NEMO-binding Domain Research Articles

NEMO-Binding Domain/IKKγ Inhibitory Peptide Alleviates Neuronal Pyroptosis in Spinal Cord Injury by Inhibiting ASMase-Induced Lysosome Membrane Permeabilization.

A short peptide termed NEMO-binding domain (NBD) peptide has an inhibitory effect on nuclear factor kappa-B (NF-κB). Despite its efficacy in inhibiting inflammatory responses, the precise neuroprotective mechanisms of NBD peptide in spinal cord injury (SCI) remain unclear. This study aims to determine whether the pyroptosis-related aspects involved in the neuroprotective effects of NBD peptide post-SCI.Using RNA sequencing, the molecular mechanisms of NBD peptide in SCI are explored. The evaluation of functional recovery is performed using the Basso mouse scale, Nissl staining, footprint analysis, Masson's trichrome staining, and HE staining. Western blotting, enzyme-linked immunosorbent assays, and immunofluorescence assays are used to examine pyroptosis, autophagy, lysosomal membrane permeabilization (LMP), acid sphingomyelinase (ASMase), and the NF-κB/p38-MAPK related signaling pathway.NBD peptide mitigated glial scar formation, reduced motor neuron death, and enhanced functional recovery in SCI mice. Additionally, NBD peptide inhibits pyroptosis, ameliorate LMP-induced autophagy flux disorder in neuron post-SCI. Mechanistically, NBD peptide alleviates LMP and subsequently enhances autophagy by inhibiting ASMase through the NF-κB/p38-MAPK/Elk-1/Egr-1 signaling cascade, thereby mitigating neuronal death. NBD peptide contributes to functional restoration by suppressing ASMase-mediated LMP and autophagy depression, and inhibiting pyroptosis in neuron following SCI, which may have potential clinical application value.

Adipose-derived stem cell modulate tolerogenic dendritic cell-induced T cell regulation is correlated with activation of Notch-NFκB signaling

BackgroundAdipose-derived stem cells (ASCs) are recognized for their potential immunomodulatory properties. In the immune system, tolerogenic dendritic cells (DCs), characterized by an immature phenotype, play a crucial role in inducing regulatory T cells (Tregs) and promoting immune tolerance. Notch1 signaling has been identified as a key regulator in the development and function of DCs. However, the precise involvement of Notch1 pathway in ASC-mediated modulation of tolerogenic DCs and its impact on immune modulation remain to be fully elucidated. This study aims to investigate the interplay between ASCs and DCs, focusing the role of Notch1 signaling and downstream pathways in ASC-modulated tolerogenic DCs. MethodsRat bone marrow-derived myeloid DCs were directly co-cultured with ASCs to generate ASC-treated DCs (ASC-DCs). Notch signaling was inhibited using DAPT, while NFκB pathways were inhibited by NEMO binding domain peptide and si-NIK. Flow cytometry assessed DC phenotypes. Real-time quantitative PCR, Western blotting and immunofluorescence determined the expression of Notch1, Jagged1 and the p52/RelB complex in ASC- DCs. ResultsNotch1 and Jagged1 were highly expressed on both DCs and ASCs. ASC-DCs displayed significantly reduced levels of CD80, CD86 and MHC II compared to mature DCs. Inhibiting the Notch pathway with DAPT reversed the dedifferentiation effects. The percentage of induced CD25+/FOXP3+/CD4+ Tregs decreased when ASC-DCs were treated with DAPT (inhibition of the Notch pathway) and si-NIK (inhibition of the non-canonical NFκB pathway). ConclusionsASCs induce DC tolerogenicity by inhibiting maturation and promoting downstream Treg generation, involving the Notch and NFκB pathways. ASC-induced tolerogenic DCs can be a potential immunomodulatory tool for clinical application.

Prophylactic Effects of NFκB Essential Modulator-Binding Domain Peptides on Bone Infection: An Experimental Study in a Rabbit Model.

IntroductionOsteomyelitis is characterized by intensive inflammatory bone disease and remains a clinical challenge in orthopedic surgery, despite the advances made in medical and surgical therapies. Staphylococcus aureusis a major causative agent of osteomyelitis, causing the progressive inflammatory destruction of bone. Prophylaxis of osteomyelitis during orthopedic surgery is necessary. NFκB essential modulator–binding domain (NBD) peptides are cell-permeable peptide inhibitors of the IκB-kinase complex. The prophylactic effect of NBD peptides in relieving inflammation and inhibiting bone defects in osteomyelitis is still under investigation. Our purpose was to determine the preventive effect of NBD peptides in S. aureus infection–induced bone defects in osteomyelitis.MethodsAn S. aureus osteomyelitis rabbit model was used in this study. The rabbits were divided into four groups: NBD, cefazolin, control, and PBS. Clinical and laboratory indicators of erythrocyte-sedimentation rate, CRP, and TNFα levels were assessed to monitor systemic reactions. The efficacy of NBD peptides in S. aureus–induced osteomyelitis was evaluated by radiological, histological, and microbiological examinations, immunohistochemistry, immunofluorescence, and micro-CT scans.ResultsIn general, NBD peptides effectively reduced clinical signs in rabbits when compared with the control group. Radiography indicated that there was more severe osteomyelitis in the bacterium-infection control group. There was no significance between cefazolin- and NBD-group average scores. The histological results of the lesion slices further confirmed different severity among the groups. Additionally, significant pathological differences were found between the cefazolin and NBD groups, and the PBS group showed no obvious pathological changes.ConclusionProphylactic administration of NBD peptides to bone-defect areas inhibited bacterial spread and promoted bone regeneration, making NBD peptides a possible treatment option for prophylaxis in bone infections.

