Downregulation of lncRNA MALAT1 contributes to renal functional improvement after duodenal-jejunal bypass in a diabetic rat model

Ameliorated renal function has been reported after bariatric surgery, but the mechanisms underlying this phenomenon are not well-studied. To investigate whether the long non-coding RNA (lncRNA) MALAT1 mediates the amelioration of diabetic nephropathy after duodenal-jejunal bypass (DJB) surgery, rats were assigned randomly into four groups: diabetic (DM) group, DM with DJB surgery group, DM with sham surgery group, and healthy control group. Food intake, body weight, oral glucose tolerance test (OGTT), urine albumin excretion rate (UAER), and glomerular filtration rate (GFR) were measured and histological examination of renal sections was performed. For in vitro study, HK-2 cells were cultured under various glucose concentrations following MALAT1 siRNA transfection. Expression levels of MALAT1, SAA3, IL-6, and TNF-α in rat renal tissues or HK-2 cell lines were evaluated by qRT-PCR and/or ELISA. Results showed DJB surgery improved the renal function of diabetic rats, as indicated by ameliorated UAER and GFR and attenuated glomerular hypertrophy. Expression of MALAT1 and its downstream target SAA3 was significantly downregulated in renal tissues after DJB, which in turn decreased the expression of the pro-inflammatory cytokines IL-6 and TNF-α. Knockdown of MALAT1 in HK-2 cell lines further confirmed that expression levels of SAA3, IL-6, and TNF-α were regulated by MALAT1 under both low- and high-glucose conditions. Our findings suggest that MALAT1 is implicated in the improvement of renal function after DJB through regulation of its downstream targets SAA3, IL-6, and TNF-α.     

Activation of cultured human endothelial cells by recombinant lymphotoxin: comparison with tumor necrosis factor and interleukin 1 species

Recombinant human lymphotoxin (LT) was compared with recombinant human tumor necrosis factor (TNF) for direct actions on cultured human endothelial cells (HEC). At equivalent half-maximal concentrations (based on L929 cytotoxicity units) LT and TNF each caused rapid and transient induction (peak 4 to 6 hr) of an antigen associated with leukocyte adhesion (detected by monoclonal antibody H4/18), a rapid but sustained increased expression (plateau 24 hr) of a lymphocyte adhesion structure (ICAM-1), a gradual (plateau 4 to 6 days) increase in expression of HLA-A,B antigens, and gradual (4 to 6 days) conversion of HEC culture morphology from epithelioid to fibroblastoid, an effect enhanced by immune interferon (IFN-gamma). Induction of H4/18 binding by maximal concentrations of LT or TNF could not be augmented by addition of the other cytokine, and 24 hr pretreatment with LT or TNF produced hyporesponsiveness to both mediators for reinduction. H4/18 binding can be transiently induced by tumor-promoting phorbol esters. Pretreatment with either LT or TNF also fully inhibited induction of H4/18 binding by phorbol ester, whereas phorbol ester pretreatment only variably and partially inhibited reinduction by LT or TNF. These actions of LT on endothelium shared with TNF may serve in vivo to promote lymphocyte and inflammatory leukocyte adhesion and transendothelial migration. Recombinant human interleukin 1 species (IL 1 alpha and IL 1 beta) shared many of the actions of LT and TNF and were indistinguishable from each other. However, IL 1 species could be distinguished from LT/TNF by their relative inability to enhance HLA-A,B expression, by their ability to augment H4/18 binding caused by maximally effective concentrations of LT or TNF, and by their inability to inhibit reinduction of H4/18 binding by LT or TNF. In contrast to the actions of LT or TNF, pretreatment with IL 1 alpha or IL 1 beta only partially inhibited induction of H4/18 binding by phorbol ester, and phorbol ester pretreatment consistently, albeit partially, inhibited induction by IL 1 species. These studies suggest that activated T cells through the secretion of LT can in turn activate the local endothelial lining so as to promote homing and extravasation of inflammatory cells. Furthermore, these LT actions can be augmented or complemented by other locally produced mediators such as IFN-gamma or IL 1.     

