Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.     

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‐arthritis activity of cationic materials

Cationic materials exhibit remarkable anti‐inflammatory activity in experimental arthritis models. Our aim was to confirm this character of cationic materials and investigate its possible mechanism. Adjuvant‐induced arthritis (AIA) models were used to test cationic materials for their anti‐inflammatory activity. Cationic dextran (C‐dextran) with different cationic degrees was used to investigate the influence of the cationic elements of materials on their anti‐inflammatory ability. Peritoneal macrophages and spleen cells were used to test the expression of cytokines stimulated by cationic materials. Interferon (IFN)‐γ receptor‐deficient mice and macrophage‐depleted rats were used to examine the possible mechanisms of the anti‐inflammatory activity of cationic materials. In AIA models, different cationic materials shared similar anti‐inflammatory characters. The anti‐inflammatory activity of C‐dextran increased with as the cationic degree increased. Cationic materials stimulated interleukin (IL)‐12 expression in peritoneal macrophages, and strong stimulation of IFN‐γ secretion was subsequently observed in spleen cells. In vivo experiments revealed that circulating IL‐12 and IFN‐γ were enhanced by the cationic materials. Using IFN‐γ receptor knockout mice and macrophage‐depleted rats, we found that IFN‐γ and macrophages played key roles in the anti‐inflammatory activity of the materials towards cells. We also found that neutrophil infiltration at inflammatory sites was reduced when AIA animals were treated with C‐dextran. We propose that cationic signals act through an unknown receptor on macrophages to induce IL‐12 secretion, and that IL‐12 promotes the expression of IFN‐γ by natural killer cells (or T cells). The resulting elevated systemic levels of IFN‐γ inhibit arthritis development by preventing neutrophil recruitment to inflammatory sites.     

Attenuated accumulation of regulatory T cells and reduced production of interleukin 10 lead to the exacerbation of tissue injury in a mouse model of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a pathological condition that involves diffuse lung injury and severe hypoxemia caused by pulmonary and systemic diseases. We have established a mouse model of severe ARDS, developed by intratracheal injection of α‐galactosylceramide (α‐GalCer), an activator of natural killer T (NKT) cells, followed by LPS. In the present study, we used this model to investigate the regulatory mechanism in the early inflammatory response during acute lung injury. In α‐GalCer/LPS‐treated mice, the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and the expression of a Treg cell‐tropic chemokine, secondary lymphoid‐tissue chemokine (SLC), in the lungs was significantly lower than in mice treated with LPS alone. Giving recombinant (r)SLC increased the number of Treg cells in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced the expression of SLC and the accumulation of Treg cells in the lungs of α‐GalCer/LPS‐treated mice, whereas giving recombinant (r)IFN‐γ reduced the number of Treg cells in mice treated with LPS alone. IL‐10 production was significantly lower in α‐GalCer/LPS‐treated mice than in mice treated with LPS alone. Giving rIL‐10 prolonged survival and attenuated lung injury as a result of reduced production of inflammatory cytokines (such as IL‐1β, IL‐6, TNF‐α, and IFN‐γ) and chemokines (including MCP‐1, RANTES, IP‐10, Mig, MIP‐2, and KC) in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced IL‐10 production in α‐GalCer/LPS‐treated mice. These results suggest that the attenuated accumulation of Treg cells may be involved in the development of severe ARDS through a reduction in the synthesis of IL‐10.