Regulatory subunit NEMO promotes polyubiquitin-dependent induction of NF-κB through a targetable second interaction with upstream activator IKK2

Canonical NF-κB signaling through the inhibitor of κB kinase (IKK) complex requires induction of IKK2/IKKβ subunit catalytic activity via specific phosphorylation within its activation loop. This process is known to be dependent upon the accessory ubiquitin (Ub)-binding subunit NF-κB essential modulator (NEMO)/IKKγ as well as poly-Ub chains. However, the mechanism through which poly-Ub binding serves to promote IKK catalytic activity is unclear. Here, we show that binding of NEMO/IKKγ to linear poly-Ub promotes a second interaction between NEMO/IKKγ and IKK2/IKKβ, distinct from the well-characterized interaction of the NEMO/IKKγ N terminus to the “NEMO-binding domain” at the C terminus of IKK2/IKKβ. We mapped the location of this second interaction to a stretch of roughly six amino acids immediately N-terminal to the zinc finger domain in human NEMO/IKKγ. We also showed that amino acid residues within this region of NEMO/IKKγ are necessary for binding to IKK2/IKKβ through this secondary interaction in vitro and for full activation of IKK2/IKKβ in cultured cells. Furthermore, we identified a docking site for this segment of NEMO/IKKγ on IKK2/IKKβ within its scaffold-dimerization domain proximal to the kinase domain–Ub-like domain. Finally, we showed that a peptide derived from this region of NEMO/IKKγ is capable of interfering specifically with canonical NF-κB signaling in transfected cells. These in vitro biochemical and cell culture–based experiments suggest that, as a consequence of its association with linear poly-Ub, NEMO/IKKγ plays a direct role in priming IKK2/IKKβ for phosphorylation and that this process can be inhibited to specifically disrupt canonical NF-κB signaling.

Andrographolide sulfonate attenuates alveolar hypercoagulation and fibrinolytic inhibition partly via NF-κB pathway in LPS-induced acute respiratory distress syndrome in mice

BackgroundAlveolar hypercoagulation and fibrinolytic inhibition are important characteristics during acute respiratory distress syndrome (ARDS), and NF-κB p65 signaling pathway is involved to regulate these pathophysiologies. We hypothesize that targeting NF-κB signal pathway could ameliorate alveolar hypercoagulation and fibrinolyitc inhibition, thus attenuating lung injury in ARDS. PurposeWe explore the efficacy and the potential mechanism of andrographolide sulfonate (Andro-S) on alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS in mice. MethodsARDS was made by lipopolysaccharide (LPS) inhalation in C57BLmice. Andrographolide sulfonate (2.5, 5 and 10 mg/kg) was intraperitoneally given to the mice (once a day for three consecutive days) before LPS administration. NEMO binding domain peptide (NBD), an inhibitor of NF-κB, was used as the positive control and it replaced Andro-S in mice of NBD group. Mice in normal control received saline instead of LPS. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis of alveolar coagulation, fibrinolytic inhibition as well as of pulmonary inflammatory response after 8 h of LPS inhalation. NF-κB signal pathway in lung tissue was simultaneously determined. ResultsAndro-S dose-dependently inhibited tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 expressions either in mRNA or in protein in lung tissue of ARDS mice, and it also decreased the concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) while promoting the production of activated protein C (APC) in BALF. Meanwhile, Andro-S effectively inhibited inflammatory response (interleukin 1β and myeloperoxidase) induced by LPS. LPS stimulation dramatically activated NF-κB signal pathway, indicated by increased expressions of phosphorylation of p65 (p-p65), p-IKKα/β and p-IκBα and the higher p65-DNA binding activity, which were all dose-dependently reversed by Andro-S. Andro-S and NBD presented similar efficacies. ConclusionsAndro-S treatment improves alveolar hypercoagulation and fibrinolytic inhibition and attenuates pulmonary inflammation in LPS-induced ARDS in mice partly through NF-κB pathway inactivation. The drug is expected to be an effective choice for ARDS.