The Pros1/Tyro3 axis protects against periodontitis by modulating STAT/SOCS signalling

Periodontitis, an oral inflammatory disease caused by periodontal pathogen infection, is the most prevalent chronic inflammatory disease and a major burden on healthcare. The TAM receptor tyrosine kinases (Tyro3, Axl and Mertk) and their ligands (Gas6 and Pros1) play a pivotal role in the resolution of inflammation and have been associated with chronic inflammatory and autoimmune diseases. In this study, we evaluated the effects of exogenous Pros1 in in vitro and in vivo models of periodontitis. We detected higher Pros1 but lower Tyro3 levels in inflamed gingival specimens of periodontitis patients compared with healthy controls. Moreover, Pros1 was mostly localized in the gingival epithelium of all specimens. In cultured human gingival epithelial cells (hGECs), Porphyromonas gingivalis LPS (p.g‐LPS) stimulation down‐regulated Pros1 and Tyro3. Exogenous Pros1 inhibited p.g‐LPS–induced production of TNF‐α, IL‐6, IL‐1β, MMP9/2 and RANKL in a Tyro3‐dependent manner as revealed by PCR, Western blot analysis, ELISA and gelatin zymography. Pros1 also restored Tyro3 expression down‐regulated by p.g‐LPS in hGECs. In rats treated with ligature and p.g‐LPS, administration of Pros1 attenuated periodontitis‐associated gingival inflammation and alveolar bone loss. Our mechanistic studies implicated SOCS1/3 and STAT1/3 as mediators of the in vitro and in vivo anti‐inflammatory effects of Pros1. Collectively, the findings from this work supported Pros1 as a novel anti‐inflammatory therapy for periodontitis.     

Activation of human bronchial epithelial cells by inflammatory cytokines IL‐27 and TNF‐α: Implications for immunopathophysiology of airway inflammation

Interleukin (IL)‐27 is a member of IL‐6/IL‐12 family cytokines produced by antigen‐presenting cells in immune responses. IL‐27 can drive the commitment of naive T cells to a T helper type 1 (Th1) phenotype and inhibit inflammation in later phases of infection. Human bronchial epithelial cells have been shown to express IL‐27 receptor complex. In this study, we investigated the in vitro effects of IL‐27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)‐α on the pro‐inflammatory activation of human primary bronchial epithelial cells and the underlying intracellular signaling mechanisms. IL‐27 was found to enhance intercellular adhesion molecule 1 (ICAM‐1) expression on the surface of human bronchial epithelial cells, and a synergistic effect was observed in the combined treatment of IL‐27 and TNF‐α on the expression of ICAM‐1. Although IL‐27 did not alter the basal IL‐6 secretion from bronchial epithelial cells, it could significantly augment TNF‐α‐induced IL‐6 release. These synergistic effects on the up‐regulation of ICAM‐1 and IL‐6 were partially due to the elevated expression of TNF‐α receptor (p55TNFR) induced by IL‐27. Further investigations showed that the elevation of ICAM‐1 and IL‐6 in human bronchial epithelial cells stimulated by IL‐27 and TNF‐α was differentially regulated by phosphatidylinositol 3‐OH kinase (PI3K)‐Akt, p38 mitogen‐activated protein kinase, and nuclear factor‐κB pathways. Our results therefore provide a new insight into the molecular mechanisms involved in airway inflammation. J. Cell. Physiol. 223:788–797, 2010. © 2010 Wiley‐Liss, Inc.     

