ABSTRACT
RIG-I-like receptors and NOD-like receptors play pivotal roles in recognizing microbe-associated molecular patterns and initiating immune responses. The LGP2 and NOD2 proteins are important members of the RIG-I-like receptor and NOD-like receptor families, recognizing viral RNA and bacterial peptidoglycan (PGN), respectively. However, in some instances bacterial infections can induce LPG2 expression via a mechanism that remains largely unknown. In the current study, we found that LGP2 can compete with NOD2 for PGN binding and inhibit antibacterial immunity by suppressing the NOD2-RIP2 axis. Recombinant CiLGP2 (Ctenopharyngodon idella LGP2) produced using either prokaryotic or eukaryotic expression platform can bind PGN and bacteria in pull-down and ELISA assays. Comparative protein structure models and intermolecular interaction prediction calculations as well as pull-down and colocalization experiments indicated that CiLGP2 binds PGN via its EEK motif with species and structural specificity. EEK deletion abolished PGN binding of CiLGP2, but insertion of the CiLGP2 EEK motif into zebrafish and mouse LGP2 did not confer PGN binding activity. CiLGP2 also facilitates bacterial replication by interacting with CiNOD2 to suppress expression of NOD2-RIP2 pathway genes. Sequence analysis and experimental verification demonstrated that LGP2 having EEK motif that can negatively regulate antibacterial immune function is present in Cyprinidae and Xenocyprididae families. These results show that LGP2 containing EEK motif competes with NOD2 for PGN binding and suppresses antibacterial immunity by inhibiting the NOD2-RIP2 axis, indicating that LGP2 plays a crucial negative role in antibacterial response beyond its classical regulatory function in antiviral immunity.
Subject(s)
Nod2 Signaling Adaptor Protein , Peptidoglycan , Animals , Nod2 Signaling Adaptor Protein/metabolism , Nod2 Signaling Adaptor Protein/immunology , Nod2 Signaling Adaptor Protein/genetics , Peptidoglycan/metabolism , Peptidoglycan/immunology , Fish Proteins/immunology , Fish Proteins/genetics , Fish Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Carps/immunology , Mice , Protein Binding , Signal Transduction/immunology , Humans , Amino Acid Motifs , Zebrafish/immunologyABSTRACT
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.
Subject(s)
Inflammation , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Signal Transduction , Humans , Inflammation/drug therapy , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolismABSTRACT
BACKGROUND AND AIMS: Diabetic kidney disease (DKD) is one of the most common complications of diabetes. It is reported that mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) may have great clinical application potential for the treatment of DKD, but the underlying mechanism has not been illustrated. To clarify the effect of MSC-Exo on NOD2 signaling pathway in podocytes under high glucose (HG) and DKD, we conduct this study. METHODS: We co-cultured podocytes and MSCs-Exo under 30 mM HG and injected MSCs-Exo into DKD mice, then we detected the NOD2 signaling pathway by western blot, qRT-PCT, immunofluorescence, transmission electron microscopy and immunohistochemistry both in vitro and in vivo. RESULTS: In vitro, HG lead to the apoptosis, increased the ROS level and activated the NOD2 signaling pathway in podocytes, while MSCs-Exo protected podocytes from injury reduced the expression of inflammatory factors including TNF-α, IL-6, IL-1ß, and IL-18 and alleviated the inflammatory response, inhibited the activation of NOD2 signaling pathway and the expression of it's downstream protein p-P65, p-RIP2, prevented apoptosis, increased cell viability in podocytes caused by HG. In vivo, MSCs-Exo alleviated renal injury in DKD mice, protected renal function, decreased urinary albumin excretion and inhibited the activation of NOD2 signaling pathway as well as the inflammation in renal tissue. CONCLUSION: MSCs-Exo protected the podocytes and DKD mice from inflammation by mediating NOD2 pathway, MSCs-Exo may provide a new target for the treatment of DKD.
