ABSTRACT
A mounting evidence exists for the despicable role of the aberrant immune response in the pathogenesis of rheumatoid arthritis (RA), where toll-like receptor 4 (TLR4) can activate synovial fibroblasts that lead to the chronic inflammation and joint destruction, thus making TLR4 a potent drug target in RA. We report that novel TLR4-antagonizing peptide, PIP2, inhibits the induction of inflammatory biomarkers in vitro as well as in vivo. Systemically, PIP2 inhibits the lipopolysaccharide (LPS)-elicited TNF-α, IL-6, and IL-12p40 in a mouse model. The rationally designed cyclic derivative, cPIP2, is capable of inhibiting LPS-induced proinflammatory cytokines at significantly lower concentration as compared to PIP2 (PIP2 IC50 = 20 µM, cPIP2 IC50 = 5 µM). Finally, cPIP2 was able to relieve the inflammatory symptoms and synovial tissue destruction in the RA rat model. Cumulatively, these data suggest that PIP2 and cPIP2 hold strong promise for the development of peptide-based immunotherapeutics that could be of great value in curbing TLR-related immune complications including RA.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Peptides/therapeutic use , Toll-Like Receptor 4/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/chemistry , Arthritis, Rheumatoid/immunology , Drug Design , Lipopolysaccharides/immunology , Male , Mice , Peptides/chemistry , RAW 264.7 Cells , Rats , Rats, Inbred Lew , Signal Transduction/drug effects , Toll-Like Receptor 4/immunology , Tumor Necrosis Factor-alpha/immunologyABSTRACT
Toll-like receptors (TLRs) recognize pathogen/damage-associated molecular patterns and initiate inflammatory signaling cascades. Occasionally, overexpression of TLRs leads to the onset of numerous inflammatory diseases, necessitating the development of selective inhibitors to allow a protective yet balanced immune response. Here, we demonstrate that a novel peptide (TIP1) derived from Toll/interleukin-1 receptor (TIR) domain-containing adapter protein inhibited multiple TLR signaling pathways (MyD88-dependent and MyD88-independent) in murine and human cell lines. TIP1 also inhibited NLRP3-mediated IL-1ß secretion, as we validated at both the protein and mRNA levels. Biophysical experiments confirmed that TIP1 specifically binds to the BB loop of the TLR4-TIR domain. Animal studies revealed that TIP1 inhibited the secretion of lipopolysaccharide (LPS)-induced proinflammatory cytokines in collagen-induced arthritis (CIA) and kaolin/carrageenan-induced arthritis (K/C) rodent models. TIP1 also rescued animals from sepsis and from LPS-induced kidney/liver damage. Importantly, TIP1 ameliorated the symptoms of rheumatoid arthritis in CIA and K/C rodent models, suggesting that TIP1 has therapeutic potential for the treatment of TLR-mediated autoimmune/inflammatory diseases.
Subject(s)
Toll-Like Receptors/metabolism , Animals , Blotting, Western , Cell Survival/genetics , Cell Survival/physiology , Interferon-beta/metabolism , Interleukin-1beta/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Confocal , Peptides/pharmacology , RAW 264.7 Cells , Rats, Sprague-Dawley , Signal Transduction/genetics , Signal Transduction/physiology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , Toll-Like Receptors/geneticsABSTRACT
Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.
