Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence

Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.     

Changes in the Expression of the Toll-Like Receptor System in the Aging Rat Kidneys

Background

The mechanisms of kidney aging are not yet clear. Studies have shown that immunological inflammation is related to kidney aging. Toll-like receptors (TLRs) are one of the receptor types of the body''s innate immune system. The function of the TLR system and the mechanisms by which it functions in renal aging remain unclear. In the present study, we, for the first time, systematically investigated the role of the TLR system and the inflammation responses activated by TLRs during kidney aging.

Methods

We used western blot and immunohistochemistry to systematically analyze the changes in the expression and activation of the endogenous TLR ligands HSP70 and HMGB1, the TLRs (TLR1–TLR11), their downstream signaling pathway molecules MyD88 and Phospho-IRF-3, and the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα (NF-κB inhibition factor α), NF-κBp65, and Phospho-NF-κBp65 (activated NF-κB p65) in the kidneys of 3 months old (youth group), 12 months old (middle age group), and 24 months old (elderly group) rats. We used RT-qPCR to detect the mRNA expression changes of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b in the rat renal tissues of the various age groups.

Results

We found that during kidney aging, the HSP70 and HMGB1 expression levels were significantly increased, and the expression levels of TLR1, 2, 3, 4, 5, and 11 and their downstream signaling pathway molecules MyD88 and Phospho-IRF-3 were markedly elevated. Further studies have shown that in the aging kidneys, the expression levels of the NF-κB signaling pathway molecules Phospho-IKKβ, Phospho-IκBα, NF-κBp65, and Phospho-NF-κBp65 were obviously increased, and those of the proinflammatory cytokines CCL3, CCL4, CCL5, CD80, TNF-α, and IL-12b were significantly upregulated.

Conclusions

These results showed that the TLR system might play an important role during the kidney aging process maybe by activating the NF-κB signaling pathway and promoting the high expression of inflammation factors.     

Reciprocal inhibition between miR-26a and NF-κB regulates obesity-related chronic inflammation in chondrocytes

Obesity is causally linked to osteoarthritis (OA), with the mechanism being not fully elucidated. miRNAs (miRs) are pivotal regulators of various diseases in multiple tissues, including inflammation in the chondrocytes. In the present study, we for the first time identified the expression of miR-26a in mouse chondrocytes. Decreased level of miR-26a was correlated to increased chronic inflammation in the chondrocytes and circulation in obese mouse model. Mechanistically, we demonstrated that miR-26a attenuated saturated free fatty acid-induced activation of NF-κB (p65) and production of proinflammatory cytokines in chondrocytes. Meanwhile, NF-κB (p65) also suppressed miR-26a production by directly binding to a predicted NF-κB binding element in the promoter region of miR-26a. Finally, we observed a negative correlation between NF-κB and miR-26a in human patients with osteoarthritis. Thus, we identified a reciprocal inhibition between miR-26a and NF-κB downstream of non-esterified fatty acid (NEFA) signalling in obesity-related chondrocytes. Our findings provide a potential mechanism linking obesity to cartilage inflammation.     

Crotepoxide Chemosensitizes Tumor Cells through Inhibition of Expression of Proliferation,Invasion, and Angiogenic Proteins Linked to Proinflammatory Pathway

Crotepoxide (a substituted cyclohexane diepoxide), isolated from Kaempferia pulchra (peacock ginger), although linked to antitumor and anti-inflammatory activities, the mechanism by which it exhibits these activities, is not yet understood. Because nuclear factor κB (NF-κB) plays a critical role in these signaling pathways, we investigated the effects of crotepoxide on NF-κB-mediated cellular responses in human cancer cells. We found that crotepoxide potentiated tumor necrosis factor (TNF), and chemotherapeutic agents induced apoptosis and inhibited the expression of NF-κB-regulated gene products involved in anti-apoptosis (Bcl-2, Bcl-xL, IAP1,² MCl-1, survivin, and TRAF1), apoptosis (Bax, Bid), inflammation (COX-2), proliferation (cyclin D1 and c-myc), invasion (ICAM-1 and MMP-9), and angiogenesis (VEGF). We also found that crotepoxide inhibited both inducible and constitutive NF-κB activation. Crotepoxide inhibition of NF-κB was not inducer-specific; it inhibited NF-κB activation induced by TNF, phorbol 12-myristate 13-acetate, lipopolysaccharide, and cigarette smoke. Crotepoxide suppression of NF-κB was not cell type-specific because NF-κB activation was inhibited in myeloid, leukemia, and epithelial cells. Furthermore, we found that crotepoxide inhibited TAK1 activation, which led to suppression of IκBα kinase, abrogation of IκBα phosphorylation and degradation, nuclear translocation of p65, and suppression of NF-κB-dependent reporter gene expression. Overall, our results indicate that crotepoxide sensitizes tumor cells to cytokines and chemotherapeutic agents through inhibition of NF-κB and NF-κB-regulated gene products, and this may provide the molecular basis for crotepoxide ability to suppress inflammation and carcinogenesis.     

