Summary ‘Circulating’ T follicular helper cells (Tfh), characterized by their surface phenotypes CD4+chemokine receptor 5 (CXCR5)+ inducible co‐stimulatory molecule (ICOS)+, have been identified as the CD4+ T cell subset specialized in supporting the activation, expansion and differentiation of B cells. Fibroblast‐like synoviocytes (FLS) are critical in promoting inflammation and cartilage destruction in rheumatoid arthritis (RA), and the interaction between FLS and T cells is considered to facilitate FLS activation and T cell recruitment. However, it remains unknown whether RA‐FLS co‐cultured with activated peripheral blood mononuclear cells (PBMC) has immunoregulatory effects on peripheral Tfh. In the present study, we co‐cultured RA‐FLS with or without anti‐CD3/CD28‐stimulated PBMC. The results showed that RA‐FLS co‐cultured with stimulated PBMC could increase the numbers of CD4+CXCR5+ICOS+ T cells of RA PBMC possibly via the production of interleukin (IL)‐6, a critical cytokine involved in the differentiation of Tfh cells. We also observed increased reactive oxygen species (ROS) levels in the co‐culture system of RA‐FLS and PBMC. The percentage of CD4+CXCR5+ICOS+ T cells was decreased when ROS production was inhibited by N‐acetyl‐L‐cysteine (NAC), a specific inhibitor which can decrease ROS production. In addition, we showed that the higher levels of tumour necrosis factor (TNF)‐α and IL‐1β in the co‐culture system and the blocking of TNF receptor 2 (TNF‐R2) and IL‐1β receptor (IL‐1βR) both decreased the numbers of CD4+CXCR5+ICOS+ T cells. Our study reveals a novel mechanistic insight into how the interaction of RA‐FLS and PBMC participates in the RA pathogenesis, and also provides support for the biologicals application for RA. In the RA‐FLS and activated PBMC coculture system, we found that the numbers of CD4+CXCR5+ICOS+ T cells in RA PBMC were increased possibly via the production of IL‐6 and ROS. In addition, it showed the higher levels of TNF‐α and IL‐1β in the coculture system, and the blocking of TNFR2 and IL‐1βR both decreased the numbers of CD4+CXCR5+ICOS+ T cells.
Abstract Background Cell therapy for cardiovascular disease has been limited by low engraftment of administered cells and modest therapeutic effects. Bone marrow (BM) -derived CD31+ cells are a promising cell source owing to their high angiovasculogenic and paracrine activities. Objectives This study sought to identify culture conditions that could augment the cell adhesion, angiogenic, and anti-inflammatory activities of BM-derived CD31+ cells, and to determine whether these cultured CD31+ cells are effective for cardiac and vascular repair. Methods CD31+ cells were isolated from human BM by magnetic-activated cell sorting and cultured for 10 days under hematopoietic stem cell, mesenchymal stem cell, or endothelial cell culture conditions. These cells were characterized by adhesion, angiogenesis, and inflammatory assays. The best of the cultured cells were implanted into myocardial infarction (MI) and hindlimb ischemia (HLI) models to determine therapeutic effects and underlying mechanisms. Results The CD31+ cells cultured in endothelial cell medium (EC-CD31+ cells) showed the highest adhesion and angiogenic activities and lowest inflammatory properties in vitro compared with uncultured or other cultured CD31+ cells. When implanted into mouse MI or HLI models, EC-CD31+ cells improved cardiac function and repaired limb ischemia to a greater extent than uncultured CD31+ cells. Histologically, injected EC-CD31+ cells exhibited higher retention, neovascularization, and cardiomyocyte proliferation. Importantly, cell retention and endothelial transdifferentiation was sustained up to 1 year. Conclusions Short-term cultured EC-CD31+ cells have higher cell engraftment, vessel-formation, cardiomyocyte proliferation, and anti-inflammatory potential, are highly effective for both cardiac and peripheral vascular repair, and enhance survival of mice with heart failure. These cultured CD31+ cells may be a promising source for treating ischemic cardiovascular diseases.
