Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.

Shikonin attenuates cerebral ischemia/reperfusion injury via inhibiting NOD2/RIP2/NF-κB-mediated microglia polarization and neuroinflammation.

OBJECTIVES: Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, which still lacks effective therapeutic agents. Shikonin possesses anti-inflammatory and neuroprotective properties. However, its underlying mechanism remains elusive. This study aimed to investigate whether Shikonin confers protection against cerebral ischemia/reperfusion (I/R) injury by modulating microglial polarization and elucidate the associated mechanisms. METHODS: This study employed an oxygen-glucose deprivation and reoxygenation (OGD/R) BV2 microglial cellular model and a middle cerebral artery occlusion/reperfusion (MCAO/R) animal model to investigate the protection and underlying mechanism of Shikonin against ischemic stroke. RESULTS: The results demonstrated that Shikonin treatment significantly reduced brain infarction volume and improved neurological function in MCAO/R rats. Simultaneously, Shikonin treatment significantly reduced microglial proinflammatory phenotype and levels of proinflammatory markers (inducible-NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6), increased microglial anti-inflammatory phenotype and levels of anti-inflammatory markers (Arginase-1 (Arg1), transforming growth factor-beta (TGF-ß), and IL-10), reversed the expression of Nucleotide-binding oligomerization domain 2 (NOD2) and phosphorylation receptor interacting protein 2 (p-RIP2), and suppressed nuclear factor kappa-B (NF-κB) signaling activation in the ischemic penumbra regions. These effects of Shikonin were further corroborated in OGD/R-treated BV2 cells. Furthermore, overexpression of NOD2 markedly attenuated the neuroprotective effects of Shikonin treatment in MCAO/R rats. NOD2 overexpression also attenuated the regulatory effects of Shikonin on neuroinflammation, microglial polarization, and NF-κB signaling activation. CONCLUSION: This study illustrates that Shikonin mitigates inflammation mediated by microglial proinflammatory polarization by inhibiting the NOD2/RIP2/NF-κB signaling pathway, thereby exerting a protective role. The findings uncover a potential molecular mechanism for Shikonin in treating ischemic stroke.

Design, synthesis and evaluation of novel thieno[2,3d]pyrimidine derivatives as potent and specific RIPK2 inhibitors.

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2.

OTUD1 ameliorates cerebral ischemic injury through inhibiting inflammation by disrupting K63-linked deubiquitination of RIP2.

BACKGROUND: Inflammatory response triggered by innate immunity plays a pivotal element in the progress of ischemic stroke. Receptor-interacting kinase 2 (RIP2) is implicated in maintaining immunity homeostasis and regulating inflammatory response. However, the underlying mechanism of RIP2 in ischemic stroke is still not well understood. Hence, the study investigated the role and the ubiquitination regulatory mechanism of RIP2 in ischemic stroke. METHODS: Focal cerebral ischemia was introduced by middle cerebral artery occlusion (MCAO) in wild-type (WT) and OTUD1-deficient (OTUD1-/-) mice, oxygen glucose deprivation and reoxygenation (OGD/R) models in BV2 cells and primary cultured astrocytes were performed for monitoring of experimental stroke. GSK2983559 (GSK559), a RIP2 inhibitor was intraventricularly administered 30 min before MCAO. Mice brain tissues were collected for TTC staining and histopathology. Protein expression of RIP2, OTUD1, p-NF-κB-p65 and IκBα was determined by western blot. Localization of RIP2 and OTUD1 was examined by immunofluorescence. The change of IL-1ß, IL-6 and TNF-α was detected by ELISA assay and quantitative real-time polymerase chain reaction. Immunoprecipitation and confocal microscopy were used to study the interaction of RIP2 and OTUD1. The activity of NF-κB was examined by dual-luciferase assay. RESULTS: Our results showed upregulated protein levels of RIP2 and OTUD1 in microglia and astrocytes in mice subjected to focal cerebral ischemia. Inhibition of RIP2 by GSK559 ameliorated the cerebral ischemic outcome by repressing the NF-κB activity and the inflammatory response. Mechanistically, OTUD1 interacted with RIP2 and sequentially removed the K63-linked polyubiquitin chains of RIP2, thereby inhibiting NF-κB activation. Furthermore, OTUD1 deficiency exacerbated cerebral ischemic injury in response to inflammation induced by RIP2 ubiquitination. CONCLUSIONS: These findings suggested that RIP2 mediated cerebral ischemic lesion via stimulating inflammatory response, and OTUD1 ameliorated brain injury after ischemia through inhibiting RIP2-induced NF-κB activation by specifically cleaving K63-linked ubiquitination of RIP2.