Selective targeting of the TLR2/MyD88/NF-\u03baB pathway reduces \u03b1-synuclein spreading in vitro and in vivo

Pathways to control the spreading of α-synuclein (α-syn) and associated neuropathology in Parkinson’s disease (PD), multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) are unclear. Here, we show that preformed α-syn fibrils (PFF) increase the association between TLR2 and MyD88, resulting in microglial activation. The TLR2-interaction domain of MyD88 (wtTIDM) peptide-mediated selective inhibition of TLR2 reduces PFF-induced microglial inflammation in vitro. In PFF-seeded A53T mice, the nasal administration of the wtTIDM peptide, NEMO-binding domain (wtNBD) peptide, or genetic deletion of TLR2 reduces glial inflammation, decreases α-syn spreading, and protects dopaminergic neurons by inhibiting NF-κB. In summary, α-syn spreading depends on the TLR2/MyD88/NF-κB pathway and it can be reduced by nasal delivery of wtTIDM and wtNBD peptides.

Pterostilbene-isothiocyanate inhibits breast cancer metastasis by selectively blocking IKK-β/NEMO interaction in cancer cells

Metastasis, the main cause of breast cancer-associated fatalities, relies on many regular pathways involved in normal cell physiology and metabolism, thus, making it challenging to identify disease-specific therapeutic target(s). Chemically synthesized anti-metastatic agents are preferred for their fast and robust actions. However, these agents have adverse side effects, thus, increasingly favouring the identification of phytocompounds as suitable alternatives. Resveratrol and pterostilbene have long been established as potent anti-cancer agents. Earlier studies from our laboratory documented the anti-cancer activities associated with pterostilbene-isothiocyanate (PTER-ITC), a derivative of pterostilbene. The current study focuses on evaluating the anti-metastatic property of PTER-ITC and the underlying mechanism, by employing in silico, in vitro, and in vivo approaches. The significant anti-metastatic activity of PTER-ITC was observed in vitro against breast cancer metastatic cell line (MDA-MB-231) and in vivo in the 4T1 cell-induced metastatic mice model. Epithelial-mesenchymal transition (EMT), a hallmark of metastasis regulated by the transcription factors, Snail1 and Twist, was found to be reverted in vitro by PTER-ITC treatment. PTER-ITC blocked the activation of NF-κB/p65 and its concomitant nuclear translocation, resulting in the transcriptional repression of its target genes, Snail1 and Twist. PTER-ITC prevented the formation of IKK complex, central to NF-κB activation, by binding to the NEMO-binding domain (NBD) of IKK-β and inhibiting its interaction with NEMO (NF-κB essential modulator). According to our observations, PTER-ITC attenuated NF-κB activation selectively in cancerous cells. In conclusion, this study demonstrated that PTER-ITC is a potent anti-metastatic agent capable of targeting physiologically important pathways in a cancer-specific manner.

NF-κB and neutrophil extracellular traps cooperate to promote breast cancer progression and metastasis

Aberrant NF-κB activation and neutrophil extracellular traps (NETs) are associated with breast cancer progression. How NF-κB and NETs modulate each other in breast cancer development remains unclear. Here, we found that NETs induced by phorbol 12-myristate 13-acetate promote breast cancer cell progression. In turn, cancer cells–derived factors, such as IL-8 and granulocyte colony-stimulating factor, stimulate neutrophils to form NETs. Mechanistically, NETs increased the interaction of NF-κB essential modifier (NEMO) with IκB kinase (IKK)α/β and enhanced NF-κB activation. We then employed a cell-permeable peptide corresponding to the NEMO-binding domain (NBD) of IKKα/β, termed NBD peptide, which disrupts NETs-mediated NEMO interaction with IKKα/β and abolished NF-κB activation in vitro. NBD peptide also reduced IL-8 level and NETs formation, and suppressed primary tumor growth and/or lung metastasis in human breast cancer mouse xenograft models and mouse spontaneous breast cancer model. Blockade of NET formation using a peptidylarginine deiminase 4 (PAD4) pharmacologic inhibitor decreased NF-κB activation and tumor metastasis. Collectively, these data suggest that NF-κB associates with NETs to form a positive loop facilitating breast tumor progression and metastasis, and that selective inhibition of NF-κB and PAD4-dependent NETs provides an effective therapeutic approach for treating breast cancer.

