LINC00313 alleviates osteoarthritis progression in mice through promoting TRAF1 promoter methylation and inhibiting the ASK1/JNK signaling pathway.

OBJECTIVES: This study aimed to explore the underlying role and mechanism of LINC00313 in osteoarthritis (OA) progression. METHODS: CHON-001 chondrocytes were treated with interleukin (IL)-1ß to induce OA in vitro, and then transfected with LINC00313 overexpression plasmids (pcDNA-LINC00313) or small interfering RNA against tumor necrosis factor (TNF) receptor-associated factor 1 (si-TRAF1). Cell viability, apoptosis, levels of inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-6 and IL-8, and expression of extracellular matrix (ECM) degradation related proteins in CHON-001 cells were determined. TRAF1 promoter methylation were was detected with methylation-specific polymerase chain reaction (MSP) assay. Furthermore, a c-Jun N-terminal kinase (JNK) signaling activator was used to confirm whether the apoptosis signal-regulating kinase 1 (ASK1)/JNK signaling pathway was involved in the function of LINC00313/TRAF1 axis in chondrocytes. In addition, an OA mouse model was established and lentivirus LINC00313 overexpression vector (Lv-LINC00313) was injected, and then inflammatory cytokine levels, ECM protein expression, and pathological changes in cartilage tissues were detected. RESULTS: LINC00313 was downregulated and TRAF1 was upregulated in OA cartilage tissues. LINC00313 overexpression or TRAF1 silencing attenuated IL-1ß-induced viability inhibition, apoptosis, inflammation and ECM degradation in CHON-001 cells. Moreover, LINC00313 inhibited TRAF1 expression through promoting DNA methyltransferase 1 (DNMT1) mediated promoter methylation. TRAF1 overexpression reversed the effects of LINC00313 on IL-1ß-induced chondrocyte injury. LINC00313 overexpression inhibited the ASK1/JNK signaling pathway, and JNK activator reversed the effect. In addition, Lv-LINC00313 treatment alleviated cartilage tissue damage and cartilage matrix degradation in OA mice. CONCLUSIONS: LINC00313 alleviated OA progression through inhibiting TRAF1 expression and the ASK1/JNK signaling pathway.

Abr, a negative regulator of Rac, attenuates cockroach allergen-induced asthma in a mouse model.

Abr deactivates Ras-related C3 botulinum toxin substrate (Rac), a master molecular switch that positively regulates many immune cell functions, by converting it to its GDP-bound conformation. In this article, we report that, in the absence of Abr function, cockroach allergen (CRA)-immunized mice experienced a fatal asthma attack when challenged with CRA. The asthma in abr(-/-) mice was characterized by increased pulmonary mucus production, elevated serum IgE, and leukocyte airway infiltration. Decreased pulmonary compliance was further documented by increased airway resistance upon methacholine challenge. Peribronchial and bronchoalveolar lavage eosinophils, key cells associated with allergic asthma, were increased in abr(-/-) mice, but adoptive transfer of this cell type from immunized mice to naive controls, followed by CRA challenge, showed that eosinophils are not primarily responsible for differences in airway resistance between controls and abr-null mutants. CD4(+) T cell numbers in the airways of CRA-challenged abr(-/-) mice also were significantly increased compared with controls, as were the Th2 T cell-secreted cytokines IL-4 and IL-5 in total lung. Interestingly, when control and abr(-/-) CD4(+) T cells from CRA-immunized mice were transferred to wild-type animals, airway resistance upon challenge with CRA was significantly higher in mice transplanted with T cells lacking Abr function. CD4(+) T cells from CRA-immunized and challenged abr(-/-) mice contained elevated levels of activated GTP-bound Rac compared with wild-type controls. Functionally, abr(-/-) CD4(+) T cells from CRA-exposed mice showed significantly enhanced chemotaxis toward CCL21. These results identify Abr-regulated CD4(+) T cell migration as an important component of severe CRA-evoked allergic asthma in mice.

Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

BACKGROUND: Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined. METHODOLOGY/PRINCIPAL FINDINGS: Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia. CONCLUSIONS: Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

Increased surfactant protein D fails to improve bacterial clearance and inflammation in serpinB1-/- mice.