Receptor Interacting Ser/Thr-Protein Kinase 2 as a New Therapeutic Target.

Receptor interacting serine/threonine protein kinase 2 (RIPK2) is a downstream signaling molecule essential for the activation of several innate immune receptors, including the NOD-like receptors (NOD1 and NOD2). Recognition of pathogen-associated molecular pattern proteins by NOD1/2 leads to their interaction with RIPK2, which induces release of pro-inflammatory cytokines through the activation of NF-κB and MAPK pathways, among others. Thus, RIPK2 has emerged as a key mediator of intracellular signal transduction and represents a new potential therapeutic target for the treatment of various conditions, including inflammatory diseases and cancer. In this Perspective, first, an overview of the mechanisms that underlie RIPK2 function will be presented along with its role in several diseases. Then, the existing inhibitors that target RIPK2 and different therapeutic strategies will be reviewed, followed by a discussion on current challenges and outlook.

RIPK2 inhibitors for disease therapy: Current status and perspectives.

Receptor-interacting protein kinase 2 (RIPK2) belongs to the receptor-interacting protein family (RIPs), which is mainly distributed in the cytoplasm. RIPK2 is widely expressed in human tissues, and its mRNA level is highly expressed in the spleen, leukocytes, placenta, testis, and heart. RIPK2 is a dual-specificity kinase with multiple domains, which can interact with tumor necrosis factor receptor (TNFR), and participate in the Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD) signaling pathways. It is considered as a vital adapter molecule involved in the innate immunity, adaptive immunity, and apoptosis. Functionally, RIPK2 and its targeted small molecules are of great significance in inflammatory responses, autoimmune diseases and tumors. The present study reviews the molecule structure and biological functions of RIPK2, and its correlation between human diseases. In addition, we focus on the structure-activity relationship of small molecule inhibitors of RIPK2 and their therapeutic potential in human diseases.

Exploring positions 6 and 7 of a quinazoline-based scaffold leads to changes in selectivity and potency towards RIPK2/3 kinases.

Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research.

NOD1 cooperates with HAX-1 to promote cell migration in a RIPK2- and NF-ĸB-independent manner.

The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway.

Characterization of the Inflammatory Response Evoked by Bacterial Membrane Vesicles in Intestinal Cells Reveals an RIPK2-Dependent Activation by Enterotoxigenic Escherichia coli Vesicles.

Although the immunomodulatory potency of bacterial membrane vesicles (MVs) is widely acknowledged, their interactions with host cells and the underlying signaling pathways have not been well studied. Herein, we provide a comparative analysis of the proinflammatory cytokine profile secreted by human intestinal epithelial cells exposed to MVs derived from 32 gut bacteria. In general, outer membrane vesicles (OMVs) from Gram-negative bacteria induced a stronger proinflammatory response than MVs from Gram-positive bacteria. However, the quality and quantity of cytokine induction varied between MVs from different species, highlighting their unique immunomodulatory properties. OMVs from enterotoxigenic Escherichia coli (ETEC) were among those showing the strongest proinflammatory potency. In depth analyses revealed that the immunomodulatory activity of ETEC OMVs relies on a so far unprecedented two-step mechanism, including their internalization into host cells followed by intracellular recognition. First, OMVs are efficiently taken up by intestinal epithelial cells, which mainly depends on caveolin-mediated endocytosis as well as the presence of the outer membrane porins OmpA and OmpF on the MVs. Second, lipopolysaccharide (LPS) delivered by OMVs is intracellularly recognized by novel caspase- and RIPK2-dependent pathways. This recognition likely occurs via detection of the lipid A moiety as ETEC OMVs with underacylated LPS exhibited reduced proinflammatory potency but similar uptake dynamics compared to OMVs derived from wild-type (WT) ETEC. Intracellular recognition of ETEC OMVs in intestinal epithelial cells is pivotal for the proinflammatory response as inhibition of OMV uptake also abolished cytokine induction. The study signifies the importance of OMV internalization by host cells to exercise their immunomodulatory activities. IMPORTANCE The release of membrane vesicles from the bacterial cell surface is highly conserved among most bacterial species, including outer membrane vesicles (OMVs) from Gram-negative bacteria as well as vesicles liberated from the cytoplasmic membrane of Gram-positive bacteria. It is becoming increasingly evident that these multifactorial spheres, carrying membranous, periplasmic, and even cytosolic content, contribute to intra- and interspecies communication. In particular, gut microbiota and the host engage in a myriad of immunogenic and metabolic interactions. This study highlights the individual immunomodulatory activities of bacterial membrane vesicles from different enteric species and provides new mechanistic insights into the recognition of ETEC OMVs by human intestinal epithelial cells.