Triptolide dose-dependently improves LPS-induced alveolar hypercoagulation and fibrinolysis inhibition through NF-κB inactivation in ARDS mice

BackgroundAlveolar hypercoagulation and fibrinolysis inhibition were associated with the refractory hypoxemia and the high mortality in patient with acute respiratory distress syndrome (ARDS), and NF-κB pathway was confirmed to contribute to the process. Triptolide (TP) significantly inhibited NF-κB pathway and thus depressed accessive inflammatory response in ARDS. We speculate that TP could improve alveolar hypercoagulation and fibrinolytic inhibition in LPS-induced ARDS via NF-κB inactivation. PurposeThe aim of this experiment was to explore the efficacy and potential mechanism of TP on alveolar hypercoagulation and fibrinolysis inhibition in LPS-induced ARDS in mice. Methods50 μl of LPS (5 mg/ml) was inhalationally given to C57BL/6 mice to set up ARDS model. Male mice were randomly accepted with LPS, LPS + TP (1 μg/kg, 10 μg/kg, 50 μg/kg respectively), or with NEMO Binding domain peptide (NBD), an inhibitor of NF-κB. TP (1 μg/kg, 10 μg/kg, 50 μg/kg) were intraperitoneally injected or 10 μg/50 μl of NBD solution were inhaled 30 min before LPS inhalation. A same volume of normal saline (NS) substituted for TP in mice in control. The endpoint of experiment was at 8 hours after LPS stimulation. Pulmonary tissues were taken for hematoxylin-eosin (HE) staining, wet / dry ratio and for lung injury scores (LIS). Tissue factor (TF) and plasminogen activator inhibitor (PAI)-1 in lung tissue were detected by Western-blotting and by quantitative Real-time PCR(qPCR) respectively. Concentrations of TF, PAI-1, thrombin-antithrombin complex (TAT), procollagen peptide type Ⅲ (PⅢP) and activated protein C (APC) in bronchoalveolar lavage fluid (BALF) were measured by ELISA. NF-κB activation and p65-DNA binding activity in pulmonary tissue were simultaneously determined. ResultsLPS stimulation resulted in pulmonary edema, neutrophils infiltration, obvious alveolar collapse, interstitial congestion, with high LIS, which were all dose-dependently ameliorated by Triptolide. LPS also dramatically promoted the expressions of TF and PAI-1 either in mRNA or in protein in lung tissue, and significantly stimulated the secretions of TF, PAI-1, TAT, PⅢP but inhibited APC production in BALF, which were all reversed by triptolide treatment in dose-dependent manner. TP dose-dependently inhibited the activation of NF-κB pathway induced by LPS, indicated by the changes of phosphorylations of p65 (p-p65), p-IKKα/β and p-IκBα, and weakened p65-DNA binding activity. TP and NBD had same efficacies either on alveolar hypercoagulation and fibrinolysis inhibition or on NF-κB signalling pathway in ARDS mice. ConclusionsTP dose-dependently improves alveolar hypercoagulation and fibrinolysis inhibition in ARDS mice through inhibiting NF-κB signaling pathway. Our data demonstrate that TP is expected to be an effective selection in ARDS.

Impact and mechanism of NEMO binding domain peptide on pulmonary inflammation and apoptosis of lung tissues in mice with acute respiratory distress syndrome

To investigate the effect of NEMO binding domain peptide (NBDP) on lung inflammation and apoptosis in mice with acute respiratory distress syndrome (ARDS) and its mechanism. Thirty-six male BALB/c mice were divided into normal saline (NS) control group, ARDS model group, NBDP negative control group and 6, 12 and 18 μg NBDP pretreatment group by random number table method, with 6 mice in each group. ARDS mouse model was reproduced by aerosol inhalation lipopolysaccharide (LPS) 50 μL. An equivalent among of NS was inhaled in NS control group. The mice in NBDP negative control group were inhaled the materials similar to the non-functional NBDP 30 minutes before the aerosol inhalation LPS; 6, 12 and 18 μg of NBDP 50 μL were respectively inhaled in NBDP pretreatment groups. After inhalation of LPS for 6 hours, mice were sacrificed to get lung tissue and observe the degree of pathological injury and edema. Western blotting was used to detect the phosphorylation of nuclear factor-κB (NF-κB) pathway related proteins [NF-κB inhibitor (IκB) kinaseα/β(IKKα/β), IκBα and NF-κB p65; p-IKKα/β, p-IκBα, p-p65] and the expression of caspase-3 in lung tissue. The bronchoalveolar lavage fluid (BALF) was collected and the levels of inflammatory markers such as myeloperoxidase (MPO), interleukins (IL-1β, IL-8), and tumor necrosis factor-α (TNF-α) were detected by enzyme linked immunosorbent assay (ELISA). ARDS model group had severe edema and hemorrhage, alveolar structure destruction, pulmonary hemorrhage and hyaline membrane formation etc. under light microscope, consistent with the pathological characteristics of ARDS lung tissue, suggesting that the ARDS model was successfully reproduced. ELISA showed that MPO, IL-1β, IL-8 and TNF-α levels of BALF in ARDS model group were obviously higher than those in NS control group. There were no significant differences in the above inflammatory indicators between NBDP negative control group and ARDS model group. The levels of MPO, IL-1β, IL-8 and TNF-α in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner, especially in 18 μg NBDP, the differences were statistically significant as compared with ARDS model group [MPO (ng/L): 393.32±19.35 vs. 985.87±101.50, IL-1β (ng/L): 43.05±5.11 vs. 97.68±10.88, IL-8 (ng/L): 84.64±2.32 vs. 204.00±17.37, TNF-α (ng/L): 229.13±17.03 vs. 546.73±62.72, all P < 0.05]. Western blotting showed that p-IKKα/β, p-IκBα, p-p65 and caspase-3 protein expressions in ARDS model group were significantly higher than those in NS control group. There was no significant difference in above NF-κB pathway and apoptosis-related protein expression between the NBDP negative control group and ARDS model group. The p-IKKα/β, p-IκBα, p-p65 and caspase-3 protein expression in NBDP pretreatment groups were significantly lower than those in ARDS model group in a dose-dependent manner, especially in 18 μg NBDP, the differences were statistically significant as compared with ARDS model group [p-IKKα/β protein (p-IKKα/β/β-actin): 0.15±0.02 vs. 0.42±0.04, p-IκBα protein (p-IκBα/β-actin): 0.10±0.01 vs. 0.93±0.30, p-p65 protein (p-p65/β-actin): 0.22±0.05 vs. 1.37±0.21, all P < 0.05]. NBDP can inhibit inflammatory response and apoptosis in ARDS lung tissue in a dose-dependent manner, and its mechanism is associated with interference NF-κB signaling pathway transduction.