     

The anti‐seizure drugs vinpocetine and carbamazepine,but not valproic acid,reduce inflammatory IL‐1β and TNF‐α expression in rat hippocampus

In the present study, the effects of the two classical anti‐epileptic drugs, carbamazepine and valproic acid, and the non‐classical anti‐seizure drug vinpocetine were investigated on the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in the hippocampus of rats by PCR or western blot after the administration of one or seven doses. Next, the effects of the anti‐seizure drugs were investigated on the rise in cytokine expression induced by lipopolysaccharides (LPS) inoculation in vivo. To validate our methods, the changes induced by the pro‐convulsive agents 4‐aminopyridine, pentylenetetrazole and pilocarpine were also tested. Finally, the effect of the anti‐seizure drugs on seizures and on the concomitant rise in pro‐inflammatory cytokine expression induced by 4‐aminopyridine was explored. Results show that vinpocetine and carbamazepine reduced the expression of IL‐1β and TNF‐α from basal conditions, and the increase in both pro‐inflammatory cytokines induced by LPS. In contrast, valproic acid failed to reduce both the expression of the cytokines from basal conditions and the rise in IL‐1β and TNF‐α expression induced by LPS. Tonic‐clonic seizures induced either by 4‐aminopyridine, pentylenetetrazole or pilocarpine increased the expression of IL‐1β and TNF‐α markedly. 4‐aminopyridine‐induced changes were reduced by all the tested anti‐seizure drugs, although valproic acid was less effective. We conclude that the anti‐seizure drugs, vinpocetine and carbamazepine, whose mechanisms of action involve a decrease in ion channels permeability, also reduce cerebral inflammation.

     

Effect of an acute moderate‐exercise session on metabolic and inflammatory profile of PPAR‐α knockout mice

Peroxisome proliferator‐activated receptors (PPARs) play a major role in metabolism and inflammatory control. Exercise can modulate PPAR expression in skeletal muscle, adipose tissue, and macrophages. Little is known about the effects of PPAR‐α in metabolic profile and cytokine secretion after acute exercise in macrophages. In this context, the aim of this study was to understand the influence of PPAR‐α on exercise‐mediated immune metabolic parameters in peritoneal macrophages. Mice C57BL/6 (WT) and PPAR‐α knockout (KO) were examined in non‐exercising control (n = 4) or 24 hours after acute moderate exercise (n = 8). Metabolic parameters (glucose, non‐esterified fatty acids, total cholesterol [TC], and triacylglycerol [TG]) were assessed in serum. Cytokine concentrations (IL‐1β, IL‐6, IL‐10, TNF‐α, and MCP‐1) were measured from peritoneal macrophages cultured or not with LPS (2.5 μg/mL) and Rosiglitazone (1 μM). Exercised KO mice exhibited low glucose concentration and higher TC and TG in serum. At baseline, no difference in cytokine production between the genotypes was observed. However, IL‐1β was significantly higher in KO mice after LPS stimulus. IL‐6 and IL‐1β had increased concentrations in KO compared with WT, even after exercise. MCP‐1 was not restored in exercised KO LPS group. Rosiglitazone was not able to reduce proinflammatory cytokine production in KO mice at baseline level or associated with exercise. Acute exercise did not alter mRNA expression in WT mice. Conclusion: PPAR‐α seems to be needed for metabolic glucose homeostasis and anti‐inflammatory effect of acute exercise. Its absence may induce over‐expression of pro‐inflammatory cytokines in LPS stimulus. Moreover, moderate exercise or PPAR‐γ agonist did not reverse this response.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Compressive mechanical force augments osteoclastogenesis by bone marrow macrophages through activation of c‐Fms‐mediated signaling

Little is known about the effects of mechanical forces on osteoclastogenesis by bone marrow macrophages (BMMs) in the absence of mechanosensitive cells, including osteoblasts and fibroblasts. In this study, we examined the effects of mechanical force on osteoclastogenesis by applying centrifugal force to BMMs using a horizontal microplate rotor. Our findings, as measured by an in vitro model system, show that tumor necrosis factor (TNF)‐α is capable of inducing osteoclast differentiation from BMMs and bone resorption in the presence of macrophage‐colony stimulating factor (M‐CSF) and is further facilitated by receptor activator of nuclear factor‐kappaB (NF‐κB) ligand (RANKL). Application of force to BMMs accelerated TNF‐α‐induced osteoclastogenesis; this was inhibited either by anti‐TNF‐α or anti‐TNF‐α receptor but not by OPG. TNF‐α also increased c‐Fms expression at both mRNA and protein levels in BMMs. An anti‐c‐Fms antibody completely inhibited osteoclast differentiation and bone resorption induced by TNF‐α but partially blocked osteoclastogenesis stimulated in combination with RANKL. These results suggest that TNF‐α (in the presence of M‐CSF) is capable of inducing osteoclastogenesis from BMMs, and that osteoclastogenesis is significantly stimulated by force application through the activation of c‐Fms‐mediated signaling. Overall, the present study reveals the facilitating effect of mechanical force on osteoclastic differentiation from BMMs without the addition of mechanosensitive cells. J. Cell. Biochem. 111: 1260–1269, 2010. © 2010 Wiley‐Liss, Inc.     