RIPK2 promotes the progression of colon cancer by regulating BIRC3-mediated ubiquitination of IKBKG.

Colon cancer is a cancer with high morbidity and mortality worldwide. Receptor interacting serine/threonine kinase 2 (RIPK2) has been identified as a proto-oncogene, but its role in colon cancer is largely unknown. Herein, we found that RIPK2 interference could inhibit the proliferation and invasion of colon cancer cells, and promote apoptosis. Baculoviral IAP repeat containing 3 (BIRC3) is an E3 ubiquitin ligase, which was found highly expressed in colon cancer cells. Co-immunoprecipitation (Co-IP) experiments showed that RIPK2 could directly bind with BIRC3. Then, we demonstrated that RIPK2 overexpression promoted the expression of BIRC3, BIRC3 interference could eliminate RIPK2-dependent cell proliferation and invasion, and BIRC3 overexpression rescued the suppressive effect of RIPK2 interference on cell proliferation and invasion. We further identified IKBKG, an inhibitor of nuclear factor kappa B, as a ubiquitination substrate targeted by BIRC3. IKBKG interference could eliminate the inhibitory effect of BIRC3 interference on cell invasion. RIPK2 could promote BIRC3-mediated ubiquitination of IKBKG, inhibit the expression of IKBKG protein, and promote the expression of NF-κB subunits p50 and p65 proteins. In addition, DLD-1 cells transfected with sh-RIPK2 or/and sh-BIRC3 were injected into mice to establish a tumor xenograft model, and we found that administration of sh-RIPK2 or sh-BIRC3 impeded the growth of xenograft tumors in vivo, and co-administration displayed a better inhibitory effect. In general, RIPK2 promotes the progression of colon cancer by promoting BIRC3-mediated ubiquitination of IKBKG and activating the NF-κB signaling pathway.

Paired protein kinases PRKCI-RIPK2 promote pancreatic cancer growth and metastasis via enhancing NF-κB/JNK/ERK phosphorylation.

BACKGROUND: Protein kinases play a pivotal role in the malignant evolution of pancreatic cancer (PC) through mediating phosphorylation. Many kinase inhibitors have been developed and translated into clinical use, while the complex pathology of PC confounds their clinical efficacy and warrants the discovery of more effective therapeutic targets. METHODS: Here, we used the Gene Expression Omnibus (GEO) database and protein kinase datasets to map the PC-related protein kinase-encoding genes. Then, applying Gene Expression and Profiling Interactive Analysis (GEPIA), GEO and Human Protein Atlas, we evaluated gene correlation, gene expression at protein and mRNA levels, as well as survival significance. In addition, we performed protein kinase RIPK2 knockout and overexpression to observe effects of its expression on PC cell proliferation, migration and invasion in vitro, as well as cell apoptosis, reactive oxygen species (ROS) production and autophagy. We established PC subcutaneous xenograft and liver metastasis models to investigate the effects of RIPK2 knockout on PC growth and metastasis. Co-immunoprecipitation and immunofluorescence were utilized to explore the interaction between protein kinases RIPK2 and PRKCI. Polymerase chain reaction and immunoblotting were used to evaluate gene expression and protein phosphorylation level. RESULTS: We found fourteen kinases aberrantly expressed in human PC and nine kinases with prognosis significance. Among them, RIPK2 with both serine/threonine and tyrosine activities were validated to promote PC cells proliferation, migration and invasion. RIPK2 knockout could inhibit subcutaneous tumor growth and liver metastasis of PC. In addition, RIPK2 knockout suppressed autophagosome formation, increased ROS production and PC cell apoptosis. Importantly, another oncogenic kinase PRKCI could interact with RIPK2 to enhance the phosphorylation of downstream NF-κB, JNK and ERK. CONCLUSION: Paired protein kinases PRKCI-RIPK2 with multiple phosphorylation activities represent a new pathological mechanism in PC and could provide potential targets for PC therapy.