Previously, we described the protective role of the neutrophil serine protease inhibitor serpinB1 in preventing early mortality of Pseudomonas aeruginosa lung infection by fostering bacterial clearance and limiting inflammatory cytokines and proteolytic damage. Surfactant protein D (SP-D), which maintains the antiinflammatory pulmonary environment and mediates bacterial removal, was degraded in infected serpinB1-deficient mice. Based on the hypothesis that increased SP-D would rescue or mitigate the pathological effects of serpinB1 deletion, we generated two serpinB1(-/-) lines overexpressing lung-specific rat SP-D and inoculated the mice with P. aeruginosa. Contrary to predictions, bacterial counts in the lungs of SP-D(low)serpinB1(-/-) and SP-D(high) serpinB1(-/-) mice were 4 logs higher than wild-type and not different from serpinB1(-/-) mice. SP-D overexpression also failed to mitigate inflammation (TNF-α), lung injury (free protein, albumin), or excess neutrophil death (free myeloperoxidase, elastase). These pathological markers were higher for infected SP-D(high)serpinB1(-/-) mice than for serpinB1(-/-) mice, although the differences were not significant after controlling for multiple comparisons. The failure of transgenic SP-D to rescue antibacterial defense of serpinB1-deficient mice occurred despite 5-fold or 20-fold increased expression levels, largely normal structure, and dose-dependent bacteria-aggregating activity. SP-D of infected wild-type mice was intact in 43-kD monomers by reducing SDS-PAGE. By contrast, proteolytic fragments of 35, 17, and 8 kD were found in infected SP-D(low)serpinB1(-/-), SP-D(high) serpinB1(-/-) mice, and serpinB1(-/-) mice. Thus, although therapies to increase lung concentration of SP-D may have beneficial applications, the findings suggest that therapy with SP-D may not be beneficial for lung inflammation or infection if the underlying clinical condition includes excess proteolysis.

TGFß signaling plays a critical role in promoting alternative macrophage activation.

BACKGROUND: Upon stimulation with different cytokines, macrophages can undergo classical or alternative activation to become M1 or M2 macrophages. Alternatively activated (or M2) macrophages are defined by their expression of specific gene products and play an important role in containing inflammation, removing apoptotic cells and repairing tissue damage. Whereas it is well-established that IL-4 can drive alternative activation, if lack of TGFß signaling at physiological levels affects M2 polarization has not been addressed. RESULTS: Vav1-Cre x TßRIIfx/fx mice, lacking TßRII function in hematopoietic cells, exhibited uncontrolled pulmonary inflammation and developed a lethal autoimmune syndrome at young age. This was accompanied by significantly increased numbers of splenic neutrophils and T cells as well as elevated hepatic macrophage infiltration and bone marrow monocyte counts. TßRII-/- CD4+ and CD8+ T-cells in the lymph nodes and spleen expressed increased cell surface CD44, and CD69 was also higher on CD4+ lymph node T-cells. Loss of TßRII in bone marrow-derived macrophages (BMDMs) did not affect the ability of these cells to perform efferocytosis. However, these cells were defective in basal and IL-4-induced arg1 mRNA and Arginase-1 protein production. Moreover, the transcription of genes that are typically upregulated in M2-polarized macrophages, such as ym1, mcr2 and mgl2, was also decreased in peritoneal macrophages and IL-4-stimulated TßRII-/- BMDMs. We found that cell surface and mRNA expression of Galectin-3, which also regulates M2 macrophage polarization, was lower in TßRII-/- BMDMs. Very interestingly, the impaired ability of these null mutant BMDMs to differentiate into IL-4 polarized macrophages was Stat6- and Smad3-independent, but correlated with reduced levels of phospho-Akt and ß-catenin. CONCLUSIONS: Our results establish a novel biological role for TGFß signaling in controlling expression of genes characteristic for alternatively activated macrophages. We speculate that lack of TßRII signaling reduces the anti-inflammatory M2 phenotype of macrophages because of reduced expression of these products. This would cause defects in the ability of the M2 macrophages to negatively regulate other immune cells such as T-cells in the lung, possibly explaining the systemic inflammation observed in Vav1-Cre x TßRIIfx/fx mice.

Critical role of serpinB1 in regulating inflammatory responses in pulmonary influenza infection.

