Human mesenchymal stromal cells modulate T‐cell responses through TNF‐α‐mediated activation of NF‐κB

Although mesenchymal stromal cells (MSCs) possess the capacity to modulate immune responses, little is known about the mechanisms that underpin these processes. In this study, we show that immunosupression is mediated by activation of nuclear factor kappa B (NF‐κB) in human MSCs. This pathway is activated by TNF‐α that is generated following TCR stimulation of T cells. Inhibition of NF‐κB through silencing of IκB kinase β or the TNF‐α receptor abolishes the immunosuppressive capacity of MSCs. Our data also indicate that MSC‐associated NF‐κB activation primarily leads to inhibition of T‐cell proliferation with little effect on expression of the activation markers CD69 and CD25. Thus, our data support the hypothesis that the TNF‐α/NF‐κB signalling pathway is required for the initial priming of immunosuppressive function in human MSCs. Interestingly, drugs that interfere with NF‐κB activation significantly antagonise the immunoregulatory effect of MSCs, which could have important implications for immunosuppression regimens in the clinic.     

Vitamin K3 attenuates lipopolysaccharide‐induced acute lung injury through inhibition of nuclear factor‐κB activation

Vitamin K is a family of fat‐soluble compounds including phylloquinone (vitamin K₁), menaquinone (vitamin K₂) and menadione (vitamin K₃). Recently, it was reported that vitamin K, especially vitamins K₁ and K₂, exerts a variety of biological effects, and these compounds are expected to be candidates for therapeutic agents against various diseases. In this study, we investigated the anti‐inflammatory effects of vitamin K₃ in in vitro cultured cell experiments and in vivo animal experiments. In human embryonic kidney (HEK)293 cells, vitamin K₃ inhibited the tumour necrosis factor (TNF)‐α‐evoked translocation of nuclear factor (NF)‐κB into the nucleus, although vitamins K₁ and K₂ did not. Vitamin K₃ also suppressed the lipopolysaccharide (LPS)‐induced nuclear translocation of NF‐κB and production of TNF‐α in mouse macrophage RAW264·7 cells. Moreover, the addition of vitamin K₃ before and after LPS administration attenuated the severity of lung injury in an animal model of acute lung injury/acute respiratory distress syndrome (ARDS), which occurs in the setting of acute severe illness complicated by systemic inflammation. In the ARDS model, vitamin K₃ also suppressed the LPS‐induced increase in the serum TNF‐α level and inhibited the LPS‐evoked nuclear translocation of NF‐κB in lung tissue. Despite marked efforts, little therapeutic progress has been made, and the mortality rate of ARDS remains high. Vitamin K₃ may be an effective therapeutic strategy against acute lung injury including ARDS.     

IRF‐5 and NF‐κB p50 co‐regulate IFN‐β and IL‐6 expression in TLR9‐stimulated human plasmacytoid dendritic cells

Synthetic oligonucleotides (ODN) expressing CpG motifs mimic the ability of bacterial DNA to trigger the innate immune system via TLR9. Plasmacytoid dendritic cells (pDCs) make a critical contribution to the ensuing immune response. This work examines the induction of antiviral (IFN‐β) and pro‐inflammatory (IL‐6) cytokines by CpG‐stimulated human pDCs and the human CAL‐1 pDC cell line. Results show that interferon regulatory factor‐5 (IRF‐5) and NF‐κB p50 are key co‐regulators of IFN‐β and IL‐6 expression following TLR9‐mediated activation of human pDCs. The nuclear accumulation of IRF‐1 was also observed, but this was a late event that was dependant on type 1 IFN and unrelated to the initiation of gene expression. IRF‐8 was identified as a novel negative regulator of gene activation in CpG‐stimulated pDCs. As variants of IRF‐5 and IRF‐8 were recently found to correlate with susceptibility to certain autoimmune diseases, these findings are relevant to our understanding of the pharmacologic effects of “K” ODN and the role of TLR9 ligation under physiologic, pathologic, and therapeutic conditions.     