Sodium salicylate modulates inflammatory responses through AMP‐activated protein kinase activation in LPS‐stimulated THP‐1 cells

Sodium salicylate (NaSal) is a nonsteroidal anti‐inflammatory drug. The putative mechanisms for NaSal's pharmacologic actions include the inhibition of cyclooxygenases, platelet‐derived thromboxane A2, and NF‐κB signaling. Recent studies demonstrated that salicylate could activate AMP‐activated protein kinase (AMPK), an energy sensor that maintains the balance between ATP production and consumption. The anti‐inflammatory action of AMPK has been reported to be mediated by promoting mitochondrial biogenesis and fatty acid oxidation. However, the exact signals responsible for salicylate‐mediated inflammation through AMPK are not well‐understood. In the current study, we examined the potential effects of NaSal on inflammation‐like responses of THP‐1 monocytes to lipopolysaccharide (LPS) challenge. THP‐1 cells were stimulated with or without 10 ug/mL LPS for 24 h in the presence or absence of 5 mM NaSal. Apoptosis was measured by flow cytometry using Annexin V/PI staining and by Western blotting for the Bcl‐2 anti‐apoptotic protein. Cell proliferation was detected by EdU incorporation and by Western blot analysis for proliferating cell nuclear antigen (PCNA). Secretion of pro‐inflammatory cytokines (TNF‐α, IL‐1β, IL‐6) was determined by enzyme‐linked immunosorbent assay (ELISA). We observed that the activation of AMPK by NaSal was accompanied by induction of apoptosis, inhibition of cell proliferation, and increasing secretion of TNF‐α and IL‐1β. These effects were reversed by Compound C, an inhibitor of AMPK. In addition, NaSal/AMPK activation inhibited LPS‐induced STAT3 phosphorylation, which was reversed by Compound C treatment. We conclude that AMPK activation is important for NaSal‐mediated inflammation by inducing apoptosis, reducing cell proliferation, inhibiting STAT3 activity, and producing TNF‐α and IL‐1β.     

Interferon‐γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)‐1 and transforming growth factor (TGF) β2

Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN‐α or IFN‐γ downregulated Fli1 and VE‐cadherin. In contrast, IFN‐α and IFN‐γ exerted opposite effects on the expression of α‐SMA, CTGF, ET‐1, and TGFβ2, with IFN‐α downregulating and IFN‐γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN‐γ‐mediated changes in Fli1, VE‐cadherin, CTGF, and ET‐1 levels, whereas upregulation of α‐SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN‐γ, including downregulation of α‐SMA and TGFβ2, suggesting that activation of the ET‐1 pathway plays a main role in the IFN‐γ responses in HDMECs. IFN‐γ induced expression of selected genes related to endothelial‐to‐mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN‐γ on EndoMT was mediated via TGFβ2 and ET‐1 signaling pathways. This study demonstrates distinct effects of IFN‐α and IFN‐γ on the biology of vascular endothelial cells. IFN‐γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT. J. Cell. Physiol. 228: 1774–1783, 2013. © 2013 Wiley Periodicals, Inc.     

