Th17 can regulate silica‐induced lung inflammation through an IL‐1β‐dependent mechanism

Silicosis is an occupational lung disease caused by the inhalation of silica dust and characterized by lung inflammation and fibrosis. Interleukin (IL)‐1β is induced by silica and functions as the key pro‐inflammatory cytokine in this process. The Th17 response, which is induced by IL‐1β, has been reported very important in chronic human lung inflammatory diseases. To elucidate the underlying mechanisms of IL‐1β and IL‐17 in silicosis, we used anakinra and an anti‐IL‐17 monoclonal antibody (mAb) to block the receptor of IL‐1β (IL‐RI) and IL‐17, respectively, in a mouse model of silicosis. We observed increased IL‐1β expression and an enhanced Th17 response after silica instillation. Treatment with an IL‐1 type I receptor (IL‐1RI) antagonist anakinra substantially decreased silica‐induced lung inflammation and the Th17 response. Lung inflammation and the accumulation of inflammatory cells were attenuated in the IL‐17‐neutralized silicosis group. IL‐17 may promote lung inflammation by modulating the differentiation of Th1 and regulatory T cells (Tregs) and by regulating the production of IL‐22 and IL‐1β during the lung inflammation of silicosis. Silica may induce IL‐1β production from alveolar macrophages and promote inflammation by initiating a Th17 response via an IL‐1β/IL‐1RI‐dependent mechanism. The Th17 response could induce lung inflammation during the pathogenesis of silicosis by regulating the homoeostasis of the Th immune responses and affecting the production of IL‐22 and IL‐1β. This study describes a potentially important inflammatory mechanism of silicosis that may bring about novel therapies for this inflammatory and fibrotic disease.     

IL‐1α and IL‐1β have different effects on formation and activity of large osteoclasts

Interleukin 1 (IL‐1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL‐1α and IL‐1β, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2–5 nuclei). Moreover, large OCs express higher levels of the IL‐1 activating receptor IL‐1RI, integrins αv and β3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL‐1RII. We hypothesized that IL‐1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL‐1α or IL‐1β (1 and 10 ng/ml). In the presence of sRANKL, both IL‐1α and IL‐1β increased total OC number and resorptive activity of large OCs. IL‐1α stimulated formation of large OCs and increased the number of resorption pits, while IL‐1β changed the morphology of large OCs and integrin‐β3 phosphorylation. No effects were seen in small OCs in response to either IL‐1 isoform. These results demonstrate that IL‐1 predominantly affects large OCs. The dissimilarity of responses to IL‐1α and IL‐1β suggests that these isoforms activate different signaling pathways within the two OC populations. J. Cell. Biochem. 109: 975–982, 2010. © 2010 Wiley‐Liss, Inc.     

NDR1/STK38 potentiates NF‐κB activation by its kinase activity

Human NDR1/STK38 belongs to the nuclear‐Dbf2‐related (NDR) family of Ser/Thr kinases. It has been implicated to function in centrosome duplication, control of cell cycle and apoptosis. However, the mechanism of NDR1 signaling pathway remains largely elusive. Here, we report a novel role of NDR1 in NF‐κB activation. By overexpression, NDR1 potentiates NF‐κB activation induced by TNFα, whereas knockdown of NDR1 expression inhibits NF‐κB activation induced by TNFα. Coimmunoprecipitation shows that NDR1 interacts with multiple signal components except p65 in NF‐κB signaling pathway. Furthermore, both phosphorylation and kinase dead mutants of NDR1 lose their synergistic effects on TNFα‐induced NF‐κB activation. siRNA oligo against NDR1 and kinase dead mutant as well mainly block the NF‐κB activation induced by TRAF2 but not RIP1. Furthermore, kinase dead mutant of NDR1 fails to interact with TRAF2. Taken together, our findings suggest an unknown function of NDR1, which may regulate NF‐κB activation by its kinase activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Glucosamine inhibits IL‐1β‐mediated IL‐8 production in prostate cancer cells by MAPK attenuation