β‐carotene suppresses Porphyromonas gingivalis lipopolysaccharide‐mediated cytokine production in THP‐1 monocytes cultured with high glucose condition

Periodontitis is associated with development of diabetes mellitus. Although lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a major pathogen of periodontitis, may lead the progression of diabetes complications, the precise mechanisms are unclear. We, therefore, investigated the effects of β‐carotene on production of Pg LPS‐induced inflammatory cytokines in human monocytes cultured high glucose (HG) condition. THP‐1 cells were cultured under 5.5 mM or 25 mM glucose conditions, and cells were stimulated with Pg LPS. To investigate the productivity of TNF‐α, IL‐6, and MCP‐1, cell supernatants were collected for ELISA. To examine the effects of NF‐kB signals on cytokine production, Bay11‐7082 was used. HG enhanced Pg LPS‐induced production of TNF‐α, IL‐6, and MCP‐1 via NF‐kB signals in THP‐1. β‐carotene suppressed the enhancement of the Pg LPS‐induced cytokine production in THP‐1 via NF‐κB inactivation. Our results suggest that β‐carotene might be a potential anti‐inflammatory nutrient for circulating Pg LPS‐mediated cytokine production in diabetic patients with periodontitis.     

Long non‐coding RNA MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis

Endometriosis is a common gynecological disease characterized by diminished apoptosis, sustained ectopic survival of dysfunctional endometrial cells. Hypoxia has been implicated as a crucial microenvironmental factor that contributes to endometriosis. It has been reported that long non‐coding RNA MALAT1 (lncRNA‐MALAT1) highly expressed in endometriosis and up‐regulated by hypoxia. Hypoxia may also induce autophagy, which might act as cell protective mechanism. However, the relationship between lncRNA‐MALAT1 and autophagy under hypoxia conditions in endometriosis remains unknown. In the present study, we found that both lncRNA‐MALAT1 and autophagy level were up‐regulated in ectopic endometrium from patients with endometriosis, and its expression level correlates positively with that of hypoxia‐inducible factor‐1α (HIF‐1α). In cultured human endometrial stromal cells, both lncRNA‐MALAT1 and autophagy were induced by hypoxia in a time‐dependent manner and lncRNA‐MALAT1 up‐regulation was dependent on HIF‐1α signalling. Our analyses also show that knockdown of lncRNA‐MALAT1 suppressed hypoxia induced autophagy. Furthermore, inhibiting autophagy with specific inhibitor 3‐Methyladenine (3‐MA) and Beclin1 siRNA enhanced apoptosis of human endometrial stromal cells under hypoxia condition. Collectively, our findings identify that lncRNA‐MALAT1 mediates hypoxia‐induced pro‐survival autophagy of endometrial stromal cells in endometriosis.     

Cerebral microvascular IL15 is a novel mediator of TNF action

The blood-brain barrier is a gatekeeper and modulatory interface for the CNS. Cerebral endothelial cells are the major component of the blood-brain barrier, and they modify inflammatory signals from the circulation to the CNS by production and secretion of secondary substances. The inflammatory mediators induced by tumor necrosis factor α (TNF) were determined by microarray analysis of RBE4 cerebral endothelial cells, at 0, 6, 12, or 24 h after TNF treatment. Interleukin (IL)-15 and its receptors were among the most robustly up-regulated genes. This was confirmed by real-time RT-PCR and western blotting. The three subunits of the IL15 receptor complex (IL15Rα, IL2Rβ, and IL2Rγ) showed differential regulation by TNF in their time course and amplitude of increased expression. Consistent with increased expression of the specific high affinity receptor IL15Rα, TNF increased cellular uptake of ¹²⁵I-IL15 and enhanced the fluorescent intensity of Alexa568-IL15 in RBE4 cells. TNF treatment in mice also increased the level of expression of IL15 receptors in enriched cerebral microvessels. We conclude that the cerebral microvascular IL15 system is a novel inflammatory mediator that transduces the actions of TNF.     