Vitamin D is a regulator of host defense against infections and induces expression of the antimicrobial peptide hCAP18/LL-37. Vitamin D deficiency is associated with chronic inflammatory lung diseases and respiratory infections. However, it is incompletely understood if and how (chronic) airway inflammation affects vitaminD metabolism and action. We hypothesized that long-term exposure of primary bronchial epithelial cells to proinflammatory cytokines alters their vitamin D metabolism, antibacterial activity, and expression of hCAP18/LL-37. To investigate this, primary bronchial epithelial cells were differentiated at the air-liquid interface for 14 days in the presence of the proinflammatory cytokines, TNF-alpha and IL-1 beta (TNF-alpha/IL-1 beta), and subsequently exposed to vitamin D (inactive 25(OH) D3 and active 1,25(OH)(2)D-3). Expression of hCAP18/LL-37, vitamin D receptor, and enzymes involved in vitamin D metabolism (CYP24A1 and CYP27B1) was determined using quantitative PCR, Western blot, and immunofluorescence staining. Furthermore, vitamin D-mediated antibacterial activity was assessed using nontypeable Haemophilus influenzae. We found that TNF-alpha/IL-1 beta treatment reduced vitaminD-induced expression of hCAP18/LL-37 and killing of nontypeable H. influenzae. In addition, CYP24A1 (a vitamin D-degrading enzyme) was increased by TNF-alpha/IL-1 beta, whereas CYP27B1 (that converts 25(OH) D-3 to its active form) and vitaminDreceptor expression remained unaffected. Furthermore, we have demonstrated that the TNF-alpha/IL1 beta-mediated induction of CYP24A1 was, at least in part, mediated by the transcription factor specific protein 1, and the epidermal growth factor receptor- mitogen-activated protein kinase pathway. These findings indicate that TNF-alpha/IL-1 beta decreases vitamin D-mediated antibacterial activity and hCAP18/LL-37 expression via induction of CYP24A1 and suggest that chronic inflammation impairs protective responses induced by vitamin D.
► Regulation of astrocytic e-5NT by TNF-α, LPS, IFN-γ, Glu and H O is investigated. ► LPS, IFN-γ, Glu and H O decrease, whereas TNF-α increases e-5NT activity. ► LPS, IFN-γ and Glu down-regulate e-5NT, while H O modulates its enzymatic efficiancy. ► TNF-α increases e-5NT protein abundance, without alteration in mRNA expression level. Ecto-5′-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H O ) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H O decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.
Background: Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer's disease (AD). AD is associated to oligomers of the amyloid beta peptide (A beta o) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and A beta o in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. Methods: Ca2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short-and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and A beta o on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4. Results: LPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with A beta o increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death. Conclusions: Aging and amyloid beta oligomers, the neurotoxin involved in Alzheimer's disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.
The inflammasomes are cytoplasmic multiprotein complexes that are responsible for activation of inflammatory reactions. In principle, there are four individual inflammasome branches (NLRP1, NLRP3, NLRC4/NALP4, and AIM2) that mediate the cleavage and activation of Caspase-1 and IL-1β that in turn lead to a complex network of cellular reactions initiating local and systemic inflammatory reactions. We have recently shown that NLRP3 expression is virtually absent in primary cultured hepatocytes and that the stimulation of hepatocytes with lipopolysaccharides results in strong activation of NLRP3 expression. We here demonstrate that this activation can be blocked by the NF-κB activation inhibitor QNZ or by infection with an adenoviral expression vector constitutively expressing a superrepressor of NF-κB. We show that QNZ blocks NF-κB-dependent expression of TNF-α, IL-1β and NLRP3. Likewise, the superrepressor of NF-κB prevents expression of NLRP3 and significantly reduces expression of inflammatory marker genes in liver cells. In a primary murine hepatoma cells, the concomitant depletion of NEMO and Caspase-8 resulted in a significant suppression of NLRP3 expression after Lipopolysaccharide challenge. Moreover, we demonstrate that a 1.3-kbp fragment located in close proximity of the most upstream transcriptional start site of the human gene that harbours one putative octamer NF-κB binding site renders LPS sensitivity in reporter gene assay. We conclude that NF-κB signalling is a necessary prerequisite for proper activation of the NLRP3 inflammasome in primary hepatocytes.
