cells using Trizol (Invitrogen). cDNA was generated from 2鈥壩糶 RNA using MMLV Reverse Transcriptase (Bioscript Bioline) and real-time PCR analyses were performed with SensiMix SYBR Master mix (Bioline) using an Applied Biosystems StepOnePlus鈩?Real-Time PCR System according to the manufacturer鈥檚 instructions. The abundance of each mRNA was normalized relative to PCR of the housekeeping gene Hypoxanthine-guanine phosphoribosyltransferase (Hprt). Mouse Il1尾, forward, CGGCACACCCACCCTG, and reverse, AAACCGTTTTTCCATCTTCTTCT; mouse Il6, forward, ACAACCACGGCCTTCCCTAC, and reverse, TCCACGATTTCCCAGAGAACA; The abundance of each mRNA was normalized relative to PCR of the housekeeping gene Hprt with the following primers: forward, GTCCCAGCGTCGTGATTAGC, and reverse, TGGCCTCCCATCTCCTTCA.LPS septic shock modelMice aged 8鈥?2 weeks were administered LPS 20鈥塵g/kg by intraperitoneal injection. Serum was collected after 6鈥塰 and various cytokine assayed by ELISA. For survival analysis, mice were administered LPS 50鈥塵g/kg intraperitoneally and monitored every 6鈥塰 for 72鈥塰. P. aeruginosa infectionMale WT and Peli2鈭?鈭?/i> mice were inoculated intraperitoneally with P. aeruginosa (PAO1 strain, kindly provided by Prof Scott Bell, the University of Queensland) (100鈥壜祃; 1.5鈥壝椻€?07 CFU), suspended in sterile endotoxin-free PBS, or sham inoculated with sterile endotoxin-free PBS. At 10鈥塰 post-inoculation the animals were killed by cervical dislocation. Peritoneal lavage was collected using 5鈥塵l of ice cold endotoxin-free PBS, centrifuged at 1000鈥塯 for 10鈥塵in and the supernatants assayed for levels of IL-1尾, IL-18, and IL-6. For cell-based infections, bacteria were grown for 18鈥塰 at 37鈥壜癈 in LB Broth. Bacteria were then diluted 1/5 in LB broth and cultured for a further 2鈥塰. Indicated cells were infected at a multiplicity of infection (MOI) of 100:1 for 6鈥塰. Cell supernatants and lysates were then collected for ELISA and western blot analysis.ASC oligomerization and fluorescence microscopyASC oligomers were analyzed as previously described54. Cells were thus treated with LPS with or without ATP or Nigericin for the indicated times. Cells were then washed in PBS and lysed in NP-40 buffer on ice. NP-40 soluble fractions were retained and insoluble pellets were washed twice in PBS. Insoluble pellets were then reconstituted in PBS and cross-linked in 2鈥塵M Suberic acid for 1鈥塰 at room temperature with gentle agitation. Cross-linked pellets were washed twice in PBS and lysed in 2鈥壝椻€塻ample buffer for western blot analysis of ASC oligomers. Coverslips were coated with BMDMs for fluorescence microscopic analysis of ASC specks. Cell were fixed for 5鈥塵in with methanol (鈭?0鈥壜癈) and washed three times with PBS. Samples were incubated with anti-rabbit ASC (1:200) overnight, followed by incubation with anti-rabbit Alexa Fluor 568 (5鈥壩糶/ml) and mounting in DAPI-containing media. Images were captured using the 脳20 objective of a fluorescent microscope. Image analysis was performed using Olympus cellSens Dimension 1.9 softwareMitochondrial ROSPrimary BMDMs were treated as indicated and assayed for mROS production using Mitosox (LifeTechnologies) according to the manufacturer鈥檚 instructions. Briefly, cells were washed twice in PBS and then incubated with 2.5鈥塵M Mitosox in PBS for 15鈥塵in at 37鈥壜癈. Cells were then washed twice in cold PBS (1鈥塵l) followed by flow cytometric analysis of Mitosox-reactive cells using an AcuriC6 flow cytometer and Flowjo softwarePreparation and transfection of siRNAHuman Pellino2-specific siRNAs were from the LifeTechnologies (AM16708, 133379). siRNA was delivered to THP1 cells via transfection with Lipfectamine2000 according to the manufacturer\'s instructions and allowed to recover for 48鈥塰 prior to experiments.Retroviral rescue assayMyc-tagged murine Pellino2 (mPeli2), Pellino2-C337A/334A (mPeli2-RING), and Pellino2-R106A/R136A (mPel2-FHA) were sub-cloned into the retroviral MSCV2.2-IRES-GFP vector. The empty MSCV vector (as control) or MSCV containing the indicated Pellino2 construct (10鈥壩糶) was co-transfected with the packaging vector 蠄 (5鈥壩糶) and VSV-g envelope vector (5鈥壩糶) into a T75鈥塩m2 flask of HEK293T cells in DMEM medium (10鈥塵l). After 48鈥塰, the retrovirus-containing medium was collected. 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Med. 214, 1725鈥?736 (2017).CAS聽 Article聽 PubMed聽 PubMed Central聽Google Scholar聽 Download referencesAcknowledgementsThis publication has emanated from research conducted with the financial support of the Science Foundation Ireland (SFI) under Grant Numbers SFI/16/IA/4622 and SFI/12/IA/1736.Author informationAuthor notesThese authors contributed equally: Fiachra Humphries, Ronan Bergin.AffiliationsInstitute of Immunology, Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, IrelandFiachra Humphries,聽Ronan Bergin,聽Ruaidhri Jackson,聽Nezira Delagic,聽Bingwei Wang,聽Shuo Yang聽 聽Paul N. MoynaghDepartment of Immunology, Nanjing Medical University, Nanjing, Jiangsu Province, ChinaShuo YangCentre for Experimental Medicine, Queen鈥檚 University Belfast, Belfast, UKAlice V. Dubois,聽Rebecca J. Ingram聽 聽Paul N. MoynaghAuthorsFiachra HumphriesView author publicationsYou can also search for this author in PubMed聽Google ScholarRonan BerginView author publicationsYou can also search for this author in PubMed聽Google ScholarRuaidhri JacksonView author publicationsYou can also search for this author in PubMed聽Google ScholarNezira DelagicView author publicationsYou can also search for this author in PubMed聽Google ScholarBingwei WangView author publicationsYou can also search for this author in PubMed聽Google ScholarShuo YangView author publicationsYou can also search for this author in PubMed聽Google ScholarAlice V. DuboisView author publicationsYou can also search for this author in PubMed聽Google ScholarRebecca J. IngramView author publicationsYou can also search for this author in PubMed聽Google ScholarPaul N. MoynaghView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsF.H. and R.B. developed the concept, designed, and performed experiments, analyzed data and prepared the figures; F.H. also assisted with writing the manuscript. R.J. designed and performed experiments and analyzed data; N.D. assisted with experiments, interpreted experimental data and advised on manuscript preparation. B.W. generated expression constructs and managed the breeding program for mice. S.Y generated Pellino2 expression constructs. A.V.D. and R.J.I. designed, performed, analyzed, and supervised the P. aeruginosa studies. P.N.M. conceived the study, supervised the overall project, analyzed data, and wrote the manuscript.Corresponding authorCorrespondence to Paul N. Moynagh.Ethics declarations Competing interests The authors declare no competing interests. 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