RIPK2 as a promising druggable target for autoimmune diseases.

Receptor Interacting Serine/Threonine Kinase 2 (RIPK2) is an essential regulator of the inflammatory process and immune response. In innate immunity, the NOD-RIPK2 signaling axis is an important pathway that directly mediates inflammation and immune response. In adaptive immunity, RIPK2 may affect T cell proliferation, differentiation and cellular homeostasis thereby involving T cell-driven autoimmunity, but the exact mechanism remains unclear. Recent advances suggest a key role of RIPK2 in diverse autoimmune diseases (ADs) such as inflammatory bowel diseases, rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, and Behcet's disease. This review aims to provide valuable therapeutic direction for ADs by focusing on the function and modulation of RIPK2 in innate and adaptive immunity, its involvement with various ADs and the application of RIPK2-related drugs in ADs. We raise the notion that drug targeting RIPK2 could be a promising therapeutic strategy for the treatment of ADs, though much work remains to be done for clinical application.

Ubiquitin-Specific Peptidase 8 Is Critical for the Onset of Infectious Osteomyelitis by Targeting RIPK2 Ubiquitination.

Receptor-interacting serine/threonine kinase (RIPK) is associated with cellular inflammation and immune regulation. The current study explored the role of RIPK2 in osteomyelitis and the potential upstream targets of RIPK2. A Staphylococcus aureus-induced osteomyelitis mouse model was established using wild-type (WT) and ubiquitin-specific peptidase 8 (USP8)-deficient (USP-/-) mice, and the osteomyelitis-related symptoms were evaluated. Bone marrow-derived macrophages (BMDMs) were isolated from the WT and USP-/- mice. Enzyme-linked immunosorbent assays, quantitative polymerase chain reaction, and immunoblot analysis were used to determine the levels of target biomarkers, which were induced by lipopolysaccharide (LPS), CpG, or PAM3CSK4. USP8 promoted RIPK2-mediated NF-κB activation. USP8 is indispensable for RIPK2-mediated LPS-induced NF-κB activation in BMDMs. USP8 is required for the production of inflammatory cytokines induced by LPS, CpG, or PAM3CSK4 in BMDMs. In addition, USP-/- mice exhibited ameliorated symptoms, including less body weight and cortical bone loss, and reduced bacterial load and reactive bone formation in the S. aureus-induced osteomyelitis mouse model. USP8 is critical in the S. aureus-induced osteomyelitis mouse model by targeting RIPK2 ubiquitination.

RIP2 inhibition alleviates lipopolysaccharide-induced septic cardiomyopathy via regulating TAK1 signaling.

PURPOSE: RIP2 is a member of the receptor-interacting protein family that has been associated with various pathophysiological processes, including immunity, apoptosis, and autophagy. However, no studies have hitherto reported the role of RIP2 in lipopolysaccharide (LPS)-induced septic cardiomyopathy (SCM). This study was designed to illustrate the role of RIP2 in LPS-induced SCM. METHODS: C57 and RIP2 knockout mice received intraperitoneal injections of LPS to establish models of SCM. Echocardiography was used to assess the cardiac function of the mice. Real-time-PCR, cytometric bead array and immunohistochemical staining were used to detect the inflammatory response. Immunoblotting was used to determine the protein expression of relevant signaling pathways. Our findings were validated by treatment with a RIP2 inhibitor. Neonatal rats cardiomyocytes (NRCMs) and cardiac fibroblasts (CFs) were transfected with Ad-RIP2 to further explore the role of RIP2 in vitro. RESULTS: RIP2 expression was upregulated in our mice models of septic cardiomyopathy and LPS-stimulated cardiomyocytes and fibroblasts. RIP2 knockout or RIP2 inhibitors attenuated LPS-induced cardiac dysfunction and reduced the inflammatory response in mice. Overexpression of RIP2 in vitro enhanced the inflammatory response, and TAK1 inhibitors attenuated the inflammatory response caused by overexpression of RIP2. CONCLUSION: Our findings substantiate that RIP2 induces an inflammatory response by regulating the TAK1/IκBα/NF-κB signaling pathway. RIP2 inhibition by genetic or pharmacological approaches has huge prospects for application as a potential treatment strategy for inhibiting inflammation, alleviating cardiac dysfunction, and improving survival.

