Enteroendocrine cells express functional Toll-like receptors

Intestinal epithelial cells (IECs) provide a physical and immunological barrier against enteric microbial flora. Toll-like receptors (TLRs), through interactions with conserved microbial patterns, activate inflammatory gene expression in cells of the innate immune system. Previous studies of the expression and function of TLRs in IECs have reported varying results. Therefore, TLR expression was characterized in human and murine intestinal sections, and TLR function was tested in an IEC line. TLR1, TLR2, and TLR4 are coexpressed on a subpopulation of human and murine IECs that reside predominantly in the intestinal crypt and belong to the enteroendocrine lineage. An enteroendocrine cell (EEC) line demonstrated a similar expression pattern of TLRs as primary cells. The murine EEC line STC-1 was activated with specific TLR ligands: LPS or synthetic bacterial lipoprotein. In STC-1 cells stimulated with bacterial ligands, NF-κB and MAPK activation was demonstrated. Furthermore, the expression of TNF and macrophage inhibitory protein-2 were induced. Additionally, bacterial ligands induced the expression of the anti-inflammatory gene transforming growth factor-β. LPS triggered a calcium flux in STC-1 cells, resulting in a rapid increase in CCK secretion. Finally, conditioned media from STC-1 cells inhibited the production of nitric oxide and IL-12 p40 by activated macrophages. In conclusion, human and murine IECs that express TLRs belong to the enteroendocrine lineage. Using a murine EEC model, a broad range of functional effects of TLR activation was demonstrated. This study suggests a potential role for EECs in innate immune responses

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-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ 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-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ 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

Spontaneous DNA damage to the nuclear genome promotes senescence, T 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-/Δ mice with reduced expression of ERCC1-XPF endonuclease have impaired capacity to repair the nuclear genome. Ercc1-/Δ mice accumulated spontaneous, oxidative DNA damage more rapidly than wild-type (WT) mice. As a consequence, senescent cells accumulated more rapidly in Ercc1-/Δ mice compared to repair-competent animals. However, the levels of DNA damage and senescent cells in Ercc1-/Δ mice never exceeded that observed in old WT mice. Surprisingly, levels of reactive oxygen species (ROS) were increased in tissues of Ercc1-/Δ 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-/Δ and aged WT mice. Chronic treatment of Ercc1-/Δ 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

Murine lupus is neutrophil elastase-independent in the MRL.Faslpr model.

Loss of tolerance to nuclear antigens and multisystem tissue destruction is a hallmark of systemic lupus erythematosus (SLE). Although the source of autoantigen in lupus remains elusive, a compelling hypothetical source is dead cell debris that drives autoimmune activation. Prior reports suggest that neutrophil extracellular traps (NETs) and their associated death pathway, NETosis, are sources of autoantigen in SLE. However, others and we have shown that inhibition of NETs by targeting the NADPH oxidase complex and peptidylarginine deiminase 4 (PADI4) did not ameliorate disease in spontaneous murine models of SLE. Furthermore, myeloperoxidase and PADI4 deletion did not inhibit induced lupus. Since NET formation may occur independently of any one mediator, to address this controversy, we genetically deleted an additional important mediator of NETs and neutrophil effector function, neutrophil elastase (ELANE), in the MRL.Faslpr model of SLE. ELANE deficiency, and by extension ELANE-dependent NETs, had no effect on SLE nephritis, dermatitis, anti-self response, or immune composition in MRL.Faslpr mice. Taken together with prior data from our group and others, these data further challenge the paradigm that NETs and neutrophils are pathogenic in SLE

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\u27s 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. Copyright © 2007 by The American Association of Immunologists, Inc

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. © Society for Leukocyte Biology

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