Lipopolysaccharide accelerates collagen‐induced arthritis in association with rapid and continuous production of inflammatory mediators and anti‐type II collagen antibody

Collagen‐induced arthritis (CIA) is an animal model for rheumatoid arthritis (RA). Lipopolysaccharide (LPS) is known to accelerate CIA; however, the pathogenetic mechanisms are not yet fully understood. In this study, type II collagen (CII)‐immunized mice were found to have marked increases in degree of expression of mRNA of inflammatory mediators such as tumor necrosis factor alpha (TNF‐α), interleukin (IL)‐1β, and macrophage inflammatory protein‐2 (MIP‐2) in their arthritic paws and of serum anti‐CII antibody concentration before the onset of arthritis induced by LPS injection. The gene expression was rapid and continuous after direct activation of nuclear factor κB. The amounts of mRNA of TNF‐α, IL‐1β, and MIP‐2, as well as of matrix metalloproteinases and the receptor activator of nuclear factor κB ligand, increased with the development of arthritis, correlated positively with clinical severity and corresponded with histopathological changes. Moreover, anti‐TNF‐α neutralizing antibody inhibited the development of LPS‐accelerated CIA and a single injection of recombinant mouse TNF‐α induced increases in anti‐CII antibody concentrations, suggesting TNF‐α may contribute to the development of arthritis by both initiation of inflammation and production of autoantibodies. These data suggest that exacerbation of RA by LPS is associated with rapid and continuous production of inflammatory mediators and autoantibodies.     

Anti‐inflammatory effects of activated protein C on human dendritic cells

Activated protein C (APC) has an anticoagulant action and plays an important role in blood coagulation homeostasis. In addition to its anticoagulant action, APC is known to have cytoprotective effects, such as anti‐apoptotic action and endothelial barrier protection, on vascular endothelial cells and monocytes. However, the effects of APC on DCs have not been clarified. To investigate the effects of APC on human DCs, monocytes were isolated from peripheral blood and DC differentiation induced with LPS. APC significantly inhibited the production of inflammatory cytokines TNF‐α and IL‐6 during differentiation of immature DCs to mature DCs, but did not inhibit the production of IL‐12 and anti‐inflammatory cytokine IL‐10. Interestingly, treatment with 5 μg/mL, but not 25 μg/mL, of APC significantly enhanced production of IL‐10. In addition, protein C, which is the zymogen of APC, did not affect production of these cytokines. On the other hand, flow cytometric analysis of DC's surface molecules indicated that APC does not significantly affect expression of CD83, a marker of mDC differentiation, and the co‐stimulatory molecules CD40, CD80 and CD86. These results suggest that APC has anti‐inflammatory effects on human DCs and may be effective against some inflammatory diseases in which the pathogenesis involves TNF‐α and/or IL‐6 production.     

5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone attenuates LPS‐induced inflammation and ROS production in EA.hy926 cells via HMBOX1 induction

Inflammation and reactive oxygen species (ROS) are important factors in the pathogenesis of atherosclerosis (AS). 5,2′‐dibromo‐2,4′,5′‐trihydroxydiphenylmethanone (TDD), possess anti‐atherogenic properties; however, its underlying mechanism of action remains unclear. Therefore, we sought to understand the therapeutic molecular mechanism of TDD in inflammatory response and oxidative stress in EA.hy926 cells. Microarray analysis revealed that the expression of homeobox containing 1 (HMBOX1) was dramatically upregulated in TDD‐treated EA.hy926 cells. According to the gene ontology (GO) analysis of microarray data, TDD significantly influenced the response to lipopolysaccharide (LPS); it suppressed the LPS‐induced adhesion of monocytes to EA.hy926 cells. Simultaneously, TDD dose‐dependently inhibited the production or expression of IL‐6, IL‐1β, MCP‐1, TNF‐α, VCAM‐1, ICAM‐1 and E‐selectin as well as ROS in LPS‐stimulated EA.hy926 cells. HMBOX1 knockdown using RNA interference attenuated the anti‐inflammatory and anti‐oxidative effects of TDD. Furthermore, TDD inhibited LPS‐induced NF‐κB and MAPK activation in EA.hy926 cells, but this effect was abolished by HMBOX1 knockdown. Overall, these results demonstrate that TDD activates HMBOX1, which is an inducible protective mechanism that inhibits LPS‐induced inflammation and ROS production in EA.hy926 cells by the subsequent inhibition of redox‐sensitive NF‐κB and MAPK activation. Our study suggested that TDD may be a potential novel agent for treating endothelial cells dysfunction in AS.     