NF-κB Blockade by NEMO Binding Domain Peptide Ameliorates Inflammation and Neurobehavioral Sequelae After Cranial Radiation Therapy in Juvenile Mice

Cranial radiation therapy (CRT) is a common treatment for pediatric brain tumor patients. However, side effects include significant neurobehavioral dysfunction in survivors. This dysfunction may in part be caused by inflammation, including increased production of tumor necrosis factor alpha (TNFα) and its receptor TNFR1, which can activate the nuclear factor kappa light-chain enhancer of activated B cells (NF-κB). The TNFα blockade abrogates this inflammatory response, although it presents immunologic risks. Thus, modulation of pathway subsets may be preferable. Here, we test whether inhibition of NF-κB activation using an NF-κB essential modulator binding domain (NBD) peptide mitigates CRT-induced neuroinflammation and improves behavioral outcomes. Male C57BL/6J 28-day old mice were randomized to saline (sham), 5 Gy whole-brain CRT, or CRT + NBD-peptide. Brain tissue was collected after 4 hours or 3 months for Western blot or immunohistochemistry. The cortex, corpus callosum (CC), and dentate gyrus were variably imaged for NF-κB-p65, IκBα, proliferation, apoptosis, necroptosis, TNFα, TNFR1, IBA-1, doublecortin, CD11c, and GFAP. Neurobehavioral changes were assessed by open field and elevated plus maze tests 3 months post-CRT. NF-κB expression increased in whole and nuclear fractions 4 hours after CRT and was abrogated by NBD treatment. Cell death increased and proliferation decreased after CRT, including within neuronal progenitors, with some loss mitigated by NBD. Increased levels of TNFα, IBA-1, and GFAP were found in the CC and cortex months after CRT and were limited by NBD. The anti-NF-κB peptide also improved neurobehavioral assessments, yielding improvements in anxiety and exploration. Results suggest a role for NF-κB modulation by NBD peptide in the reduction of neuroinflammation and mitigation of behavioral complications after pediatric radiation therapy.

Abstract IA46: Molecular and genetic profiling in canine lymphoma unravels targets for the human counterpart