Inhibitory effect of co‐administration of atorvastatin and endothelin‐1 receptor antagonist on the progression of atherosclerosis in rabbit

Atorvastatin, a hydroxymethylglutaryl‐CoA reductase inhibitor, and endothelin‐1 (ET‐1) receptor antagonist have been separately indicated to ameliorate disease progression in atherosclerosis. However, no study has evaluated the effect of their combination on atherosclerosis. The objective of the current study was to evaluate the direct in vivo effects of a combination regimen of atorvastatin and ET‐1 receptor antagonist on male New Zealand white rabbit models of atherosclerosis (injury‐induced). Thirty‐two atherosclerotic rabbits were divided into four experimental groups: (a) injury group – fed high‐fat diet; (b) ET‐1 receptor antagonist preventive group – fed high‐fat diet, but with intragastric administration of the ET‐1 receptor antagonist, darusentan; (c) combined preventive group – fed high‐fat diet, but with intragastric administration of both darusentan and atorvastatin; and (d) treatment group – fed high‐fat diet for the first 8 weeks, followed by normal diet and intragastric administration of both darusentan and atorvastatin up to 16 weeks. A further eight non‐atherosclerotic rabbits were fed normal diet and classified as the control group. At the end of 8 and 16 weeks, compared with the injury group, the combined preventive group had significant reduction in both the concentration of serum lipids and inflammatory factors and atherosclerosis formation, indicative of a multifaceted anti‐atherosclerotic impact. The relative area of atherosclerotic lesions in the injury group (30.84%) was significantly higher than the control group (4.62%; p < 0.05). The combined preventive group showed a significantly robust effect on lowering serum lipid, inflammatory cytokines, and maintained homeostatic balance of free radicals, and important downstream effectors like ET‐1 and matrixmetalloproteinase‐9. Our data show that atorvastatin and ET‐1 receptor antagonist co‐administration may decrease lipid levels, stabilize plaques and relieve vascular inflammation. By reducing the plaque burden, this regimen may minimize the risk of atherosclerotic plaque rupture or arterial occlusion.     

Wogonin prevents lipopolysaccharide‐induced acute lung injury and inflammation in mice via peroxisome proliferator‐activated receptor gamma‐mediated attenuation of the nuclear factor‐kappaB pathway

Acute lung injury (ALI) from a variety of clinical disorders, characterized by diffuse inflammation, is a cause of acute respiratory failure that develops in patients of all ages. Previous studies reported that wogonin, a flavonoid‐like chemical compound which was found in Scutellaria baicalensis, has anti‐inflammatory effects in several inflammation models, but not in ALI. Here, the in vivo protective effect of wogonin in the amelioration of lipopolysaccharide (LPS) ‐induced lung injury and inflammation was assessed. In addition, the in vitro effects and mechanisms of wogonin were studied in the mouse macrophage cell lines Ana‐1 and RAW264.7. In vivo results indicated that wogonin attenuated LPS‐induced histological alterations. Peripheral blood leucocytes decreased in the LPS‐induced group, which was ameliorated by wogonin. In addition, wogonin inhibited the production of several inflammatory cytokines, including tumour necrosis factor‐α, interleukin‐1β (IL‐1β) and IL‐6, in the bronchoalveolar lavage fluid and lung tissues after LPS challenge, while the peroxisome proliferator‐activated receptor γ (PPARγ) inhibitor GW9662 reversed these effects. In vitro results indicated that wogonin significantly decreased the secretion of IL‐6, IL‐1β and tumour necrosis factor‐α in Ana‐1 and RAW264.7 cells, which was suppressed by transfection of PPARγ small interfering RNA and GW9662 treatment. Moreover, wogonin activated PPARγ, induced PPARγ‐mediated attenuation of the nuclear translocation and the DNA‐binding activity of nuclear factor‐κB in vivo and in vitro. In conclusion, all of these results showed that wogonin may serve as a promising agent for the attenuation of ALI‐associated inflammation and pathology by regulating the PPARγ‐involved nuclear factor‐κB pathway.     

Cytosolic phospholipase A2α has a crucial role in the pathogenesis of DSS‐induced colitis in mice

Colitis, an inflammation of the colon, is a well‐characterized massive tissue injury. Cytosolic phospholipase A₂α (cPLA₂α) upregulation plays an important role in the development of several inflammatory diseases. The aim of the present study was to define the role of cPLA₂α upregulation in the development of colitis. We used a mouse model of dextran sulfate sodium induced colitis. Immunoblotting analysis showed that cPLA₂α and NF‐κB were upregulated and activated in the colon from day 2 of colitis induction. This molecular event preceded the development of the disease, as determined by Disease Activity Index score, body weight, colon length, and the expression of colonic inflammatory markers, including neutrophil infiltration detected by myeloperoxidase and by NIMP‐R14, ICAM‐1, COX‐2, iNOS upregulation and LTB₄ and TNF‐α secretion. Prevention of cPLA₂α upregulation and activity in the colon by i.v. administration of specific antisense oligonucleotides against cPLA₂α 1 day prior and every day of exposure to dextran sulfate sodium significantly impeded the development of the disease and prevented NF‐κB activation, neutrophils infiltration into the colonic mucosa, and expression of proinflammatory proteins in the colon. Our results demonstrate a critical role of cPLA₂α upregulation in inflammation and development of murine colitis.     

