Jeremy S. Tilstra

NF-κB inhibition delays DNA damage–induced senescence and aging in mice

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Muscle-derived stem/progenitor cell dysfunction limits healthspan and lifespan in a murine progeria model

With ageing, there is a loss of adult stem cell function. However, there is no direct evidence that this has a causal role in ageing-related decline. We tested this using muscle-derived stem/progenitor cells (MDSPCs) in a murine progeria model. Here we show that MDSPCs from old and progeroid mice are defective in proliferation and multilineage differentiation. Intraperitoneal administration of MDSPCs, isolated from young wild-type mice, to progeroid mice confer significant lifespan and healthspan extension. The transplanted MDSPCs improve degenerative changes and vascularization in tissues where donor cells are not detected, suggesting that their therapeutic effect may be mediated by secreted factor(s). Indeed, young wild-type-MDSPCs rescue proliferation and differentiation defects of aged MDSPCs when co-cultured. These results establish that adult stem/progenitor cell dysfunction contributes to ageing-related degeneration and suggests a therapeutic potential of post-natal stem cells to extend health.

Ethyl pyruvate decreases HMGB1 release and ameliorates murine colitis

Signals from stressed cells and the enteric microbiota activate macrophages and dendritic cells and mediate intestinal inflammation. HMGB1 serves as an immunogenic stimuli causing release of inflammatory cytokines by myeloid cells. Ethyl pyruvate inhibits secretion of HMGB1 and improves survival in models of endotoxemia and hemorrhagic shock. We reasoned that ethyl pyruvate may be protective in colitis, which involves similar inflammatory pathways. In IL‐10−/− mice with established chronic colitis, ethyl pyruvate administration ameliorated colitis and reduced intestinal cytokine production. IL‐10−/− mice demonstrated increased intestinal HMGB1 expression and decreased expression of RAGE compared with wild‐type mice. Fecal HMGB1 levels were decreased in ethyl pyruvate‐treated mice. Furthermore, ethyl pyruvate induced HO‐1 expression in intestinal tissue. In TNBS‐induced colitis, intrarectal administration of ethyl pyruvate resulted in amelioration of colitis and reduced intestinal cytokine production. In LPS‐activated murine macrophages, ethyl pyruvate decreased expression of IL‐12 p40 and NO production but did not affect IL‐10 levels. Ethyl pyruvate did not inhibit nuclear translocation of NF‐κB family members but attenuated NF‐κB DNA binding. Additionally, ethyl pyruvate induced HO‐1 mRNA and protein expression and HO‐1 promoter activation. Moreover, ethyl pyruvate prevented nuclear‐to‐cytoplasmic translocation of HMGB1. In conclusion, the HMGB1/RAGE pathway has pathophysiologic and diagnostic significance in experimental colitis. Ethyl pyruvate and other strategies to inhibit HMGB1 release and function represent promising interventions in chronic inflammatory diseases.

Amelioration of Chronic Murine Colitis by Peptide-Mediated Transduction of the IκB Kinase Inhibitor NEMO Binding Domain Peptide

The NF-κB family of transcription factors is a central regulator of chronic inflammation. The phosphorylation of IκB proteins by the IκB kinase (IKK) complex (IKKα, IKKβ, and NF-κB essential modulator or NEMO) is a key step in NF-κB activation. Peptides corresponding to the NEMO binding domain (NBD) of IKK blocks NF-κB activation without inhibiting basal NF-κB activity. In this report, we determined the effects of the IKK inhibitor peptide (NBD) in a model of spontaneously occurring chronic murine colitis, the IL-10-deficient (IL-10−/−) mouse. Using a novel cationic peptide transduction domain (PTD) consisting of eight lysine residues (8K), we were able to transduce the NBD peptide into cells and tissues. In a NF-κB reporter system, 8K-NBD dose-dependently inhibits TNF-induced NF-κB activation. Furthermore, 8K-NBD inhibited nuclear translocation of NF-κB family members. In NF-κBEGFP knock-in mice, 8K-NBD inhibited LPS-activated NF-κB (EGFP activity) in the ileum but did not inhibit basal NF-κB in Peyer’s patches. IL-10−/− mice treated systemically with 8K-NBD demonstrate amelioration of established colitis, decreased NF-κB activation in the lamina propria, and a reduction in spontaneous intestinal IL-12 p40, TNF, IFN-γ, and IL-17 production. These results demonstrate that inhibitors of IKK, in particular a PTD-NBD peptide, may be therapeutic in the treatment of inflammatory bowel disease.