Abstract Canine lymphoma represents an excellent cancer model for the human counterpart and is the most widely investigated tumor both in veterinary and comparative oncology. Knowledge of the diagnosis, molecular biology, genetic, and epigenetic has progressively grown in recent years, but prognosis in both B- and T-cell lymphoma remains poor. Several lymphoma subtypes are recognized in canine species and diffuse large B-cell lymphoma (DLBCL) is the most frequent, followed by marginal zone lymphoma (MZL) and a peculiar T-cell malignancy named T-zone lymphoma. DLBCL resembles in many elements the human counterpart, including frequency, molecular abnormalities, and similar therapeutic responses to chemotherapy. However, many other aspects are still scarcely known, and a complete characterization becomes fundamental when including dogs with this lymphoma in clinical trials for new molecules. Indeed, the inability to accurately predict outcome poses several limitations when testing efficacy of new drugs. With NGS advent and the dramatic reduction of costs, researchers in veterinary medicine have started to describe canine lymphoma in a similar fashion to rodent models. The aims of this presentation are to provide an overview of the most significant results in canine lymphoma that have been published in the last decade, focusing on molecular and therapeutic aspects of this tumor. About B-cell lymphoma, transcriptomic studies in canine DLBCL, MZL, and follicular lymphoma (FL) have revealed similarities with the human histotypes. Genes involved in B-cell receptor signaling were found to be enriched both in canine DLBCL and MZL, and several Toll-like receptors were overexpressed, suggesting mechanisms associated with human activated B-cell (ABC)-like subtype DLBCL pathogenesis. The aberrant expression of these genes seems to contribute to enhancing proliferation and protection of canine B cells from apoptosis by stimulating NF-κB activity. The biologic role of NF-kB was tested in dogs with DLBCL using NEMO-binding domain peptide in two clinical trials and the compound resulted in safe blocking of NF-kB activity. Previously, treatment with Ibrutinib was found effective in dogs with B-cell lymphoma. Also, genes coding for proteins involved in MYC signaling and PI3K/AKT/mTOR pathway were found to be overexpressed in both DLBCL and MZL. A peculiar MYC deregulation controlled by LIN28B/Let-7 axis was identified only in DLBCL and LIN28B resulted the most upregulated gene both in vivo and in vitro, revealing an important functional relevance and opening new scenarios for treatment of this tumor in dogs. Within this context, the pan-BET inhibitor OTX015, targeting BRD4, was able to reduce proliferation and expression of MYC-associated genes in vitro. When investigating the cell of origin of canine DLBCL, a study from our group showed a segregation of this lymphoma in two transcriptomic subgroups that were further characterized by distinct methylation profiles and associated with survival. The dogs with inferior survival showed a higher expression of transcripts involved in T-cell and macrophage regulation (CD163, CD96, PD-1, PDL-1, CTLA4, CD8a, CD4). Recently, a study explored the mutation profile of canine B- and T-cell lymphoma in three pure breeds. B-cell lymphomas were categorized based on their mutation profile, and TRAF3, BIRC3, and MAP3K14 were found to be frequently mutated. For TRAF3, a combination of somatic frameshift mutations and truncating SNVs in causing down-expression was identified and germ-line mutations were retrieved in some dogs, possibly indicating that inherited mutations in this gene contribute to genetic predisposition to the tumor. In future, we expect an increasing number of preclinical trials including data from dogs with different lymphoma histotypes, but a detailed pathologic and molecular classification of the tumors is definitively needed to avoid biases related to the heterogeneity of this disease. Inclusion of dogs with lymphoma as part of preclinical testing or in parallel with human trials will be a serious advantage both for human and veterinary medicine. However, limitations, including that dogs are from private owners and toxicities, should always be considered. Citation Format: Luca Aresu. Molecular and genetic profiling in canine lymphoma unravels targets for the human counterpart [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA46.

Fish IKKα from Japanese eel (Anguilla japonica) can activate NF-κB, AP1, and type I IFN signaling pathways

Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays a pivotal role in the activation of nuclear factor kappa-B (NF-κB) pathway in response to pathogens infections in mammals, but the information about IKKα in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IKKα homologue, AjIKKα, was cloned by 5′ and 3′ SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIKKα GFP fusion protein and the induction of AjIKKα in the activation of NF-κB, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIKKα has typical conserved domains, including an N-terminal kinase domain, an ubiquitin-like domain, a scaffold dimerization domain, and a C-terminal NEMO-binding domain. The predicted three-dimensional structure of AjIKKα is similar to that of human IKKα. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjIKKα in a wide range of tissues, with the highest expression in the liver, followed by the intestine, gills, and spleen, and with a lower expression in the muscle and heart. The AjIKKα expressions in the liver and kidney were significantly induced following injection with the viral mimic poly I:C and Aeromonas hydrophila infection, whereas the bacterial mimic LPS down-regulated the expression of AjIKKα in the spleen. In vitro, the AjIKKα transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of different concentration of Aeromonas hydrophila (1 × 106 cfu/mL, 1 × 107 cfu/mL, and 1 × 108 cfu/mL). Luciferase assays demonstrated that AjIKKα expression could significantly induce NF-κB, AP-1 and type I IFN promoter activation in a dose-dependent manner. Additionally, subcellular localization studies showed that AjIKKα was evenly distributed in the cytoplasm in the natural state, but AjIKKα was found to aggregate into spots in the cytoplasm after the stimulation of LPS and poly I:C. These results collectively indicated that AjIKKα plays an important role in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB, AP1and type I IFN signaling pathway.