The purinergic P2×7 receptor is expressed on monocytes in Behçet's disease and is modulated by TNF‐α

The P2×7 receptor (P2×7r) is expressed in innate immune cells (e.g. monocyte/macrophages), playing a key role in IL‐1β release. Since innate immune activation and IL‐1β release seem to be implicated in Behçet's disease (BD), a systemic immune‐inflammatory disorder of unknown origin, we hypothesized that P2×7r is involved in the pathogenesis of the disease. Monocytes were isolated from 18 BD patients and 17 healthy matched controls. In BD monocytes, an increased P2×7r expression and Ca²⁺ permeability induced by the selective P2×7r agonist 2′‐3′‐O‐(4‐benzoylbenzoyl)ATP (BzATP) was observed. Moreover, IL‐1β release from LPS‐primed monocytes stimulated with BzATP was markedly higher in BD patients than in controls. TNF‐α‐incubated monocytes from healthy subjects almost reproduced the findings observed in BD patients, as demonstrated by the increase in P2×7r expression and BzATP‐induced Ca²⁺ intake. Our results provide evidence that in BD monocytes both the expression and function of the P2×7r are increased compared with healthy controls, as the possible result, at least in part, of a positive modulating effect of TNF‐α on the receptor. These data indicate P2×7r as a new potential therapeutic target for the control of BD, further supporting the rationale for the use of anti‐TNF‐α drugs in the treatment of the disease.     

The Role of TLR4, TNF‐α and IL‐1β in Type 2 Diabetes Mellitus Development within a North Indian Population

This study investigated the role of IL‐1β‐511 (rs16944), TLR4‐896 (rs4986790) and TNF‐α‐308 (rs1800629) polymorphisms in type 2 diabetes mellitus (T2DM) among an endogamous Northern Indian population. Four hundred fourteen participants (204 T2DM patients and 210 nondiabetic controls) were genotyped for IL‐1β‐511, TLR4‐896 and TNF‐α‐308 loci. The C allele of IL‐1β‐511 was shown to increase T2DM susceptibility by 75% (OR: 1.75 [CI 1.32–2.33]). Having two parents affected by T2DM increased susceptibility by 5.7 times (OR: 5.693 [CI 1.431–22.648]). In this study, we have demonstrated a conclusive association with IL‐1β‐511 locus and IL‐1β‐511‐TLR4‐896 diplotype (CC‐AA) and T2DM, which warrants further comprehensive analyses in larger cohorts.     

Lipopolysaccharide (LPS)‐induced Interferon (IFN)‐gamma Production by Decidual Mononuclear Cells (DMNC) is Interleukin (IL)‐2 and IL‐12 Dependent

Citation Negishi M, Izumi Y, Aleemuzzaman S, Inaba N, Hayakawa S. Lipopolysaccharide (LPS)‐induced interferon (IFN)‐gamma production by decidual mononuclear cells (DMNC) is interleukin (IL)‐2 and IL‐12 dependent. Am J Reprod Immunol 2011; 65: 20–27 Problem Th1‐shifted immune response is believed to be harmful for successful pregnancy because of activation of maternal cytotoxic T lymphocytes and natural killer cells. However, its effects on Toll‐like receptor (TLR)‐mediated innate immune response are so far unknown and this study has been undertaken to address the issue. Method of study Decidual tissues were obtained from 16 pregnant women undergoing elective termination during the first trimester pregnancy for socioeconomic reasons. Decidual Mononuclear Cells (DMNC) were stimulated with suboptimal doses of IL‐2 and IL‐12 with/without LPS, considered to be a TLR4 ligand, for 48 hr. Productions of IFN‐γ and tumor necrosis factor (TNF)‐α in culture supernatant were measured with ELISA. Results (i) IFN‐γ production was induced with LPS alone which was strongly up‐regulated in the presence of IL‐2 and IL‐12. (ii) TNF‐α was also induced by LPS but was not affected by the presence of IL‐2 and IL‐12. Conclusion IL‐2 and IL‐12 up‐regulated the production of IFN‐γ in DMNC through increasing their susceptibility to LPS. TNF‐α production is independent of such a mechanism.