Protein transduction: identification, characterization and optimization

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly employed to deliver biologically active agents to a variety of cell types in vitro and in vivo. In addition to the previously characterized arginine-rich PTDs, including Tat (transactivator of transcription), Antp (Antennapedia) and PTD-5, we have demonstrated that lysine and ornithine, as well as arginine, homopolymers are able to mediate transduction of a wide variety of agents. To screen for optimal PTDs, we have used as a therapeutic cargo a peptide derived from IKK {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase} beta, able to bind to the IKK regulatory subunit [NEMO (NF-kappaB essential modulator)], preventing formation of an active kinase complex. This peptide, termed NBD, is able to block activation of NF-kappaB, but not basal activity. We demonstrate that PTD-mediated delivery of NBD using certain PTDs, in particular 8K (octalysine), is therapeutic following systemic delivery in murine models of inflammatory bowel disease, diabetes and muscular dystrophy. In addition, we have developed a peptide phage display library screening method for novel transduction peptides able to facilitate tissue-specific internalization of marker protein complexes. Using this approach, we have identified transduction peptides that are able to facilitate internalization of large protein complexes into tumours, airway epithelia, synovial fibroblasts, cardiac tissue and HEK-293 (human embryonic kidney) cells in culture and/or in vivo.

NF-κB Negatively Impacts the Myogenic Potential of Muscle-derived Stem Cells

Inhibition of the inhibitor of kappa B kinase (IKK)/nuclear factor-kappa B (NF-κB) pathway enhances muscle regeneration in injured and diseased skeletal muscle, but it is unclear exactly how this pathway contributes to the regeneration process. In this study, we examined the role of NF-κB in regulating the proliferation and differentiation of muscle-derived stem cells (MDSCs). MDSCs isolated from the skeletal muscles of p65(+/-) mice (haploinsufficient for the p65 subunit of NF-κB) had enhanced proliferation and myogenic differentiation compared to MDSCs isolated from wild-type (wt) littermates. In addition, selective pharmacological inhibition of IKKβ, an upstream activator of NF-κB, enhanced wt MDSC differentiation into myotubes in vitro. The p65(+/-) MDSCs also displayed a higher muscle regeneration index than wt MDSCs following implantation into adult mice with muscular dystrophy. Additionally, using a muscle injury model, we observed that p65(+/-) MDSC engraftments were associated with reduced inflammation and necrosis. These results suggest that inhibition of the IKK/NF-κB pathway represents an effective approach to improve the myogenic regenerative potential of MDSCs and possibly other adult stem cell populations. Moreover, our results suggest that the improved muscle regeneration observed following inhibition of IKK/NF-κB, is mediated, at least in part, through enhanced stem cell proliferation and myogenic potential.

ISSLS prize winner: inhibition of NF-κB activity ameliorates age-associated disc degeneration in a mouse model of accelerated aging.