Intercellular delivery of NF-κB inhibitor peptide utilizing small extracellular vesicles for the application of anti-inflammatory therapy

Excessive activation of NF-κB in macrophages contributes to the onset and exacerbation of inflammatory disorders. The NEMO binding domain (NBD) peptide is an NF-κB inhibitor peptide that binds to NEMO, one of components of the IκB kinase (IKK) complex, and inhibits the IKK kinase activity, in the cytosol. Because of this property, the NBD peptide is expected to inhibit NF-κB activation in macrophages. In this study, we developed a delivery carrier for NBD based on small extracellular vesicles (sEVs), which are membrane vesicles released from cells. We constructed fusion proteins comprising Gag (an sEV tropic protein) and one, three, or six repeats of NBD peptide (Gag-1NBD, Gag-3NBD, and Gag-6NBD, respectively) to load the NBD peptide to the inner space of the sEVs, and attempted the intracellular delivery of the NBD peptide to macrophages using Gag-NBD-loaded sEVs (nNBD-sEVs). The nNBD-sEVs significantly inhibited LPS-induced phosphorylation of NF-κB pathway-related proteins in macrophages in a repeat number-dependent manner. Moreover, they exerted inhibitory effects on the NF-κB-dependent expression of proinflammatory mediators such as TNFα, CXCL10, iNOS, and NO. Collectively, our results indicate that NBD-containing sEVs can be used for the treatment of inflammatory diseases owing to their ability to effectively deliver peptides to the macrophage cytosol.

Targeting NF-κB by the Cell-Permeable NEMO-Binding Domain Peptide Improves Albuminuria and Renal Lesions in an Experimental Model of Type 2 Diabetic Nephropathy.

Diabetic nephropathy (DN) is a multifactorial disease characterized by hyperglycemia and close interaction of hemodynamic, metabolic and inflammatory factors. Nuclear factor-κB (NF-κB) is a principal matchmaker linking hyperglycemia and inflammation. The present work investigates the cell-permeable peptide containing the inhibitor of kappa B kinase γ (IKKγ)/NF-κB essential modulator (NEMO)-binding domain (NBD) as therapeutic option to modulate inflammation in a preclinical model of type 2 diabetes (T2D) with DN. Black and tan, brachyuric obese/obese mice were randomized into 4 interventions groups: Active NBD peptide (10 and 6 µg/g body weight); Inactive mutant peptide (10 µg/g); and vehicle control. In vivo/ex vivo fluorescence imaging revealed efficient delivery of NBD peptide, systemic biodistribution and selective renal metabolization. In vivo administration of active NBD peptide improved albuminuria (>40% reduction on average) and kidney damage, decreased podocyte loss and basement membrane thickness, and modulated the expression of proinflammatory and oxidative stress markers. In vitro, NBD blocked IKK-mediated NF-κB induction and target gene expression in mesangial cells exposed to diabetic-like milieu. These results constitute the first nephroprotective effect of NBD peptide in a T2D mouse model that recapitulates the kidney lesions observed in DN patients. Targeting IKK-dependent NF-κB activation could be a therapeutic strategy to combat kidney inflammation in DN.

NF-κB inhibitor, NEMO-binding domain peptide attenuates intervertebral disc degeneration

BACKGROUND CONTEXTNonphysiological mechanical loading and inflammation are both critically involved in intervertebral disc (IVD) degeneration, which is characterized by an increase in cytokines and matrix metalloproteases (MMPs) in the nucleus pulposus (NP). This process is known to be mediated by the NF-κB pathway. CLINICAL SIGNIFICANCECurrent clinical treatments for IVD degeneration focus on the alleviation of symptoms rather than targeting the underlying mechanism. Injection of an NF-κB inhibitor may attenuate the progression of IVD degeneration. PURPOSETo investigate the ability of the NF-κB inhibitor, NEMO binding domain peptide (NBD), to alter IVD degeneration processes by reducing IL-1β- and mechanically-induced cytokine and MMP levels in human nucleus pulposus cells in vitro, and by attenuating IVD degeneration in an in vivo rat model for disc degeneration. STUDY DESIGNExperimental in vitro and animal model. PATIENT SAMPLEDiscarded specimens of lumbar disc from 21 patients, and 12 Sprague Dawley rats. OUTCOME MEASURESGene and protein expression, cell viability, µMRI and histology. METHODSIL-1β-prestimulated human nucleus pulposus cells embedded into fibrin constructs were loaded in the Flexcell FX-5000 compression system at 5 kPa and 1 Hz for 48 hours in the presence and absence of NBD. Unloaded hNPC/fibrin constructs served as controls. Cell viability in loaded and unloaded constructs was quantified, and gene and protein expression levels determined. For in vivo testing, a rat needle disc puncture model was employed. Experimental groups included injured discs with and without NBD injection and uninjured controls. Levels of disc degeneration were determined via µMRI, qPCR and histology. Funding sources include $48,874 NASS Young Investigator Research Grant and $119,174 NIH 5K01AR071512-02. There were no applicable financial relationships or conflicts of interest. RESULTSMechanical compression of hNPC/fibrin constructs resulted in upregulation of MMP-3 and IL-8. Supplementation of media with 10 μM NBD during loading increased cell viability, and decreased MMP-3 gene and protein levels. IVD injury in rat resulted in an increase in MMP-3, IL-1β and IL-6 gene expression. Injections of 250 µg of NBD during disc injury resulted in decreased IL-6 gene expression. µMRI analysis demonstrated a reduction of disc hydration in response to disc needle injury, which was attenuated in NBD-treated IVDs. Histological evaluation showed NP and AF lesion in injured discs, which was attenuated by NBD injection. CONCLUSIONSThe results of this study show NBD peptide's capacity to reduce IL-1β- and loading-induced MMP-3 levels in hNPC/fibrin constructs while increasing the cells’ viability, and to attenuate IVD degeneration in rat, involving downregulation of IL-6. Therefore, NBD may be a potential therapeutic agent to treat IVD degeneration.

