Down-Regulated NOD2 by Immunosuppressants in Peripheral Blood Cells in Patients with SLE Reduces the Muramyl Dipeptide-Induced IL-10 Production

Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles. basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs.Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection

Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders

T-bet upregulation and subsequent interleukin 12 stimulation are essential for induction of Th1 mediated immunopathology in Crohn’s disease

Background and aims: Many lines of evidence suggest that T helper cell type 1 (Th1) immune responses predominate in Crohn’s disease (CD). Recently, a novel transcription factor T-box expressed in T cells (T-bet) has been reported as the master regulator of Th1 development. This study was designed to investigate the role of T-bet and proinflammatory cytokines in Th1 mediated immunopathology in CD. Materials: CD4+ lamina propria mononuclear cells (LPMCs) were isolated from surgically resected specimens (CD, n = 10; ulcerative colitis (UC), n = 10; normal controls (NL), n = 5). Methods: (1) T-bet expression of CD4+ LPMCs was examined by quantitative real time polymerase chain reaction and western blotting. (2) T-bet expression of LPMCs stimulated by interleukin (IL)-12/IL-18 was analysed by western blotting. (3) Interferon γ (IFN-γ) production and T-bet expression of CD4+ peripheral blood mononuclear cells (PBMCs) were examined with or without stimulation by anti-CD3/CD28 monoclonal antibodies and/or IL-12. Results: (1) T-bet expression of CD4+ LPMCs was increased in CD compared with UC and NL. (2) Synergistically, augmentation of IFN-γ production by IL-12/IL-18 was independent of T-bet expression in LPMCs. (3) T-bet was induced by T cell receptor stimulation in CD4+ PBMCs. T-bet induction correlated with IFN-γ production and with augmentation of surface expressed IL-12 receptor β2. Conclusions: T-bet induction by antigenic stimulation and subsequent stimulation by macrophage derived IL-12/IL-18 are important for establishing Th1 mediated immunopathology in CD

Missense mutations in the NF2 gene result in the quantitative loss of merlin protein and minimally affect protein intrinsic function

Neurofibromatosis type 2 (NF2) is a multiple neoplasia syndrome and is caused by a mutation of the NF2 tumor suppressor gene that encodes for the tumor suppressor protein merlin. Biallelic NF2 gene inactivation results in the development of central nervous system tumors, including schwannomas, meningiomas, ependymomas, and astrocytomas. Although a wide variety of missense germline mutations in the coding sequences of the NF2 gene can cause loss of merlin function, the mechanism of this functional loss is unknown. To gain insight into the mechanisms underlying loss of merlin function in NF2, we investigated mutated merlin homeostasis and function in NF2-associated tumors and cell lines. Quantitative protein and RT-PCR analysis revealed that whereas merlin protein expression was significantly reduced in NF2-associated tumors, mRNA expression levels were unchanged. Transfection of genetic constructs of common NF2 missense mutations into NF2 gene-deficient meningioma cell lines revealed that merlin loss of function is due to a reduction in mutant protein half-life and increased protein degradation. Transfection analysis also demonstrated that recovery of tumor suppressor protein function is possible, indicating that these mutants maintain intrinsic functional capacity. Further, increased expression of mutant protein is possible after treatment with specific proteostasis regulators, implicating protein quality control systems in the degradative fate of mutant tumor suppressor proteins. These findings provide direct insight into protein function and tumorigenesis in NF2 and indicate a unique treatment paradigm for this disorder

αB-Crystallin Is Elevated in Highly Infiltrative Apoptosis-Resistant Glioblastoma Cells

We have previously established two distinct glioma phenotypes by serial xenotransplantation of human glioblastoma (GBM) biopsies in nude rats. These tumors undergo a gradual transition from a highly invasive nonangiogenic to a less-invasive angiogenic phenotype. In a protein screen to identify molecular markers associated with the infiltrative phenotype, we identified α-basic-crystallin (αBc), a small heat-shock protein with cytoprotective properties. Its increased expression in the infiltrative phenotype was validated by immunohistochemistry and Western blots, confirming its identity to be tumor-derived and not from the host. Stereotactic human GBM biopsies taken from MRI-defined areas verified stronger αBc expression in the infiltrative edge compared to the tumor core. Cell migration assays and immunofluorescence staining showed αBc to be expressed by migrating cells in vitro. To determine αBc function, we altered its expression levels. αBc siRNA depletion caused a loss of migrating tumor cells from biopsy spheroids and delayed monolayer wound closure. In contrast, glioma cell migration in a Boyden chamber assay was unaffected by either αBc knockdown or overexpression, indicating that αBc is not functionally linked to the cell migration machinery. However, after siRNA αBc depletion, a significant sensitization of cells to various apoptotic inducers was observed (actinomycin, tumor necrosis factor α, and TNF-related apoptosis-inducing ligand [TRAIL]). In conclusion, αBc is overexpressed by highly migratory glioma cells where it plays a functional role in apoptosis resistance