Design, synthesis and biological evaluation of 4-aminoquinoline derivatives as receptor-interacting protein kinase 2 (RIPK2) inhibitors.

Receptor-interacting protein kinase 2 (RIPK2) is an essential protein kinase mediating signal transduction by NOD1 and NOD2, which play an important role in regulating immune signalling. In this study, we designed and synthesised a novel series of 4-aminoquinoline-based derivatives as RIPK2 inhibitors. In vitro, compound 14 exhibited high affinity (IC50 = 5.1 ± 1.6 nM) and excellent selectivity to RIPK2 showing in a dendrogram view of the human kinome phylogenetic tree. Bearing favourable lipophilicity and eligible lipophilic ligand efficiency (LipE), compound 14 was selected to investigate cellular anti-inflammatory effect and was identified as a potent inhibitor to reduce the secretion of MDP-induced TNF-α with a dose-dependent manner. Moreover, compound 14 showed moderate stability in human liver microsome. Given these promising results, compound 14 could serve as a favourable inhibitor of RIPK2 for further physiological and biochemical research so as to be used in therapeutic treatment.

Mechanism of RIP2 enhancing stemness of glioma cells induces temozolomide resistance.

AIMS: We aimed to investigate the role of receptor-interacting protein 2 (RIP2) in regulation of stemness of glioma cells and chemotherapy resistance. METHODS: Plasmid transfection was used to overexpress RIP2. Chemical inhibitors were used to inhibit RIP2 or NF-κB activity. Cancer stemness of glioma cells was investigated by sphere formation assays, clone formation assays, and xenograft tumor formation assays. The expression of RIP2, p-NF-κB, IκBα, CD133, or SOX-2 was detected by Western blotting and immunofluorescence. Apoptosis was detected by flow cytometry. Immunohistochemical staining was used to detect the expression of RIP2, CD133, and SOX-2 in xenograft tumor tissue. The effect of the RIP2/NF-κB pathway on temozolomide (TMZ) resistance was evaluated by xenograft tumor assay. RESULTS: Transfection with RIP2 plasmid enhanced the sphere formation capability of U251 cells, clone formation capability, and xenograft tumor formation capability. RIP2 could mediate TMZ resistance by upregulating the expression of CD133 and SOX-2 by activating the NF-κB pathway. Both RIP2 inhibitor GSK583 and the NF-κB inhibitor SC75741 could reverse the resistance of U251 cells to TMZ. CONCLUSION: RIP2 mediates TMZ resistance by regulating the maintenance of stemness in glioma cells through NF-κB. Interventions targeting the RIP2/NF-κB pathway may be a new strategy for TMZ-resistant gliomas.

The protective effects of natural product tunicatachalcone against neuroinflammation via targeting RIPK2 in microglia BV-2 cells stimulated by LPS.

Microglia-induced neuroinflammation plays a critical role in neurological diseases. At present, RIPK2 is considered to participate in inflammatory and autoimmune cellular pathways and diseases. RIPK2 is found to be a pivotal therapeutic target in neurologic disorders related to inflammation. In our research, we discovered the protective function of tunicatachalcone (TC) against neuroinflammation. TC is a natural chalcone compound derived from Pongamia pinnata, a medicinal plant. The results revealed that TC (5-20 µM) ameliorated the activation of BV-2 microglia induced by lipopolysaccharide (LPS) in a dose-dependent way, which was proved by the reduced production of inflammation-related mediators. By using SPR-LC-MS/MS analysis, we revealed the potent inhibitory function of TC against neuroinflammation mediated by microglia via targeting RIPK2. A strong binding between TC and RIPK2 was further demonstrated based on the results of SPR, MST and molecular modeling. Through applying mRNA transcriptomics and bioinformatics analysis, it was demonstrated that TC could mediate RIPK2-dependent gene transcription to exert the neuroprotective effect. In summary, our research presented that RIPK2 was a possible therapeutic target of TC.