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα.

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.

Selective Susceptibility of Human Bladder Transitional Cell Carcinoma T24 Cells towards NBD Peptide

Bladder cancer is a recurrent disease that often develops chemoresistance and is on the rise in developing countries. After diagnosis surgery followed by immunotherapy and/or chemo- radiation therapy are the available treatments. These treatments do not provide protection against disease progression or recurrence thus highlighting the need to identify and develop drugs that are capable of inhibiting bladder cancer. T24 cell line (human bladder transitional cell carcinoma) was treated with NEMO Binding Domain (NBD) peptide and tested for its cytotoxicity, cell cycle inhibition, apoptosis and migration, in vitro. The cytotoxicity of NBD peptide was tested on non-cancerous cells (NIH-3T3-L1, CHO, Vero) and the peptide localization in T24 cells was confirmed using confocal microscopy. The effects of NBD peptide and the standard drugs (cisplatin, gemcitabine) were compared with untreated control. Peptide based chemotherapy for human bladder cells was tested that include combinations of NBD and standard drugs. Statistical significance was assessed between the different treatment groups. The viability of T24 cells after NBD peptide treatment was reduced to 50% at a dose of 18.5 µM after 48 h. The NBD peptide elicited selective cytotoxicity dose-dependently in T24 cells while being non-toxic on normal (non-cancer) cell lines. Sub G0-G1 accumulation of T24 cells was seen where they underwent necrosis and lost the ability to migrate when treated with 100 µM NBD. T24 cell death increased to ~78% when treated with a combination of NBD with cisplatin and gemcitabine (standard drugs) as opposed to ~50% when treated with NBD peptide alone. NBD peptide exhibits specific anti-cancer activity on T24 cells in vitro and found to be non-cytotoxic to select normal cell lines. Combining NBD with standard drugs demonstrated excellent inhibition of malignant growth than the individual anti-neoplastic agents.

Efficacy of NEMO-binding domain peptide used to treat experimental osteomyelitis caused by methicillin-resistant Staphylococcus aureus: an in-vivo study

PurposeTreatment of chronic osteomyelitis (bone infection) remains a clinical challenge. Our previous study had demonstrated that NEMO-binding domain (NBD) peptide effectively ameliorates the inhibition of osteoblast differentiation by TNF-α in vitro. In this work, NBD peptide was evaluated in vivo for treating chronic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit model.MethodsTibial osteomyelitis was induced in 50 New Zealand white rabbits by tibial canal inoculation of MRSA strain. After 3 weeks, 45 rabbits with osteomyelitis were randomly divided into four groups that correspondingly received the following interventions: 1) Control group (9 rabbits, no treatment); 2) Van group (12 rabbits, debridement and parenteral treatment with vancomycin); 3) NBD + Van group (12 rabbits, debridement and local NBD peptide injection, plus parenteral treatment with vancomycin); 4) NBD group (12 rabbits, debridement and local NBD peptide injection). Blood samples were collected weekly for the measurement of leucocyte count, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. The rabbits in all four groups were sacrificed 6 weeks after debridement; the anti-infective efficacy was evaluated by radiological, histological, and microbiological examination, and promotion of bone remodeling was quantified by micro-CT using the newly formed bone.ResultsExcept two rabbits in the Control group and one in the NBD group that died from severe infection before the end point, the remaining 42 animals (7, 12, 12, 11 in the Control, Van, NBD + Van, and NBD group respectively) were sacrificed 6 weeks after debridement. In general, there was no significant difference in the leucocyte count, and ESR and CRP levels, although there were fluctuations throughout the follow-up period after debridement. MRSA was still detectable in bone tissue samples of all animals. Interestingly, treatment with NBD peptide plus vancomycin significantly reduced radiological and histological severity scores compared to that in other groups. The best therapeutic efficacy in bone defect repair was observed in the NBD peptide + Van group.ConclusionsIn a model of osteomyelitis induced by MRSA, despite the failure in demonstrating antibacterial effectiveness of NBD peptide in vivo, the results suggest antibiotics in conjunction with NBD peptide to possibly have promising therapeutic potential in osteomyelitis.

A Central Region of NF-κB Essential Modulator Is Required for IKKβ-Induced Conformational Change and for Signal Propagation.

NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKβ to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112-195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKβ (amino acids 701-745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKβ compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKβ. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.