STING promotes senescence,apoptosis, and extracellular matrix degradation in osteoarthritis via the NF-κB signaling pathway

Damaged deoxyribonucleic acid (DNA) is a primary pathologic factor for osteoarthritis (OA); however, the mechanism by which DNA damage drives OA is unclear. Previous research demonstrated that the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) participates in DNA damage response. As a result, the current study aimed at exploring the role STING, which is the major effector in the cGAS-STING signaling casacde, in OA progress in vitro, as well as in vivo. In this study, the expression of STING was evaluated in the human and mouse OA tissues, and in chondrocytes exposed to interleukin-1 beta (IL-1β). The influences of STING on the metabolism of the extracellular matrix (ECM), apoptosis, and senescence, were assessed in STING overexpressing and knocking-down chondrocytes. Moreover, the NF-κB-signaling casacde and its role in the regulatory effects of STING on ECM metabolism, apoptosis, and senescence were explored. The STING knockdown lentivirus was intra-articularly injected to evaluate its therapeutic impact on OA in mice in vivo. The results showed that the expression of STING was remarkably elevated in the human and mouse OA tissues and in chondrocytes exposed to IL-1β. Overexpression of STING promoted the expression of MMP13, as well as ADAMTS5, but suppressed the expression of Aggrecan, as well as Collagen II; it also enhanced apoptosis and senescence in chondrocytes exposed to and those untreated with IL-1β. The mechanistic study showed that STING activated NF-κB signaling cascade, whereas the blockage of NF-κB signaling attenuated STING-induced apoptosis and senescence, and ameliorated STING-induced ECM metabolism imbalance. In in vivo study, it was demonstrated that STING knockdown alleviated destabilization of the medial meniscus-induced OA development in mice. In conclusion, STING promotes OA by activating the NF-κB signaling cascade, whereas suppression of STING may provide a novel approach for OA therapy.Subject terms: Apoptosis, Senescence     

Polydnaviral Ankyrin Proteins Aid Parasitic Wasp Survival by Coordinate and Selective Inhibition of Hematopoietic and Immune NF-kappa B Signaling in Insect Hosts

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.     

Brap2 Regulates Temporal Control of NF-κB Localization Mediated by Inflammatory Response

Nuclear factor-kappaB (NF-κB) is critical for the expression of multiple genes involved in inflammatory responses and cellular survival. NF-κB is normally sequestered in the cytoplasm through interaction with an inhibitor of NF-κB (IκB), but inflammatory stimulation induces proteasomal degradation of IκB, followed by NF-κB nuclear translocation. The degradation of IκB is mediated by a SCF (Skp1-Cullin1-F-box protein)-type ubiquitin ligase complex that is post-translationaly modified by a ubiquitin-like molecule Nedd8. In this study, we report that BRCA1-associated protein 2 (Brap2) is a novel Nedd8-binding protein that interacts with SCF complex, and is involved in NF-κB translocation following TNF-α stimulation. We also found a putative neddylation site in Brap2 associated with NF-κB activity. Our findings suggest that Brap2 is a novel modulator that associates with SCF complex and controls TNF-α-induced NF-κB nuclear translocation.     

EVM005: An Ectromelia-Encoded Protein with Dual Roles in NF-κB Inhibition and Virulence

Poxviruses contain large dsDNA genomes encoding numerous open reading frames that manipulate cellular signalling pathways and interfere with the host immune response. The NF-κB signalling cascade is an important mediator of innate immunity and inflammation, and is tightly regulated by ubiquitination at several key points. A critical step in NF-κB activation is the ubiquitination and degradation of the inhibitor of kappaB (IκBα), by the cellular SCF^β-TRCP ubiquitin ligase complex. We show here that upon stimulation with TNFα or IL-1β, Orthopoxvirus-infected cells displayed an accumulation of phosphorylated IκBα, indicating that NF-κB activation was inhibited during poxvirus infection. Ectromelia virus is the causative agent of lethal mousepox, a natural disease that is fatal in mice. Previously, we identified a family of four ectromelia virus genes (EVM002, EVM005, EVM154 and EVM165) that contain N-terminal ankyrin repeats and C-terminal F-box domains that interact with the cellular SCF ubiquitin ligase complex. Since degradation of IκBα is catalyzed by the SCF^β-TRCP ubiquitin ligase, we investigated the role of the ectromelia virus ankyrin/F-box protein, EVM005, in the regulation of NF-κB. Expression of Flag-EVM005 inhibited both TNFα- and IL-1β-stimulated IκBα degradation and p65 nuclear translocation. Inhibition of the NF-κB pathway by EVM005 was dependent on the F-box domain, and interaction with the SCF complex. Additionally, ectromelia virus devoid of EVM005 was shown to inhibit NF-κB activation, despite lacking the EVM005 open reading frame. Finally, ectromelia virus devoid of EVM005 was attenuated in both A/NCR and C57BL/6 mouse models, indicating that EVM005 is required for virulence and immune regulation in vivo.     