Subject(s)
Apoptosis , Diabetic Nephropathies , Exosomes , Mesenchymal Stem Cells , Nod2 Signaling Adaptor Protein , Podocytes , Signal Transduction , Animals , Exosomes/metabolism , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/therapy , Mesenchymal Stem Cells/metabolism , Mice , Nod2 Signaling Adaptor Protein/metabolism , Podocytes/metabolism , Podocytes/pathology , Male , Mice, Inbred C57BL , Glucose/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Coculture Techniques , Mesenchymal Stem Cell Transplantation/methods , Reactive Oxygen Species/metabolism , Kidney/pathology , Kidney/metabolism , Diabetes Mellitus, Experimental/complicationsABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents , Infarction, Middle Cerebral Artery , NF-kappa B , Naphthoquinones , Neuroprotective Agents , Nod2 Signaling Adaptor Protein , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Reperfusion Injury , Animals , Male , Mice , Anti-Inflammatory Agents/pharmacology , Cell Line , Cytokines/metabolism , Disease Models, Animal , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/drug therapy , Inflammation Mediators/metabolism , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , Naphthoquinones/pharmacology , Neuroinflammatory Diseases/drug therapy , Neuroinflammatory Diseases/metabolism , Neuroinflammatory Diseases/pathology , Neuroprotective Agents/pharmacology , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Phenotype , Rats, Sprague-Dawley , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Reperfusion Injury/metabolism , Reperfusion Injury/drug therapy , Reperfusion Injury/pathology , Signal Transduction/drug effectsABSTRACT
Alternative splicing is the process of generating different mRNAs from the same primary transcript, which contributes to increase the transcriptome and proteome diversity. Abnormal splicing has been associated with the development of several diseases including cancer. Given that mutations and abnormal levels of the RIPK2 transcript and RIP-2 protein are frequent in tumors, and that RIP-2 modulates immune and inflammatory responses, we investigated alternative splicing events that result in partial deletions of the kinase domain at the N-terminus of RIP-2. We also investigated the structure and expression of the RIPK2 truncated variants and isoforms in different environments. In addition, we searched data throughout Supraprimates evolution that could support the biological importance of RIPK2 alternatively spliced products. We observed that human variants and isoforms were differentially regulated following temperature stress, and that the truncated transcript was more expressed than the long transcript in tumor samples. The inverse was found for the longer protein isoform. The truncated variant was also detected in chimpanzee, gorilla, hare, pika, mouse, rat, and tree shrew. The fact that the same variant has been preserved in mammals with divergence times up to 70 million years raises the hypothesis that it may have a functional significance.
Subject(s)
Alternative Splicing , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Animals , Humans , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Evolution, Molecular , Protein Isoforms/genetics , Mice , Neoplasms/genetics , RatsABSTRACT
Ischemic stroke induces a debilitating neurological insult, where inflammatory processes contribute greatly to the expansion and growth of the injury. Receptor-interacting protein kinase 2 (RIPK2) is most well-known for its role as the obligate kinase for NOD1/2 pattern recognition receptor signaling and is implicated in the pathology of various inflammatory conditions. Compared to a sham-operated control, ischemic stroke resulted in a dramatic increase in the active, phosphorylated form of RIPK2, indicating that RIPK2 may be implicated in the response to stroke injury. Here, we assessed the effects of pharmacological inhibition of RIPK2 to improve post-stroke outcomes in mice subjected to experimental ischemic stroke. We found that treatment at the onset of reperfusion with a RIPK2 inhibitor, which inhibits the phosphorylation and activation of RIPK2, resulted in marked improvements in post-stroke behavioral outcomes compared to the vehicle-administered group assessed 24 h after stroke. RIPK2 inhibitor-treated mice exhibited dramatic reductions in infarct volume, concurrent with reduced damage to the blood-brain barrier, as evidenced by reduced levels of active matrix metalloproteinase-9 (MMP-9) and leakage of blood-borne albumin in the ipsilateral cortex. To explore the protective mechanism of RIPK2 inhibition, we next pretreated mice with RIPK2 inhibitor or vehicle and examined transcriptomic alterations occurring in the ischemic brain 6 h after stroke. We observed a dramatic reduction in neuroinflammatory markers in the ipsilateral cortex of the inhibitor-treated group while also attaining a comprehensive view of the vast transcriptomic alterations occurring in the brain with inhibitor treatment through bulk RNA-sequencing of the injured cortex. Overall, we provide significant novel evidence that RIPK2 may represent a viable target for post-stroke pharmacotherapy and potentially other neuroinflammatory conditions.
Subject(s)
Ischemic Stroke , Mice, Inbred C57BL , Neuroprotective Agents , Receptor-Interacting Protein Serine-Threonine Kinase 2 , Animals , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Mice , Ischemic Stroke/drug therapy , Ischemic Stroke/metabolism , Ischemic Stroke/pathology , Neuroprotective Agents/pharmacology , Neuroprotective Agents/therapeutic use , MaleABSTRACT
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.