Inflammation is a complex process involving cytokine production to regulate host defense cascades. In contrast to the therapeutic significance of acute inflammation, a pathogenic impact of chronic inflammation on cancer development has been proposed. Upregulation of inflammatory cytokines, such as IL‐1β and IL‐8, has been noted in prostate cancer patients and IL‐8 has been shown to promote prostate cancer cell proliferation and migration; however, it is not clear whether IL‐1β regulates IL‐8 expression in prostate cancer cells. Glucosamine is widely regarded as an anti‐inflammatory agent and thus we hypothesized that if IL‐1β activated IL‐8 production in prostate cancer cells, then glucosamine ought to blunt such an effect. Three prostate cancer cell lines, DU‐145, PC‐3, and LNCaP, were used to evaluate the effects of IL‐1β and glucosamine on IL‐8 expression using ELISA and RT‐PCR analyses. IL‐1β elevated IL‐8 mRNA expression and subsequent IL‐8 secretion. Glucosamine significantly inhibited IL‐1β‐induced IL‐8 secretion. IL‐8 appeared to induce LNCaP cell proliferation by MTT assay; involvement of IL‐8 in IL‐1β‐dependent PC‐3 cell migration was demonstrated by wound‐healing and transwell migration assays. Inhibitors of MAPKs and NFκB were used to pinpoint MAPKs but not NFκB being involved in IL‐1β‐mediated IL‐8 production. IL‐1β‐provoked phosphorylation of all MAPKs was notably suppressed by glucosamine. We suggest that IL‐1β can activate the MAPK pathways resulting in an induction of IL‐8 production, which promotes prostate cancer cell proliferation and migration. In this context, glucosamine appears to inhibit IL‐1β‐mediated activation of MAPKs and therefore reduces IL‐8 production; this, in turn, attenuates cell proliferation/migration. J. Cell. Biochem. 108: 489–498, 2009. © 2009 Wiley‐Liss, Inc.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

IL‐23 drives differentiation of peripheral γδ17 T cells from adult bone marrow‐derived precursors

Pro‐inflammatory interleukin (IL)‐17‐producing γδ (γδ17) T cells are thought to develop exclusively in the thymus during fetal/perinatal life, as adult bone marrow precursors fail to generate γδ17 T cells under homeostatic conditions. Here, we employ a model of experimental autoimmune encephalomyelitis (EAE) in which hematopoiesis is reset by bone marrow transplantation and demonstrate unequivocally that Vγ4⁺ γδ17 T cells can develop de novo in draining lymph nodes in response to innate stimuli. In vitro, γδ T cells from IL‐17 fate‐mapping reporter mice that had never activated the Il17 locus acquire IL‐17 expression upon stimulation with IL‐1β and IL‐23. Furthermore, IL‐23R (but not IL‐1R1) deficiency severely compromises the induction of γδ17 T cells in EAE, demonstrating the key role of IL‐23 in the process. Finally, we show, in a composite model involving transfers of both adult bone marrow and neonatal thymocytes, that induced γδ17 T cells make up a substantial fraction of the total IL‐17‐producing Vγ4⁺ T‐cell pool upon inflammation, which attests the relevance of this novel pathway of peripheral γδ17 T‐cell differentiation.     

IL‐1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence‐associated secretory phenotype

Interleukin‐1 alpha (IL‐1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL‐1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL‐1α in these contexts is unknown. We show IL‐1α is activated by caspase‐5 or caspase‐11 cleavage at a conserved site. Caspase‐5 drives cleaved IL‐1α release after human macrophage inflammasome activation, while IL‐1α secretion from murine macrophages only requires caspase‐11, with IL‐1β release needing caspase‐11 and caspase‐1. Importantly, senescent human cells require caspase‐5 for the IL‐1α‐dependent senescence‐associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase‐11 for the SASP‐driven immune surveillance of senescent cells in vivo. Together, we identify IL‐1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL‐1α is activated during senescence. Thus, targeting caspase‐5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.     