Novel small molecule inhibition of IKK/NF‐κB activation reduces markers of senescence and improves healthspan in mouse models of aging

Constitutive NF‐κB activation is associated with cellular senescence and stem cell dysfunction and rare variants in NF‐κB family members are enriched in centenarians. We recently identified a novel small molecule (SR12343) that inhibits IKK/NF‐κB activation by disrupting the association between IKKβ and NEMO. Here we investigated the therapeutic effects of SR12343 on senescence and aging in three different mouse models. SR12343 reduced senescence‐associated beta‐galactosidase (SA‐β‐gal) activity in oxidative stress‐induced senescent mouse embryonic fibroblasts as well as in etoposide‐induced senescent human IMR90 cells. Chronic administration of SR12343 to the Ercc1 ^{−/ ∆} and Zmpste24 ^−/− mouse models of accelerated aging reduced markers of cellular senescence and SASP and improved multiple parameters of aging. SR12343 also reduced markers of senescence and increased muscle fiber size in 2‐year‐old WT mice. Taken together, these results demonstrate that IKK/NF‐κB signaling pathway represents a promising target for reducing markers of cellular senescence, extending healthspan and treating age‐related diseases.     

Celastrol,an NF-κB Inhibitor,Improves Insulin Resistance and Attenuates Renal Injury in db/db Mice

The NF-κB pathway plays an important role in chronic inflammatory and autoimmune diseases. Recently, NF-κB has also been suggested as an important mechanism linking obesity, inflammation, and metabolic disorders. However, there is no current evidence regarding the mechanism of action of NF-κB inhibition in insulin resistance and diabetic nephropathy in type 2 diabetic animal models. We investigated the effects of the NF-κB inhibitor celastrol in db/db mice. The treatment with celastrol for 2 months significantly lowered fasting plasma glucose (FPG), HbA1C and homeostasis model assessment index (HOMA-IR) levels. Celastrol also exhibited significant decreases in body weight, kidney/body weight and adiposity. Celastrol reduced insulin resistance and lipid abnormalities and led to higher plasma adiponectin levels. Celastrol treatment also significantly mitigated lipid accumulation and oxidative stress in organs including the kidney, liver and adipose tissue. The treated group also exhibited significantly lower creatinine levels and urinary albumin excretion was markedly reduced. Celastrol treatment significantly lowered mesangial expansion and suppressed type IV collagen, PAI-1 and TGFβ1 expressions in renal tissues. Celastrol also improved abnormal lipid metabolism, oxidative stress and proinflammatory cytokine activity in the kidney. In cultured podocytes, celastrol treatment abolished saturated fatty acid-induced proinflammatory cytokine synthesis. Taken together, celastrol treatment not only improved insulin resistance, glycemic control and oxidative stress, but also improved renal functional and structural changes through both metabolic and anti-inflammatory effects in the kidney. These results suggest that targeted therapy for NF-κB may be a useful new therapeutic approach for the management of type II diabetes and diabetic nephropathy.     

Sulforaphane Attenuates Muscle Inflammation in Dystrophin-deficient mdx Mice via NF-E2-related Factor 2 (Nrf2)-mediated Inhibition of NF-κB Signaling Pathway

Inflammation is widely distributed in patients with Duchenne muscular dystrophy and ultimately leads to progressive deterioration of muscle function with chronic muscle damage, oxidative stress, and reduced oxidative capacity. NF-E2-related factor 2 (Nrf2) plays a critical role in defending against inflammation in different tissues via activation of phase II enzyme heme oxygenase-1 and inhibition of the NF-κB signaling pathway. However, the role of Nrf2 in the inflammation of dystrophic muscle remains unknown. To determine whether Nrf2 may counteract inflammation in dystrophic muscle, we treated 4-week-old male mdx mice with the Nrf2 activator sulforaphane (SFN) by gavage (2 mg/kg of body weight/day) for 4 weeks. The experimental results demonstrated that SFN treatment increased the expression of muscle phase II enzyme heme oxygenase-1 in an Nrf2-dependent manner. Inflammation in mice was reduced by SFN treatment as indicated by decreased infiltration of immune cells and expression of the inflammatory cytokine CD45 and proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the skeletal muscles of mdx mice. In addition, SFN treatment also decreased the expression of NF-κB(p65) and phosphorylated IκB kinase-α as well as increased inhibitor of κB-α expression in mdx mice in an Nrf2-dependent manner. Collectively, these results show that SFN-induced Nrf2 can alleviate muscle inflammation in mdx mice by inhibiting the NF-κB signaling pathway.