BACKGROUND: Excessive inflammatory host response increases morbidity and mortality associated with seasonal respiratory influenza, and highly pathogenic virus strains are characterized by massive infiltration of monocytes and/or macrophages that produce a storm of injurious cytokines. METHODS: Here, we examined the role in respiratory influenza of serpinB1, an endogenous inhibitor of the serine proteases elastase, cathepsin G, and proteinase-3, increasingly recognized as regulators of inflammation. RESULTS: After challenge with high-dose surfactant protein-D (SP-D)-sensitive influenza A/Philadelphia/82 (H3N2), serpinB1(-/-) mice died earlier and in greater numbers than did wild-type mice. Sublethally infected animals suffered increased morbidity, delayed resolution of epithelial injury, and increased immune cell death. Viral clearance and SP-D/SP-A upregulation were unimpaired and so were early virus-induced cytokine and chemokine burst and influx of large numbers of neutrophils and monocytes. Whereas initial cytokines and chemokines rapidly cleared in wild-type mice, TNF-α, IL-6, KC/CXCL1, G-CSF, IL-17A, and MCP-1/CCL2 remained elevated in serpinB1(-/-) mice. Monocyte-derived cells were the dominant immune cells in influenza-infected lungs, and those from serpinB1(-/-) mice produced excessive IL-6 and TNF-α when tested ex vivo. Pulmonary γδ T-cells that produced IL-17A were also increased. CONCLUSIONS: Because viral clearance was unimpaired, the study highlights the critical role of serpinB1 in mitigating inflammation and restricting pro-inflammatory cytokine production in influenza infection.

Cytokine-dependent Blimp-1 expression in activated T cells inhibits IL-2 production.

After Ag activation of naive T cells in vitro, extensive growth and differentiation into effector cells depend upon IL-2. DNA microarray analysis was used to identify IL-2-dependent molecules regulating this process. In this study, we show that the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp-1) is expressed by a cytokine-dependent pathway in activated T lymphocytes. IL-2 production by activated CD4(+) and CD8(+) T cells inversely correlated with Blimp-1 levels as higher IL-2 production was associated with lower Blimp-1 expression. Furthermore, ectopic expression of Blimp-1 by activated T cells inhibited IL-2 production but enhanced granzyme B and CD25 expression. Collectively, these findings indicate that there is a negative feedback regulatory loop in activated T cells such that IL-2 inhibits its own production through induction of Blimp-1 while promoting an effector cell phenotype.

Quantitative assessment concerning the contribution of IL-2Rbeta for superantigen-mediated T cell responses in vivo.

IL-2- and IL-2R-deficient mice readily develop T cell-dependent immune responses in vivo, but the relevance of this finding is complicated by severe concurrent autoimmunity. Furthermore, the detection of such responses does not address whether under normal circumstances IL-2 dominates T cell immunity. In the present report, we investigated the extent IL-2-independent T cell growth is mediated by other cytokines in the IL-2 family and compared such responses to those generated by IL-2/IL-2R-sufficient T cells. T cell expansion and contraction to the superantigen staphylococcal enterotoxin A (SEA) by autoimmune-free IL-2Rbeta-/- CD4 and CD8 T cells were comparable to normal control mice, although their CD8+ T cells did not optimally develop into IFNgamma-producing effector cells. The proliferation by these IL-2Rbeta-deficient T cells in vivo was independent of IL-2, IL-4 and IL-15 and not blocked by mAbs to the common gamma chain. However, in co-adoptive transfer experiments, wild-type T cells exhibited somewhat more extensive proliferation than IL-2Rbeta-deficient T cells to SEA and this difference was almost entirely accounted for by CD8+ T cells. Collectively, these data indicate that substantial T cell proliferation occurs in the absence of responsiveness to cytokines in the IL-2 family, although maximal T cell proliferation and development of IFNgamma-producing effector CD8+ T cells depend upon IL-2Rbeta.

RNA stability of the E2A-encoded transcription factor E47 is lower in splenic activated B cells from aged mice.

We have demonstrated previously that DNA binding and protein expression of the E2A-encoded transcription factor E47 are lower in nuclear extracts of activated splenic B cells from old mice. In the present study, we address how E47 protein expression is regulated in aging. Results herein show that E2A mRNA levels were decreased in stimulated splenic B cells from old as compared with young mice. RNA stability assays showed that the rate of E2A mRNA decay was accelerated in stimulated splenic B cells from old mice, but E47 protein degradation rates were comparable in young vs aged B cells, indicating that the regulation of E47 expression in activated splenic B cells occurs primarily by mRNA stability. The rates of decay of other mRNAs showed that the increased mRNA degradation in aged splenic activated B cells is not a general phenomenon but restricted to a subset of mRNAs. We next investigated the signal transduction pathways controlling E2A mRNA expression and stability and found that p38 MAPK regulates E2A mRNA expression through increased mRNA stability and is down-regulated in aged activated B cells. Results show that inhibition of p38 MAPK significantly reduces E2A mRNA stability in both young and old B cells, further stressing the role of p38 MAPK in E2A RNA stabilization. These studies demonstrate that the transcription factor E2A, critical for many aspects of B cell function, is regulated by a novel mechanism in aging.