Sodium hydrosulphide restores tumour necrosis factor‐α‐induced mitochondrial dysfunction and metabolic dysregulation in HL‐1 cells

Tumour necrosis factor (TNF)‐α induces cardiac metabolic disorder and mitochondrial dysfunction. Hydrogen sulphide (H₂S) contains anti‐inflammatory and biological effects in cardiomyocytes. This study investigated whether H₂S modulates TNF‐α‐dysregulated mitochondrial function and metabolism in cardiomyocytes. HL‐1 cells were incubated with TNF‐α (25 ng/mL) with or without sodium hydrosulphide (NaHS, 0.1 mmol/L) for 24 hours. Cardiac peroxisome proliferator‐activated receptor (PPAR) isoforms, pro‐inflammatory cytokines, receptor for advanced glycation end products (RAGE) and fatty acid metabolism were evaluated through Western blotting. The mitochondrial oxygen consumption rate and adenosine triphosphate (ATP) production were investigated using Seahorse XF24 extracellular flux analyzer and bioluminescence assay. Fluorescence intensity using 2′, 7′‐dichlorodihydrofluorescein diacetate was used to evaluate mitochondrial oxidative stress. NaHS attenuated the impaired basal and maximal respiration, ATP production and ATP synthesis and enhanced mitochondrial oxidative stress in TNF‐α‐treated HL‐1 cells. TNF‐α‐treated HL‐1 cells exhibited lower expression of PPAR‐α, PPAR‐δ, phosphorylated 5′ adenosine monophosphate‐activated protein kinase‐α2, phosphorylated acetyl CoA carboxylase, carnitine palmitoyltransferase‐1, PPAR‐γ coactivator 1‐α and diacylglycerol acyltransferase 1 protein, but higher expression of PPAR‐γ, interleukin‐6 and RAGE protein than control or combined NaHS and TNF‐α‐treated HL‐1 cells. NaHS modulates the effects of TNF‐α on mitochondria and the cardiometabolic system, suggesting its therapeutic potential for inflammation‐induced cardiac dysfunction.     

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.     

Evidence for the prevention of enthesitis in HLA‐B27/hβ2m transgenic rats treated with a monoclonal antibody against TNF‐α

Transgenic rats with high expression of HLA‐B27 and human β₂‐microglobulin (B27TR) develop a multisystem inflammatory disease resembling human inflammatory bowel disease (IBD) and spondyloarthropaties (SpA). Tumour necrosis factor α (TNF‐α) has a crucial role in sustaining chronic inflammation in the gut and joints. The aim of this work was to evaluate whether TNF‐α blockade could prevent or reduce the inflammation of peripheral joints in B27TR. A first group of 9‐week‐old B27TR received an anti‐TNF‐α monoclonal antibody (mAb) or an isotypic IgG2a,k up to the age of 18 weeks. An untreated group was monitored up to the age of 18 weeks and then randomly assigned to a 9‐week treatment with anti‐TNF‐α mAb or IgG2a,k. Each rat was monitored for clinical IBD and peripheral joint manifestations. After sacrifice the colon and hind paws were examined for macroscopical and microscopical pathological changes. Early TNF‐α blockade prevented, and late treatment improved IBD signs in B27TR. Erythema, oedema, inflammatory infiltrate close to the tendons and enthesis, proliferating chondrocyte‐like cells, signs of new endochondral bone ossification and bone erosion were observed in peripheral joints of four out of six IgG2a,k‐treated B27TR, both at 18 and 27 weeks. Immunopositivity for phosphorylated Smad1/5/8 indicated that the process of joint remodelling was activated in B27TR. Some entheses showed chondroid nodules. Anti‐TNF‐α treatment reduced inflammation and preserved the enthesis organization in most animals. Occasional and transient erythema and oedema were still present in three of six of the late anti‐TNF‐α‐treated animals. Smad1/5/8 signalling was not inhibited by late anti‐TNF‐α treatment. In B27TR, articular involvement follows IBD onset and develops at entheses. Early TNF‐α blockade prevents the onset of IBD and consequently the development of enthesitis in peripheral joints in the B27TR model of human SpA.