Discovery of Potent and Selective Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) Inhibitors for the Treatment of Inflammatory Bowel Diseases (IBDs).

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) has been demonstrated to be a promising target for treating inflammatory diseases. Herein, we describe the discovery and optimization of a series of RIPK2 inhibitors derived from an FLT3 inhibitor, CHMFL-FLT3-165. Compound 10w was identified to possess an IC50 value of 0.6 nM for RIPK2 and greater than 50,000-fold selectivity over its family homologous kinase RIPK1 (IC50 > 30 µM). It exhibited high kinase selectivity and inhibited RIPK2 to prevent NOD-induced cytokine production following muramyl dipeptide (MDP) stimulation. In an acute colitis model, compound 10w exerted better therapeutic effects than the JAK inhibitor filgotinib and the RIPK2 inhibitor WEHI-345. These robust results of in vitro and in vivo pharmacodynamic experiments demonstrate that RIPK2 as a therapeutic target shows potential abilities for the treatment of inflammatory bowel diseases.

NOD2-RIP2 signaling alleviates microglial ROS damage and pyroptosis via ULK1-mediated autophagy during Streptococcus pneumonia infection.

Meningitis occurs when S. pneumonia invade the blood-brain barrier, provoking inflammatory host response and neurological injury. Nucleotide-binding oligomerization domain 2 (NOD2) has been identified to promote microglial activation and autophagy during pneumococcal meningitis, but the mechanism remains unclear. In the present study, we investigated the passway of NOD2-mediated autophagy activation and the role of autophagy in inflammatory damage of murine microglia and mouse meningitis model. We demonstrated that autophagy was activated during S. pneumonia infection, and NOD2-RIP2 signaling was involved in the process. Treatment of microglia with GSK583, the RIP2 kinase inhibitor resulted in reduced autophagy-related protein and p-ULK1, indicating that RIP2 regulated autophagy in a kinase-dependent manner by phosphorylating ULK1. In addition, microglia with ULK1 knockdown exhibited enhanced production of ROS, leading to IL-1ß and IL-18 release and cellular pyroptosis. Similar to the in vitro results, NOD2-RIP2 signaling induced autophagy in the brain in a mouse meningitis model. Moreover, ULK1 or RIP2 silencing significantly increased pyroptosis of brain and induced more inflammatory damage of pneumococcal meningitis mice. Taken together, our study demonstrate that NOD2-RIP2 signaling is involved in the activation of autophagy by promoting ULK1 phosphorylation, which alleviates microglial ROS damage and pyroptosis during S. pneumonia infection.

Assaying RIPK2 Activation by Complex Formation.

The receptor-interacting serine/threonine-protein kinase-2 (RIPK2, RIP2) is a key player in downstream signaling of nuclear oligomerization domain (NOD)-like receptor (NLR)-mediated innate immune response against bacterial infections. RIPK2 is recruited following activation of the pattern recognition receptors (PRRs) NOD1 and NOD2 by sensing bacterial peptidoglycans leading to activation of NF-κB and MAPK pathways and the production of pro-inflammatory cytokines. Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells, also called RIPosomes. These can be induced by Shigella flexneri or by the inhibition of RIPK2 by small compounds, such as GSK583 and gefitinib.In this chapter, we describe fluorescent light microscopic and Western blot approaches to analyze the cytoplasmic aggregation of RIPK2 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri, or by the treatment with RIPK2 inhibitors. This method is based on HeLa cells stably expressing eGFP-tagged RIPK2 and describes a protocol to induce and visualize RIPosome formation. The described method is useful to study the deposition of RIPK2 in speck-like structures, also in living cells, using live cell imaging and can be adopted for the study of other inhibitory proteins or to further analyze the process of RIPosome structure assembly.