Fluorofenidone attenuates pulmonary inflammation and fibrosis via inhibiting the activation of NALP3 inflammasome and IL‐1β/IL‐1R1/MyD88/NF‐κB pathway

Interleukin (IL)‐1β plays an important role in the pathogenesis of idiopathic pulmonary fibrosis. The production of IL‐1β is dependent upon caspase‐1‐containing multiprotein complexes called inflammasomes and IL‐1R1/MyD88/NF‐κB pathway. In this study, we explored whether a potential anti‐fibrotic agent fluorofenidone (FD) exerts its anti‐inflammatory and anti‐fibrotic effects through suppressing activation of NACHT, LRR and PYD domains‐containing protein 3 (NALP3) inflammasome and the IL‐1β/IL‐1R1/MyD88/NF‐κB pathway in vivo and in vitro. Male C57BL/6J mice were intratracheally injected with Bleomycin (BLM) or saline. Fluorofenidone was administered throughout the course of the experiment. Lung tissue sections were stained with haemotoxylin and eosin and Masson's trichrome. Cytokines were measured by ELISA, and α‐smooth muscle actin (α‐SMA), fibronectin, collagen I, caspase‐1, IL‐1R1, MyD88 were measured by Western blot and/or RT‐PCR. The human actue monocytic leukaemia cell line (THP‐1) were incubated with monosodium urate (MSU), with or without FD pre‐treatment. The expression of caspase‐1, IL‐1β, NALP3, apoptosis‐associated speck‐like protein containing (ASC) and pro‐caspase‐1 were measured by Western blot, the reactive oxygen species (ROS) generation was detected using the Flow Cytometry, and the interaction of NALP3 inflammasome‐associated molecules were measured by Co‐immunoprecipitation. RLE‐6TN (rat lung epithelial‐T‐antigen negative) cells were incubated with IL‐1β, with or without FD pre‐treatment. The expression of nuclear protein p65 was measured by Western blot. Results showed that FD markedly reduced the expressions of IL‐1β, IL‐6, monocyte chemotactic protein‐1 (MCP‐1), myeloperoxidase (MPO), α‐SMA, fibronectin, collagen I, caspase‐1, IL‐1R1 and MyD88 in mice lung tissues. And FD inhibited MSU‐induced the accumulation of ROS, blocked the interaction of NALP3 inflammasome‐associated molecules, decreased the level of caspase‐1 and IL‐1β in THP‐1 cells. Besides, FD inhibited IL‐1β‐induced the expression of nuclear protein p65. This study demonstrated that FD, attenuates BLM‐induced pulmonary inflammation and fibrosis in mice via inhibiting the activation of NALP3 inflammasome and the IL‐1β/IL‐1R1/MyD88/ NF‐κB pathway.     

Pulsatilla decoction and its active ingredients inhibit secretion of NO,ET‐1, TNF‐α, and IL‐1α in LPS‐induced rat intestinal microvascular endothelial cells

To investigate the pharmacological mechanism of the traditional Chinese medicine, Pulsatilla decoction (PD), the levels of nitric oxide (NO), endothelin‐1 (ET‐1), tumor necrosis factor‐α (TNF‐α), and interleukin‐1α (IL‐1α) secreted by cultured rat intestinal microvascular endothelial cells (RIMECs) were determined after treatment with PD and its seven active ingredients, namely anemoside B₄, anemonin, berberine, jatrorrhizine, palmatine, aesculin, and esculetin. RIMECs were challenged with lipopolysaccharide (LPS) at 1 µg ml^?1 for 3 h and then treated with PD at 1, 5, and 10 mg ml^?1 and its seven ingredients at 1, 5, and 10 µg ml^?1 for 21 h, respectively. The results revealed that PD, anemonin, berberine, and esculetin inhibited the production of NO; PD, anemonin, and esculetin inhibited the secretion of ET‐1; PD, anemoside B₄, berberine, jatrorrhizine, and aesculin downregulated TNF‐α expression; PD, anemoside B₄, berberine, and palmatine decreased the content of IL‐1α. It showed that PD and its active ingredients could significantly inhibit the secretion of NO, ET‐1, TNF‐α, and IL‐1α in LPS‐induced RIMECs and suggested they would reduce inflammatory response via these cytokines. Copyright © 2009 John Wiley & Sons, Ltd.     