Scope Obese adipose tissue (AT) is infiltrated by inflammatory immune cells including IL‐17A‐producing‐T (Th17) cells. It has been previously demonstrated that adipose‐derived stem cells from obese (ob‐ASCs), but not lean AT promote Th17 cells. Because n‐3 PUFAs are known to inhibit obese AT inflammation, it is tested here whether they could inhibit ob‐ASC‐mediated IL‐17A secretion. Methods and Results The n‐3 PUFA precursor, alpha‐linolenic acid (ALA), or its derivatives, eicosapentaenoic, or docosahexaenoic acid, is added to co‐cultures of human ob‐ASCs and mononuclear cells (MNCs). All three inhibited IL‐17A, but not IL‐1β, IL‐6, nor TNFα secretion. As a control, palmitic acid (PA), a saturated fatty acid, did not inhibit IL‐17A secretion. ALA also inhibited IL‐17A secretion mediated by adipocytes differentiated from ob‐ASCs. Toll‐like‐receptor 4 is shown to be involved in ob‐ASC‐mediated‐IL‐17A secretion, and to be inhibited by ALA, together with Cyclo‐Oxygenase‐2 and Signal‐Transducer‐and‐Activator‐of‐transcription‐3. In addition, ALA down‐regulated Intercellular‐Adhesion‐Molecule‐1 (ICAM‐1) expression in both monocytes and ASCs, which resulted in decreased interactions between ob‐ASCs and MNCs, and inhibition of IL‐17A secretion. Conclusion It is demonstrated herein that ALA inhibits Th17 cell promotion, through decreased ICAM‐1expression in both ob‐ASCs and monocytes. This novel mechanism may contribute to explain the beneficial effects of n‐3 PUFA in IL‐17A‐related inflammatory pathologies. n‐3 PUFAs decrease ASC‐mediated IL‐17 A secretion, through ICAM‐1 inhibition of expression. ASCs isolated from obese, but not lean adipose tissue (AT) are previously shown to promote Th17 cells, activate IL‐1β and IL‐6 secretion, and inhibit Th1 cells. Here, it is demonstrated that n‐3PUFAs inhibit ICAM‐1‐mediated cell interactions between ASCs and mononuclear cells, which results in inhibition of IL‐17A secretion.
3,3'-Diindolylmethane (DIM), a digestive metabolite originating from cruciferous vegetables, has dietary potential for the treatment of various human intestinal diseases. Although intestinal permeability dysfunction is closely related to the initiation and progression of human intestinal inflammatory diseases (IBDs), the effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM on the intestinal permeability of human intestinal cell monolayers and the animal model Caenorhabditis elegans, which were treated with IL-1 beta and Pseudomonas aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the intestinal permeability of differentiated Caco-2 cells by enhancing the expression of tight junction proteins (including occludin and ZO-1). Compared to the IL-1 beta single treatment (551.0 +/- 49.0 Omega.cm(2)), DIM (10 mu M) significantly increased the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 +/- 66.4 Omega.m(2), p < 0.001). DIM also ameliorated the impaired intestinal permeability and extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-treated worms (10.8 +/- 1.3 days) was higher than that of control-treated worms (9.7 +/- 1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment of leaky gut syndrome by improving intestinal permeability.
Peroxisome proliferator‐activated receptors (PPARs) are potentially useful for the treatment of skin diseases, because they stimulate keratinocyte differentiation, exert anti‐inflammatory effects and improve barrier function. We examined five PPAR‐γ agonists, including four thiazolidinediones (ciglitazone, troglitazone, rosiglitazone and pioglitazone) and an angiotensin‐II receptor blocker (telmisartan), for their ability to upregulate filaggrin and loricrin expression at both mRNA and protein levels in cultured normal human keratinocytes (NHKs). Troglitazone, rosiglitazone, pioglitazone and telmisartan significantly increased filaggrin expression at both mRNA and protein levels in calcium‐induced differentiated NHKs. Rosiglitazone and pioglitazone, but not troglitazone nor telmisartan, also significantly increased loricrin expression at both mRNA and protein levels in differentiated NHKs. These effects were not found in undifferentiated NHKs nor differentiated NHKs treated with ciglitazone. This study revealed differential effects of various PPAR‐γ agonists on epidermal differentiation, and the most potent of those are rosiglitazone and pioglitazone.