Significant morbidity and mortality can be attributed to inflammatory diseases; therefore, a greater understanding of the mechanisms involved in the progression of inflammation is crucial. Here, we demonstrate that p21 (WAF1/CIP1) , an established suppressor of cell cycle progression, is a inhibitor of IL-1b synthesis in macrophages. Mice deficient in p21 (p21 À/À ) display increased susceptibility to endotoxic shock, which is associated with increased serum levels of IL-1b. Administration of IL-1 receptor antagonist reduces LPS-induced lethality in p21 À/À mice. Analysis of isolated macrophages, which are one of the central producers of IL-1b, reveals that deficiency for p21 led to more IL-1b mRNA and pro-protein synthesis following TLR ligation. The increase in IL-1b pro-protein is associated with elevated secretion of active IL-1b by p21 À/À macrophages. siRNA-mediated knockdown of p21 in human macrophages results in increased IL-1b secretion as well. A peptide mapping strategy shows that the cyclin-dependent-kinase (CDK)-binding domain of p21 is sufficient to reduce the secretion of IL-1b by p21 À/À macrophages. These data suggest a novel role for p21 and specifically for the CDK-binding domain of p21 (WAF1/CIP1) in inhibiting inflammation. IntroductionThe pleiotropic cytokine IL-1b is required for the progression of numerous inflammatory disorders [1]. As key components of the innate immune response, monocytes and macrophages play an important role in the initiation and/or progression of inflammatory disease, largely through secretion of IL-1b, which contributes significantly to pathogenesis. Current treatments for diseases such as rheumatoid arthritis, gout, and periodic fever syndromes include the IL-1 receptor antagonist anakinra (Kineret s , Amgen) SHORT COMMUNICATIONÃ These authors contributed equally to this work. 820[2]; yet this therapy is ineffective in a cohort of patients [3]. Therefore, a better understanding of the mechanisms by which IL-1b production is regulated remains crucial for the development of new therapies to treat inflammatory diseases.Here, we investigate the role that p21 (WAF1/CIP1) (p21) plays in the production of IL-1b and development of inflammatory disease. As a member of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors, p21 induces blockade of cell cycle progression via inhibition of the activity of CDK/cyclin complexes as well as that of proliferating cell nuclear antigen (PCNA) [4]. p21 binds to CDK and cyclins through two distinct domains on the N-terminus and to PCNA via its C-terminus [5]. Mice deficient in p21 show no developmental or reproductive abnormalities, although the mice display sensitivity to radiation [6] and develop a form of lupus-like disease [6][7][8]. Furthermore, a paucity of p21 expression is observed in synovial tissue from rheumatoid arthritis patients compared with osteoarthritis controls [9]. Because of the implication of p21 in the pathogenesis of lupus and rheumatoid arthritis and the importance of IL-1b in these and other inflamm...

Objective. Rheumatoid arthritis (RA) is a destructive autoimmune disease characterized by an increased inflammation in the joint. Therapies that activate the apoptotic cascade may have potential for use in RA; however, few therapeutic agents fit this category. The purpose of this study was to examine the potential of Bim, an agent that mimics the action of Bcl-2 homology 3 (BH3) domain-only proteins that have shown success in preclinical studies of cancer, in the treatment of autoimmune disease.Methods. Synovial tissues from RA and osteoarthritis patients were analyzed for the expression of Bim and CD68 using immunohistochemistry. Macrophages from Bim -/-mice were examined for their response to lipopolysaccharide (LPS) using flow cytometry, realtime polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and immunoblotting. Bim -/-mice were stimulated with thioglycollate or LPS and examined for macrophage activation and cytokine production. Experimental arthritis was induced using the K/BxN serum-transfer model. A mimetic peptide corresponding to the BH3 domain of Bim (TAT-BH3) was administered as a prophylactic agent and as a therapeutic agent. Edema of the ankles and histopathologic analysis of ankle tissue sections were used to determine the severity of arthritis, its cellular composition, and the degree of apoptosis.

CD4Foxp3 regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we comprehensively analyzed the immunophenotype of Treg after DR3 signal activation, demonstrating that DR3-activated Treg (DR3-Treg) had an activated/mature phenotype. Furthermore, the CD25Foxp3 subpopulation in DR3-Treg showed stronger suppressive effects in vivo. Prophylactic treatment of αDR3 to recipient mice expanded recipient-derived Treg and reduced the severity of GVHD, whereas DR3 activation in mice with ongoing GVHD further promoted donor T-cell activation/proliferation. These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. In conclusion, our data demonstrated that DR3 signaling affects the function of Treg and T-cell activation after alloantigen exposure in a time-dependent manner. These observations provide important information for future clinical testing using human DR3 signal modulation and highlight the critical effect of the state of T-cell activation on clinical outcomes after activation of DR3.

Invariant natural killer T (iNKT) cells serve as a bridge between innate and adaptive immunity and have been shown to play an important role in immune regulation, defense against pathogens, and cancer immunity. Recent data also suggest that this compartment of the immune system plays a significant role in reducing graft-versus-host disease (GVHD) in the setting of allogeneic hematopoietic stem cell transplantation. Murine studies have shown that boosting iNKT numbers through certain conditioning regimens or adoptive transfer leads to suppression of acute or chronic GVHD. Preclinical work reveals that iNKT cells exert their suppressive function by expanding regulatory T cells in vivo, though the exact mechanism by which this occurs has yet to be fully elucidated. Human studies have demonstrated that a higher number of iNKT cells in the graft or in the peripheral blood of the recipient post-transplantation are associated with a reduction in GVHD risk, importantly without a loss of graft-versus-tumor effect. In two separate analyses of many immune cell subsets in allogeneic grafts, iNKT cell dose was the only parameter associated with a significant improvement in GVHD or in GVHD-free progression-free survival. Failure to reconstitute iNKT cells following allogeneic transplantation has also been associated with an increased risk of relapse. These data demonstrate that iNKT cells hold promise for future clinical application in the prevention of GVHD in allogeneic stem cell transplantation and warrant further study of the immunoregulatory functions of iNKT cells in this setting.

Objective The mechanism responsible for persistent inflammation of the synovium that occurs in patients with rheumatoid arthritis (RA) is unknown. Previously, we were the first to demonstrate that expression of the cyclin dependent kinase (CDK) inhibitor p21(WAF1/CIP1) is reduced in synovial tissue from RA patients compared to osteoarthritis patients and that p21 is a novel suppressor of the inflammatory response in macrophages. Here, we sought to determine the role and mechanism of p21-mediated suppression of experimental inflammatory arthritis. Methods Experimental arthritis was induced in WT or p21−/− (C57BL/6) mice using the K/BxN serum transfer induced model. p21-peptide mimetics were administered to mice as a prophylactic for arthritis development. LPS-induced cytokine and signal transduction pathways were examined in macrophages that were treated with p21-peptide mimetics using Luminex-based assays, flow cytometry, or ELISAs. Results p21−/− mice exhibit enhanced and sustained development of experimental inflammatory arthritis, which is associated with markedly increased numbers of macrophages and severe articular destruction. Administration of a p21-peptide mimetic suppresses activation of macrophages and reduces the severity of experimental arthritis only in p21-intact mice. Mechanistically, treatment with the p21-peptide mimetic leads to activation of the serine/threonine kinase Akt and subsequent reduction in the activated isoform of mitogen-activated protein kinase p38 in macrophages. Conclusion These data are the first to reveal that p21 plays an important role in limiting the activation response of macrophages in an inflammatory disease such as RA. Thus, targeting p21 in macrophages may be crucial for suppressing the development and persistence of RA.