Hydrogen sulfide reduces cell adhesion and relevant inflammatory triggering by preventing ADAM17‐dependent TNF‐α activation

H₂S is the third endogenous gaseous mediator, after nitric oxide and carbon monoxide, possessing pleiotropic effects, including cytoprotection and anti‐inflammatory action. We analyzed, in an in vitro model entailing monocyte adhesion to an endothelial monolayer, the changes induced by H₂S on various potential targets, including cytokines, chemokines, and proteases, playing a crucial role in inflammation and cell adhesion. Results show that H₂S prevents the increase in monocyte adhesion induced by tumor necrosis factor‐α (TNF‐α). Under these conditions, downregulation of monocyte chemoattractant protein‐1 (MCP‐1), chemokine C‐C motif receptor 2, and increase of cluster of differentiation 36 could be detected in monocytes. In endothelial cells, H₂S treatment reduces the increase in MCP‐1, inter‐cellular adhesion molecule‐1, vascular cell adhesion molecule‐1, and of a disintegrin and metalloproteinase metallopeptidase domain 17 (ADAM17), both at the gene expression and protein levels. Cystathionine γ‐lyase and 3‐mercaptopyruvate sulfurtransferase, the major H₂S forming enzymes, are downregulated in endothelial cells. In addition, H₂S significantly reduces activation of ADAM17 by PMA in endothelial cells, with consequent reduction of both ADAM17‐dependent TNF‐α ectodomain shedding and MCP‐1 release. In conclusion, H₂S is able to prevent endothelial activation by hampering endothelial activation, triggered by TNF‐α. The mechanism of this protective effect is mainly mediated by down‐modulation of ADAM17‐dependent TNF‐converting enzyme (TACE) activity with consequent inhibition of soluble TNF‐α shedding and its relevant MCP‐1 release in the medium. These results are discussed in the light of the potential protective role of H₂S in pro‐inflammatory and pro‐atherogenic processes, such as chronic renal failure. J. Cell. Biochem. 114: 1536–1548, 2013. © 2013 Wiley Periodicals, Inc.     

Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway

Whether dendritic cell (DC) derived exosomes play a role in the progression of endothelial inflammation and atherosclerosis remains unclear. Using a transwell system and exosome release inhibitor GW4869, we demonstrated that mature DCs contributed to endothelial inflammation and exosomes were involved in the process. To further confirm this finding, we isolated exosomes from bone marrow dendritic cell (BMDC) culture medium (named DC‐exos) and stimulated human umbilical vein endothelial cell (HUVEC) with these DC‐exos. We observed that mature DC‐exos increased HUVEC inflammation through NF‐κB pathway in a manner similar to that of lipopolysaccharide. After a protein array analysis of exosomes, we identified and confirmed tumour necrosis factor (TNF)‐α on exosome membrane being the trigger of NF‐κB pathway in HUVECs. We then performed an in vivo study and found that the aorta endothelial of mice could uptake intravenously injected exosomes and was activated by these exosomes. After a period of 12 weeks of mature DC‐exos injection into ApoE?/? mice, the atherosclerotic lesions significantly increased. Our study demonstrates that mature DCs derived exosomes increase endothelial inflammation and atherosclerosis via membrane TNF‐α mediated NF‐κB pathway. This finding extends our knowledge on how DCs affect inflammation and provides a potential method to prevent endothelial inflammation and atherosclerosis.     

Suppression of IL‐8‐Src signalling axis by 17β‐estradiol inhibits human mesenchymal stem cells‐mediated gastric cancer invasion