Study Design. NF-&kgr;B activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. Objective. To study the mediatory role of NF-&kgr;B-signaling pathway in age-dependent intervertebral disc degeneration. Summary of Background Data. Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-&kgr;B is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-&kgr;B activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-&kgr;B inhibition on IDD, using a DNA repair-deficient mouse model of accelerated aging (Ercc1−/&Dgr; mice) previously been reported to exhibit age-related IDD. Methods. Systemic inhibition of NF-&kgr;B activation was achieved either genetically by deletion of 1 allele of the NF-&kgr;B subunit p65 (Ercc1−/&Dgr;p65+− mice) or pharmacologically by chronic intraperitoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-&kgr;B, I&kgr;B Inducible Kinase (IKK), in Ercc1−/&Dgr; mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice, and untreated controls were assessed. Results. Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-&kgr;B activity were observed in discs of progeroid Ercc1−/&Dgr; mice and naturally aged wild-type mice compared with young wild-type mice. Systemic inhibition of NF-&kgr;B by the 8K-NBD peptide in Ercc1−/&Dgr; mice increased disc proteoglycan synthesis and ameriolated loss of disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1−/&Dgr;p65+/− mice. Conclusion. These findings demonstrate that the IKK/NF-&kgr;B signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.

Pharmacologic IKK/NF-κB inhibition causes antigen presenting cells to undergo TNFα dependent ROS-mediated programmed cell death.

Monocyte-derived antigen presenting cells (APC) are central mediators of the innate and adaptive immune response in inflammatory diseases. As such, APC are appropriate targets for therapeutic intervention to ameliorate certain diseases. APC differentiation, activation and functions are regulated by the NF-κB family of transcription factors. Herein, we examined the effect of NF-κB inhibition, via suppression of the IκB Kinase (IKK) complex, on APC function. Murine bone marrow-derived macrophages and dendritic cells (DC), as well as macrophage and DC lines, underwent rapid programmed cell death (PCD) after treatment with several IKK/NF-κB inhibitors through a TNFα-dependent mechanism. PCD was induced proximally by reactive oxygen species (ROS) formation, which causes a loss of mitochondrial membrane potential and activation of a caspase signaling cascade. NF-κB-inhibition-induced PCD of APC may be a key mechanism through which therapeutic targeting of NF-κB reduces inflammatory pathologies.

Spontaneous DNA damage to the nuclear genome promotes senescence, redox imbalance and aging

Accumulation of senescent cells over time contributes to aging and age-related diseases. However, what drives senescence in vivo is not clear. Here we used a genetic approach to determine if spontaneous nuclear DNA damage is sufficient to initiate senescence in mammals. Ercc1-/∆ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/∆ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/∆ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/∆ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/∆ mice to an extent identical to naturally-aged WT mice. Increased enzymatic production of ROS and decreased antioxidants contributed to the elevation in oxidative stress in both Ercc1-/∆ and aged WT mice. Chronic treatment of Ercc1-/∆ mice with the mitochondrial-targeted radical scavenger XJB-5–131 attenuated oxidative DNA damage, senescence and age-related pathology. Our findings indicate that nuclear genotoxic stress arises, at least in part, due to mitochondrial-derived ROS, and this spontaneous DNA damage is sufficient to drive increased levels of ROS, cellular senescence, and the consequent age-related physiological decline.

Kidney–infiltrating T cells in murine lupus nephritis are metabolically and functionally exhausted

While T cells are important for the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis, little is known about how T cells function after infiltrating the kidney. The current paradigm suggests that kidney-infiltrating T cells (KITs) are activated effector cells contributing to tissue damage and ultimately organ failure. Herein, we demonstrate that the majority of CD4+ and CD8+ KITs in 3 murine lupus models are not effector cells, as hypothesized, but rather express multiple inhibitory receptors and are highly dysfunctional, with reduced cytokine production and proliferative capacity. In other systems, this hypofunctional profile is linked directly to metabolic and specifically mitochondrial dysfunction, which we also observed in KITs. The T cell phenotype was driven by the expression of an “exhausted” transcriptional signature. Our data thus reveal that the tissue parenchyma has the capability of suppressing T cell responses and limiting damage to self. These findings suggest avenues for the treatment of autoimmunity based on selectively exploiting the exhausted phenotype of tissue-infiltrating T cells.