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.     

IL‐35 recombinant protein reverses inflammatory bowel disease and psoriasis through regulation of inflammatory cytokines and immune cells

Interleukin‐35 (IL‐35), a member of the IL‐12 family, functions as a new anti‐inflammatory factor involved in arthritis, psoriasis, inflammatory bowel disease (IBD) and other immune diseases. Although IL‐35 can significantly prevent the development of inflammation in many diseases, there have been no early studies accounting for the role of IL‐35 recombinant protein in IBD and psoriasis. In this study, we assessed the therapeutic potential of IL‐35 recombinant protein in three well‐known mouse models: the dextransulfate sodium (DSS)‐induced colitis mouse model, the keratin14 (K14)‐vascular endothelial growth factor A (VEGF‐A)‐transgenic (Tg) psoriasis mouse model and the imiquimod (IMQ)‐induced psoriasis mouse model. Our results indicated that IL‐35 recombinant protein can slow down the pathologic process in DSS‐induced acute colitis mouse model by decreasing the infiltrations of macrophages, CD4⁺T and CD8⁺T cells and by promoting the infiltration of Treg cells. Further analysis demonstrated that IL‐35 recombinant protein may regulate inflammation through promoting the secretion of IL‐10 and inhibiting the expression of pro‐inflammatory cytokines such as IL‐6, TNF‐α and IL‐17 in acute colitis model. In addition, lower dose of IL‐35 recombinant protein could achieve long‐term treatment effects as TNF‐α monoclonal antibody did in the psoriasis mouse. In summary, the remarkable therapeutic effects of IL‐35 recombinant protein in acute colitis and psoriasis mouse models indicated that IL‐35 recombinant protein had a variety of anti‐inflammatory effects and was expected to become an effective candidate drug for the treatment of inflammatory diseases.     

Regulation of lung fibroblast activation by annexin A1

Annexin‐A1 (AnxA1) is a glucocorticoid‐induced protein with multiple actions in the regulation of inflammatory cell activation. The contribution of AnxA1 to human cell biology is not well understood. We investigated the contribution of AnxA1 and its receptor, formyl‐peptide receptor 2 (FPR2), to the regulation of inflammatory responses in human normal lung fibroblasts (NLF). Silencing constitutive AnxA1 expression in NLF using small interfering RNA (siRNA) was associated with moderate but significant increases in tumor necrosis factor (TNF)‐induced proliferation and interleukin (IL)‐6 production, accompanied by reduction of ERK and NF‐κB activity. AnxA1 regulation of ERK and NF‐κB activation was associated with effects on proliferation. Blocking FPR2 using the specific antagonist WRW4 mimicked the effects of AnxA1 silencing on TNF‐induced proliferation, IL‐6, ERK, and NF‐κB activation. AnxA1 silencing also impaired inhibitory effects of glucocorticoid on IL‐6 production and on the expression of glucocorticoid‐induced leucine zipper (GILZ), but blocking FPR2 failed to mimic these effects of AnxA1 silencing. These data suggest that AnxA1 regulates TNF‐induced proliferation and inflammatory responses in lung fibroblasts, via effects on the ERK and NF‐κB pathways, which depend on FPR2. AnxA1 also mediates effects of glucocorticoids and GILZ expression, but these effects appear independent of FPR2. These findings suggest that mimicking AnxA1 actions might have therapeutic potential in chronic inflammatory lung diseases. J. Cell. Physiol. 228: 476–484, 2013. © 2012 Wiley Periodicals, Inc.