Epidemiologic data show the incidence of gastric cancer in men is twofold higher than in women worldwide. Oestrogen is reported to have the capacity against gastric cancer development. Endogenous oestrogen reduces gastric cancer incidence in women. Cancer patients treated with oestrogens have a lower subsequent risk of gastric cancer. Accumulating studies report that bone marrow mesenchymal stem cells (BMMSCs) might contribute to the progression of gastric cancer through paracrine effect of soluble factors. Here, we further explore the effect of oestrogen on BMMSCs‐mediated human gastric cancer invasive motility. We founded that HBMMSCs notably secrete interleukin‐8 (IL‐8) protein. Administration of IL‐8 specific neutralizing antibody significantly inhibits HBMMSCs‐mediated gastric cancer motility. Treatment of recombinant IL‐8 soluble protein confirmed the role of IL‐8 in mediating HBMMSCs‐up‐regulated cell motility. IL‐8 up‐regulates motility activity through Src signalling pathway in human gastric cancer. We further observed that 17β ‐estradiol inhibit HBMMSCS‐induced cell motility via suppressing activation of IL8‐Src signalling in human gastric cancer cells. 17β‐estradiol inhibits IL8‐up‐regulated Src downstream target proteins including p‐Cas, p‐paxillin, p‐ERK1/2, p‐JNK1/2, MMP9, tPA and uPA. These results suggest that 17β‐estradiol significantly inhibits HBMMSCS‐induced invasive motility through suppressing IL8‐Src signalling axis in human gastric cancer cells.     

Toll‐like receptor‐mediated IRE1α activation as a therapeutic target for inflammatory arthritis

In rheumatoid arthritis (RA), macrophage is one of the major sources of inflammatory mediators. Macrophages produce inflammatory cytokines through toll‐like receptor (TLR)‐mediated signalling during RA. Herein, we studied macrophages from the synovial fluid of RA patients and observed a significant increase in activation of inositol‐requiring enzyme 1α (IRE1α), a primary unfolded protein response (UPR) transducer. Myeloid‐specific deletion of the IRE1α gene protected mice from inflammatory arthritis, and treatment with the IRE1α‐specific inhibitor 4U8C attenuated joint inflammation in mice. IRE1α was required for optimal production of pro‐inflammatory cytokines as evidenced by impaired TLR‐induced cytokine production in IRE1α‐null macrophages and neutrophils. Further analyses demonstrated that tumour necrosis factor (TNF) receptor‐associated factor 6 (TRAF6) plays a key role in TLR‐mediated IRE1α activation by catalysing IRE1α ubiquitination and blocking the recruitment of protein phosphatase 2A (PP2A), a phosphatase that inhibits IRE1α phosphorylation. In summary, we discovered a novel regulatory axis through TRAF6‐mediated IRE1α ubiquitination in regulating TLR‐induced IRE1α activation in pro‐inflammatory cytokine production, and demonstrated that IRE1α is a potential therapeutic target for inflammatory arthritis.     

Z‐FA.FMK activates duodenal epithelial cell proliferation through oxidative stress,NF‐κB and IL‐1β in d‐GalN/TNF‐α‐administered mice

This study was designed to evaluate the effect of Z‐FA.FMK (benzyloxycarbonyl‐l ‐phenylalanyl‐alanine‐fluoromethylketone), a pharmacological inhibitor of cathepsin B, on the proliferation of duodenal mucosal epithelial cells and the cellular system that controls this mechanism in these cells in vivo. For this investigation, BALB/c male mice were divided into four groups. The first group received physiological saline, the second group was administered Z‐FA.FMK, the third group received d ‐GalN (d ‐galactosamine) and TNF‐α (tumour necrosis factor‐α) and the fourth group was given both d ‐GalN/TNF‐α and Z‐FA.FMK. When d ‐GalN/TNF‐α was administered alone, we observed an increase in IL‐1β‐positive and active NF‐κB‐positive duodenal epithelial cells, a decrease in PCNA (proliferative cell nuclear antigen)‐positive duodenal epithelial cells and an increase in degenerative changes in duodenum. On the other hand, Z‐FA.FMK pretreatment inhibited all of these changes. Furthermore, lipid peroxidation, protein carbonyl and collagen levels were increased, glutathione level and superoxide dismutase activity were decreased, while there was no change in catalase activity by d ‐GalN/TNF‐α injection. On the contrary, the Z‐FA.FMK pretreatment before d ‐GalN/TNF‐α blocked these effects. Based on these findings, we suggest that Z‐FA.FMK might act as a proliferative mediator which is controlled by IL‐1β through NF‐κB and oxidative stress in duodenal epithelial cells of d ‐GalN/TNF‐α‐administered mice.